# Genetic manipulation of giant viruses and their host, Acanthamoeba castellanii

Nadege Philippe, Avi Shukla, Chantal Abergel, Hugo Bisio

<figure><img src="image_1.png" /></figure>

## To cite this version:

Nadege Philippe, Avi Shukla, Chantal Abergel, Hugo Bisio. Genetic manipulation of giant viruses and their host, Acanthamoeba castellanii. Nature Protocols, 2023, 19 (1), pp.3-29. 10.1038/s41596-023-00910-y. hal-04590186

HAL Id: hal-04590186

https://amu.hal.science/hal-04590186

Submitted on 28 May 2024

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Distributed under a Creative Commons Attribution 4.0 International License
# Genetic manipulation of giant viruses and their host, Acanthamoeba castellanii

Nadege Philippe, Avi Shukla, Chantal Abergel, Hugo Bisio

► To cite this version:

Nadege Philippe, Avi Shukla, Chantal Abergel, Hugo Bisio. Genetic manipulation of giant viruses and their host, Acanthamoeba castellanii. Nature Protocols, 2023, 19 (1), pp.3-29. 10.1038/s41596-023-00910-y. hal-04590186

HAL Id: hal-04590186

https://amu.hal.science/hal-04590186v1

Submitted on 28 May 2024

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
1 **Ready for Production**

2

3 **H1 Genetic manipulation of giant viruses and their host *Acanthamoeba castellanii***

4 Nadege Philippe,¹, ² Avi Shukla,¹ Chantal Abergel,¹, * Hugo Bisio,¹, ²,*

5 ¹Aix-Marseille University, Centre National de la Recherche Scientifique, Information Genomique &

6 Structurale, Unite Mixte de Recherche 7256 (Institut de Microbiologie de la Mediterranee, FR3479, IM2B),

7 13288 Marseille Cedex 9, France.

8 ²These authors contributed equally: Hugo Bisio, Nadege Philippe.

9 *Correspondence: hugo.bisio@igs.cnrs-mrs.fr (H.B.), chantal.abergel@igs.cnrs-mrs.fr (C.A.).

10

11 **H1 Editorial Summary:** This protocol enables genetic manipulation of nuclear and cytoplasmic giant

12 viruses, and their host *Acanthamoeba castellani*, using either CRISPR/Cas9 or homologous

13 recombination.

14

15 **H1 Key references**

16 Liu, Y. et al. Virus-encoded histone doublets are essential and form nucleosome-like structures. Cell 184,

17 4237-4250 e4219 (2021). https://doi.org:10.1016/j.cell.2021.06.032

18 Bisio, H. et al. Evolution of giant pandoravirus revealed by CRISPR/Cas9. Nat Commun 14, 428 (2023).

19 https://doi.org:10.1038/s41467-023-36145-4

20 Alempic, J-M. et al. No fitness impact of the knockout of the two main components of mimivirus genomic

21 fiber and fibril layer. bioRxiv, 2023.04.28.538727 (2023). https://doi.org/10.1101/2023.04.28.538727

22

23 **H1 Key points**

24 • This protocol enables genetic manipulation of nuclear and cytoplasmic giant viruses, and their host *Acanthamoeba castellanii*, using either CRISPR/Cas9 or homologous recombination.

25 • The methodology allows the generation of stable gene modifications, which wasn't possible using RNA silencing, the only previously available reverse genetic tool.

26 **H1 ABSTRACT**

27 Giant viruses (GVs) provide an unprecedented source of genetic innovation in the viral world and are thus, besides their importance in basic and environmental virology, in the spotlight for bioengineering advances.

28 Their host, *Acanthamoeba castellanii* is an accidental human pathogen that acts as a natural host and environmental reservoir of other human pathogens. Tools for genetic manipulation of viruses and host

29 were lacking. Here, we provide a detailed method for genetic manipulation of *A. castellanii* and the GVs it

30 plays host to by utilizing either CRISPR/Cas9 and/or homologous recombination. We detail the steps of
1 **Ready for Production**

2

3 **H1 Genetic manipulation of giant viruses and their host *Acanthamoeba castellanii***

4 Nadege Philippe,¹, ² Avi Shukla,¹ Chantal Abergel,¹, * Hugo Bisio,¹, ²,*

5 ¹Aix-Marseille University, Centre National de la Recherche Scientifique, Information Genomique &

6 Structurale, Unite Mixte de Recherche 7256 (Institut de Microbiologie de la Mediterranee, FR3479, IM2B),

7 13288 Marseille Cedex 9, France.

8 ²These authors contributed equally: Hugo Bisio, Nadege Philippe.

9 *Correspondence: hugo.bisio@igs.cnrs-mrs.fr (H.B.), chantal.abergel@igs.cnrs-mrs.fr (C.A.).

10

11 **H1 Editorial Summary:** This protocol enables genetic manipulation of nuclear and cytoplasmic giant

12 viruses, and their host *Acanthamoeba castellani*, using either CRISPR/Cas9 or homologous

13 recombination.

14

15 **H1 Key references**

16 Liu, Y. et al. Virus-encoded histone doublets are essential and form nucleosome-like structures. Cell 184,

17 4237-4250 e4219 (2021). https://doi.org:10.1016/j.cell.2021.06.032

18 Bisio, H. et al. Evolution of giant pandoravirus revealed by CRISPR/Cas9. Nat Commun 14, 428 (2023).

19 https://doi.org:10.1038/s41467-023-36145-4

20 Alempic, J-M. et al. No fitness impact of the knockout of the two main components of mimivirus genomic

21 fiber and fibril layer. bioRxiv, 2023.04.28.538727 (2023). https://doi.org/10.1101/2023.04.28.538727

22

23 **H1 Key points**

24 • This protocol enables genetic manipulation of nuclear and cytoplasmic giant viruses, and their host *Acanthamoeba castellanii*, using either CRISPR/Cas9 or homologous recombination.

25 • The methodology allows the generation of stable gene modifications, which wasn't possible using RNA silencing, the only previously available reverse genetic tool.

26 **H1 ABSTRACT**

27 Giant viruses (GVs) provide an unprecedented source of genetic innovation in the viral world and are thus, besides their importance in basic and environmental virology, in the spotlight for bioengineering advances.

28 Their host, *Acanthamoeba castellanii* is an accidental human pathogen that acts as a natural host and environmental reservoir of other human pathogens. Tools for genetic manipulation of viruses and host

29 were lacking. Here, we provide a detailed method for genetic manipulation of *A. castellanii* and the GVs it

30 plays host to by utilizing either CRISPR/Cas9 and/or homologous recombination. We detail the steps of
vector preparation (4 days), transfection of amoeba cells (1 h), infection (1 h), selection (5 days for viruses, 2 weeks for amoebas), and cloning of recombinant viruses (4 days) or amoebas (2 weeks). This procedure takes about 3 weeks or a month for the generation of recombinant viruses or amoebas, respectively. This methodology allows the generation of stable gene modifications, which wasn't possible using RNA silencing, the only previously available reverse genetic tool. We also include detailed sample-preparation steps for protein localization by immunofluorescence (4 hs), western blotting (4 hs), quantification of viral particles by optical density (15 min), calculation of viral lethal dose 50 (7 days) and quantification of DNA replication by quantitative PCR (4 hs) to allow efficient broad phenotyping of recombinant organisms. This methodology allows the function of thousands of ORFan genes present in GVs, as well as the complex pathogen-host, pathogen-pathogen, or pathogen-symbiont interactions in *A. castellanii* to be studied *in vivo*.

## H1 INTRODUCTION

*Acanthamoeba spp.* is an opportunistic human pathogen with increasing importance for ocular health¹. Moreover, this organism has been largely recognized as a host and environmental reservoir for pathogenic organisms like Legionella, Mycobacterium, or Cryptococcus². More recently, *Acanthamoeba castellanii* has also been utilized as an experimental host for the discovery of several giant viruses (GVs), which contain genomes of up to 2.8 megabases and viral particles exceeding 1 µm in size³⁻⁵. Different examples of GVs include Pandoravirus salinus (linear 2.8 Mb genome, 1µ particle)⁴, Mollivirus sibericum (linear 0.65 Mb genome, 500nm particle)⁶, Pithovirus sibericum (circular 0.6 Mb genome, 1.5µM particles)⁵ and Mimivirus (linear 1.2 Mb genome, 400nm particle)³. Importantly, GV not only play key roles in ecology but also 70% of the thousands of genes encoded by these organisms are ORFan genes (unrelated to any other protein known so far)⁷. Thus, these viruses represent an unexplored resource of innovations for bioengineering advances⁸. Yet, genetic manipulation of both GV and their host *A. castellanii*, has been largely neglected due to the difficulty in implementing it on these organisms. Specifically, *A. castellanii* is a highly polyploid organism (25n approx.)⁹ with amitotic nuclear division leading to aneuploidy and random segregation of genetic material¹⁰,¹¹. Moreover, selection markers for drug resistance are scarce (currently limited to two, geneticin¹² and nourseothricin¹³). Cytoplasmic GV protect their DNA by establishing viral factories¹⁴ which segregate their DNA from protein effectors like Cas9 (ref. 15). Thus, previous gene functional assessment was mostly accomplished by RNA silencing¹⁶,¹⁷. Here we describe a protocol for rapid genetic engineering of both fully cytoplasmic and nuclear GV as well as their host *A. castellanii*. CRISPR/Cas9 can be utilized for the modification of nuclear GV and *A. castellanii*, while both cytoplasmic and nuclear GV can also be genetically engineered by recombination in combination with selection markers. Investigators should be able to obtain recombinant viruses in 3 weeks and recombinant amoebas in a month.

## H2 Overview of the genetic manipulation techniques.

A schematic overview of our protocols is provided in Figure 1. Our methods utilise both homologous recombination (HR) and non-homologous-end joining (NHEJ) for modifications of the genome of amoeba and viral DNA¹³,¹⁵,¹⁸. CRISPR-Cas (Clustered Regularly Interspersed Short Palindromic Repeats) has proved invaluable for the genetic manipulation of many organisms, based on the tunable selectivity of a dsDNA endonuclease (like Cas9) for a 20 base pair sequence-specific region of the genome¹⁹. Such selectivity is
vector preparation (4 days), transfection of amoeba cells (1 h), infection (1 h), selection (5 days for viruses, 2 weeks for amoebas), and cloning of recombinant viruses (4 days) or amoebas (2 weeks). This procedure takes about 3 weeks or a month for the generation of recombinant viruses or amoebas, respectively. This methodology allows the generation of stable gene modifications, which wasn't possible using RNA silencing, the only previously available reverse genetic tool. We also include detailed sample-preparation steps for protein localization by immunofluorescence (4 hs), western blotting (4 hs), quantification of viral particles by optical density (15 min), calculation of viral lethal dose 50 (7 days) and quantification of DNA replication by quantitative PCR (4 hs) to allow efficient broad phenotyping of recombinant organisms. This methodology allows the function of thousands of ORFan genes present in GVs, as well as the complex pathogen-host, pathogen-pathogen, or pathogen-symbiont interactions in *A. castellanii* to be studied *in vivo*.

## H1 INTRODUCTION

*Acanthamoeba spp.* is an opportunistic human pathogen with increasing importance for ocular health¹. Moreover, this organism has been largely recognized as a host and environmental reservoir for pathogenic organisms like Legionella, Mycobacterium, or Cryptococcus². More recently, *Acanthamoeba castellanii* has also been utilized as an experimental host for the discovery of several giant viruses (GVs), which contain genomes of up to 2.8 megabases and viral particles exceeding 1 µm in size³⁻⁵. Different examples of GVs include Pandoravirus salinus (linear 2.8 Mb genome, 1µ particle)⁴, Mollivirus sibericum (linear 0.65 Mb genome, 500nm particle)⁶, Pithovirus sibericum (circular 0.6 Mb genome, 1.5µM particles)⁵ and Mimivirus (linear 1.2 Mb genome, 400nm particle)³. Importantly, GV not only play key roles in ecology but also 70% of the thousands of genes encoded by these organisms are ORFan genes (unrelated to any other protein known so far)⁷. Thus, these viruses represent an unexplored resource of innovations for bioengineering advances⁸. Yet, genetic manipulation of both GV and their host *A. castellanii*, has been largely neglected due to the difficulty in implementing it on these organisms. Specifically, *A. castellanii* is a highly polyploid organism (25n approx.)⁹ with amitotic nuclear division leading to aneuploidy and random segregation of genetic material¹⁰,¹¹. Moreover, selection markers for drug resistance are scarce (currently limited to two, geneticin¹² and nourseothricin¹³). Cytoplasmic GV protect their DNA by establishing viral factories¹⁴ which segregate their DNA from protein effectors like Cas9 (ref. 15). Thus, previous gene functional assessment was mostly accomplished by RNA silencing¹⁶,¹⁷. Here we describe a protocol for rapid genetic engineering of both fully cytoplasmic and nuclear GV as well as their host *A. castellanii*. CRISPR/Cas9 can be utilized for the modification of nuclear GV and *A. castellanii*, while both cytoplasmic and nuclear GV can also be genetically engineered by recombination in combination with selection markers. Investigators should be able to obtain recombinant viruses in 3 weeks and recombinant amoebas in a month.

## H2 Overview of the genetic manipulation techniques.

A schematic overview of our protocols is provided in Figure 1. Our methods utilise both homologous recombination (HR) and non-homologous-end joining (NHEJ) for modifications of the genome of amoeba and viral DNA¹³,¹⁵,¹⁸. CRISPR-Cas (Clustered Regularly Interspersed Short Palindromic Repeats) has proved invaluable for the genetic manipulation of many organisms, based on the tunable selectivity of a dsDNA endonuclease (like Cas9) for a 20 base pair sequence-specific region of the genome¹⁹. Such selectivity is
accomplished by annealing of the DNA with a guide RNA (gRNA) which binds to the endonuclease and directs it to its targeting site¹⁹. Double strand breaks generated by Cas9 can consequently be repaired by two major mechanisms: HR and NHEJ. Particularly relevant for this protocol, we have demonstrated that CRISPR/Cas9 can be efficiently used for manipulation of the genome of *A. castellanii* and nuclear GV's like mollivirus and pandoravirus¹⁵. We have utilized this technology to generate knockouts in *A. castellanii* and nuclear GV's by inducing NHEJ repair¹⁵. In addition, HR can also be exploited in the absence of double-strand breaks to prompt modifications on the genomic DNA. Thus, when combined with selection markers for the enrichment of recombinant organisms, it can also be utilized for genetic engineering independently of CRISPR-based modifications. We have demonstrated that HR can be efficiently used for the modification of cytoplasmic GV's like marseillevirus and mimivirus¹⁸, but also for nuclear GV's like mollivirus and pandoravirus¹⁵. HR allows not only the generation of gene knockouts but also the endogenous tagging of genes (for protein localization) or the introduction of exogenous DNA (i.e. for cis-complementation)¹⁵.

## H2 Comparison with previous reverse genetic techniques.

Reverse genetics of GV's and *A. castellanii* were limited to RNA silencing before we developed genetic tools for these organisms. While silencing has proven efficient for the functional analysis of *A. castellanii* genes, particularly for genes involved in encystment¹⁶,²⁰, gene knockdowns are usually only partial and transient²¹. Thus, silencing is mostly limited to a certain window of time and the study of vulnerable genes²² (i.e. genes where low expression inhibition will result in a decrease in organismal fitness). Moreover, highly stable proteins might also be difficult to downregulate, hampering their study by RNA silencing (i.e. highly stable proteins might take days for efficient protein downregulation²³). On the other hand, gene knockout of essential genes is lethal and thus provides only partial information about the functionality of a gene. In such cases, RNA silencing might result in a better way to address the functionality of a gene. Since conditional knockout in *A. castellanii* has not been developed so far, RNA silencing is the only available technique for the study of essential genes.

Gene function has also been previously studied by RNA silencing in mimivirus (a cytoplasmic GV)¹⁷,²⁴,²⁵. While the same pros and cons that were discussed for amoeba reverse genetics apply to GV's, only 3 publications have been shown to be successful in applying this technology for the silencing of GV's genes (all in mimivirus)¹⁷,²⁴,²⁵. Reports associated to the use of RNA silencing of mimiviral genes were generated by a single research team and no other group has reported successful reverse genetics using this tool. Moreover, the study of the essential genes of GV's can be studied by generating gene knock-out viruses in trans-complementing amoeba cells¹³, eliminating the usual advantage of gene knockdown over gene knockout to study essential genes. In addition, a previous study has described the replacement of the mimivirus gene R349 by green fluorescent protein using homologous recombination²⁶. Unfortunately, even when infected cells were sorted by the presence of GFP, the strategy did not allow the isolation of stable recombinant viruses²⁶ and has not been applied since its publication. We have overcome such limitations by introducing a selection cassette to allow enrichment of the recombinant viruses¹³,¹⁵,¹⁸.

Overall, while we believe that the field will be inclined to use the tools described in this protocol, traditional silencing approaches and emerging genetic tools will likely also be necessary to unveil different aspects of viral and host biology.
accomplished by annealing of the DNA with a guide RNA (gRNA) which binds to the endonuclease and directs it to its targeting site¹⁹. Double strand breaks generated by Cas9 can consequently be repaired by two major mechanisms: HR and NHEJ. Particularly relevant for this protocol, we have demonstrated that CRISPR/Cas9 can be efficiently used for manipulation of the genome of *A. castellanii* and nuclear GV's like mollivirus and pandoravirus¹⁵. We have utilized this technology to generate knockouts in *A. castellanii* and nuclear GV's by inducing NHEJ repair¹⁵. In addition, HR can also be exploited in the absence of double-strand breaks to prompt modifications on the genomic DNA. Thus, when combined with selection markers for the enrichment of recombinant organisms, it can also be utilized for genetic engineering independently of CRISPR-based modifications. We have demonstrated that HR can be efficiently used for the modification of cytoplasmic GV's like marseillevirus and mimivirus¹⁸, but also for nuclear GV's like mollivirus and pandoravirus¹⁵. HR allows not only the generation of gene knockouts but also the endogenous tagging of genes (for protein localization) or the introduction of exogenous DNA (i.e. for cis-complementation)¹⁵.

## H2 Comparison with previous reverse genetic techniques.

Reverse genetics of GV's and *A. castellanii* were limited to RNA silencing before we developed genetic tools for these organisms. While silencing has proven efficient for the functional analysis of *A. castellanii* genes, particularly for genes involved in encystment¹⁶,²⁰, gene knockdowns are usually only partial and transient²¹. Thus, silencing is mostly limited to a certain window of time and the study of vulnerable genes²² (i.e. genes where low expression inhibition will result in a decrease in organismal fitness). Moreover, highly stable proteins might also be difficult to downregulate, hampering their study by RNA silencing (i.e. highly stable proteins might take days for efficient protein downregulation²³). On the other hand, gene knockout of essential genes is lethal and thus provides only partial information about the functionality of a gene. In such cases, RNA silencing might result in a better way to address the functionality of a gene. Since conditional knockout in *A. castellanii* has not been developed so far, RNA silencing is the only available technique for the study of essential genes.

Gene function has also been previously studied by RNA silencing in mimivirus (a cytoplasmic GV)¹⁷,²⁴,²⁵. While the same pros and cons that were discussed for amoeba reverse genetics apply to GV's, only 3 publications have been shown to be successful in applying this technology for the silencing of GV's genes (all in mimivirus)¹⁷,²⁴,²⁵. Reports associated to the use of RNA silencing of mimiviral genes were generated by a single research team and no other group has reported successful reverse genetics using this tool. Moreover, the study of the essential genes of GV's can be studied by generating gene knock-out viruses in trans-complementing amoeba cells¹³, eliminating the usual advantage of gene knockdown over gene knockout to study essential genes. In addition, a previous study has described the replacement of the mimivirus gene R349 by green fluorescent protein using homologous recombination²⁶. Unfortunately, even when infected cells were sorted by the presence of GFP, the strategy did not allow the isolation of stable recombinant viruses²⁶ and has not been applied since its publication. We have overcome such limitations by introducing a selection cassette to allow enrichment of the recombinant viruses¹³,¹⁵,¹⁸.

Overall, while we believe that the field will be inclined to use the tools described in this protocol, traditional silencing approaches and emerging genetic tools will likely also be necessary to unveil different aspects of viral and host biology.
117 **H2 Potential applications and limitations of the method.**

118 The versatility and efficiency of CRISPR/Cas9 are exploited in this protocol for the genetic manipulation of
119 *A. castellanii*. This organism possesses about 25 copies of each chromosome⁹, thus 25 alleles of every gene
120 in their genome need to be modified to achieve successful gene knockout. Regardless, we have recently
121 shown that it is possible to target multicopy genes in a single experiment, using CRISPR/Cas9 with 100%
122 efficiency¹⁵. Specifically, we targeted the three genes of the cellulose synthase using 2 guide RNAs (gRNAs)
123 simultaneously and demonstrated modification in the 125 targeting sites (1 gRNA targeting the 3 genes
124 (3x25=75 target locus) and 1 gRNA targeting 2 of the genes (2x25=50 target locus)) without traces of the
125 wild-type alleles¹⁵. Such modifications were performed by NHEJ, generating random indels (insertion-
126 deletions). Moreover, we have shown that upon Cas9-mediated cleavage of *A. castellanii* genome, small
127 indels are preferentially generated upon repair of the targeting site¹⁵, thus, making it necessary to perform
128 extensive genotyping to demonstrate non-sense modification and complete gene knockout. Importantly,
129 while this limitation of the technology needs to be addressed in every experiment by cloning of
130 recombinant amoebas and sequencing to identify amoebas with non-sense mutations, the use of
131 polycistronic gRNA expression allows users to partially bypass such issues. Specifically, the use of multiple
132 gRNAs targeting a single gene significantly decreases the chances of getting functional recombinant genes
133 by increasing the sites to be repair by NHEJ and thus, the generation of potential non-sense mutations.
134 Thus, we expect that the use of such tools for the manipulation of *A. castellanii* will allow gene functionality
135 to be studied in this human-pathogenic organism as well as the complex sets of interactions among
136 microorganisms resistant to amoeba digestion which have made *A. castellanii* their environmental host.

137 Nuclear and cytoplasmic GVs modification can be easily achieved by recombination techniques¹³,¹⁵,¹⁸. We
138 have currently set up selection cassettes that allow efficient selection of recombinant viruses in
139 pandoravirus/mollivirus¹⁵ and mimivirus/megavirus¹⁸. Importantly, promoters from pandoravirus have
140 been used for the expression of selection cassettes in different pandoravirus and mollivirus¹⁵, while
141 megavirus promoters have been used to express selection cassettes in mimivirus and megavirus¹⁸. Thus,
142 our arsenal of vectors shows a broad usage for different viruses. Regulatory elements can also easily be
143 exchanged to modify other GVs. To achieve efficient drug resistance by virally encoded drug selection
144 cassettes, we have selected promoters with early and constant expression during the infectious cycle of
145 the virus. Such traits in viral promoters should be prioritized by any investigator wishing to adapt our
146 vectors for the modification of different viruses. Recombination allows precise modifications of the
147 genome of all viruses and has shown high rates of success in our experience. The main limitation of the
148 system lies in the limited amount of selection cassettes currently available (limited to two, geneticin¹² and
149 nourseothricin¹³). Thus, we are currently limited to two modifications per genome (only one if trans-
150 complementation is needed). Finally, the modification of essential genes remains challenging. It is worth
151 mentioning that marker-free recombinant viruses cannot be generated for cytoplasmic viruses since
152 selection cassettes are indispensable to achieve selection of recombinant viruses. On the other hand,
153 nuclear viruses can be generated marker-free by combining CRISPR/Cas9 and homologous recombination
154 to repair the cleaved DNA. Another limitation of the technique involves the study of essential genes due
155 to the current lack of conditional systems for gene inactivation. In order to address this issue, we have
156 developed a trans-complementation strategy to allow the expression of the gene to be knocked-out by
157 the amoeba¹³, bypassing the essentiality of the genes. The basis of trans-complementation is related to
158 the fact that independently of where transcription occurs, viral and amoeba mRNAs are transported into
159 the cytoplasm for translation. Thus, localization of the gene coding sequence is irrelevant for protein
117 **H2 Potential applications and limitations of the method.**

118 The versatility and efficiency of CRISPR/Cas9 are exploited in this protocol for the genetic manipulation of
119 *A. castellanii*. This organism possesses about 25 copies of each chromosome⁹, thus 25 alleles of every gene
120 in their genome need to be modified to achieve successful gene knockout. Regardless, we have recently
121 shown that it is possible to target multicopy genes in a single experiment, using CRISPR/Cas9 with 100%
122 efficiency¹⁵. Specifically, we targeted the three genes of the cellulose synthase using 2 guide RNAs (gRNAs)
123 simultaneously and demonstrated modification in the 125 targeting sites (1 gRNA targeting the 3 genes
124 (3x25=75 target locus) and 1 gRNA targeting 2 of the genes (2x25=50 target locus)) without traces of the
125 wild-type alleles¹⁵. Such modifications were performed by NHEJ, generating random indels (insertion-
126 deletions). Moreover, we have shown that upon Cas9-mediated cleavage of *A. castellanii* genome, small
127 indels are preferentially generated upon repair of the targeting site¹⁵, thus, making it necessary to perform
128 extensive genotyping to demonstrate non-sense modification and complete gene knockout. Importantly,
129 while this limitation of the technology needs to be addressed in every experiment by cloning of
130 recombinant amoebas and sequencing to identify amoebas with non-sense mutations, the use of
131 polycistronic gRNA expression allows users to partially bypass such issues. Specifically, the use of multiple
132 gRNAs targeting a single gene significantly decreases the chances of getting functional recombinant genes
133 by increasing the sites to be repair by NHEJ and thus, the generation of potential non-sense mutations.
134 Thus, we expect that the use of such tools for the manipulation of *A. castellanii* will allow gene functionality
135 to be studied in this human-pathogenic organism as well as the complex sets of interactions among
136 microorganisms resistant to amoeba digestion which have made *A. castellanii* their environmental host.

137 Nuclear and cytoplasmic GV modification can be easily achieved by recombination techniques13,15,18. We
138 have currently set up selection cassettes that allow efficient selection of recombinant viruses in
139 pandoravirus/mollivirus15 and mimivirus/megavirus18. Importantly, promoters from pandoravirus have
140 been used for the expression of selection cassettes in different pandoravirus and mollivirus15, while
141 megavirus promoters have been used to express selection cassettes in mimivirus and megavirus18. Thus,
142 our arsenal of vectors shows a broad usage for different viruses. Regulatory elements can also easily be
143 exchanged to modify other GV. To achieve efficient drug resistance by virally encoded drug selection
144 cassettes, we have selected promoters with early and constant expression during the infectious cycle of
145 the virus. Such traits in viral promoters should be prioritized by any investigator wishing to adapt our
146 vectors for the modification of different viruses. Recombination allows precise modifications of the
147 genome of all viruses and has shown high rates of success in our experience. The main limitation of the
148 system lies in the limited amount of selection cassettes currently available (limited to two, geneticin12 and
149 nourseothricin13). Thus, we are currently limited to two modifications per genome (only one if trans-
150 complementation is needed). Finally, the modification of essential genes remains challenging. It is worth
151 mentioning that marker-free recombinant viruses cannot be generated for cytoplasmic viruses since
152 selection cassettes are indispensable to achieve selection of recombinant viruses. On the other hand,
153 nuclear viruses can be generated marker-free by combining CRISPR/Cas9 and homologous recombination
154 to repair the cleaved DNA. Another limitation of the technique involves the study of essential genes due
155 to the current lack of conditional systems for gene inactivation. In order to address this issue, we have
156 developed a trans-complementation strategy to allow the expression of the gene to be knocked-out by
157 the amoeba13, bypassing the essentiality of the genes. The basis of trans-complementation is related to
158 the fact that independently of where transcription occurs, viral and amoeba mRNAs are transported into
159 the cytoplasm for translation. Thus, localization of the gene coding sequence is irrelevant for protein
160 production and functionality. This allows ectopic expression of viral genes from plasmids localized at the
161 nucleus of the amoeba to produce fully functional proteins. We have experienced that early expressed
162 genes are easier to trans-complement, while genes expressed late in the infectious cycle of the virus tend
163 not to be efficiently complemented in trans. These differences are likely related to protein expression
164 levels and/or timing. Thus, the success of this strategy is gene dependent and should be tested empirically.
165 Importantly, we suggest trans-complementation only as a tool to generate recombinant viruses with
166 modifications that otherwise would impair viral replication. To confirm that the phenotype observed upon
167 gene knockout is specific to the gene modification and not an off-target effect would be accomplished by
168 cis-complementation, by inserting the gene back at the right locus with the endogenous promoter.

169 Finally, CRISPR/Cas9 can also be used for the modification of nuclear GVs¹⁵. Cytoplasmic giant viruses
170 efficiently protect their DNA, rendering the use of Cas9 impossible¹⁵. Cas9 can be used to induce double-
171 strand breaks in the genome of nuclear GVs and allow NHEJ to generate recombinant viruses¹⁵. This
172 technique allows the generation of marker-free viruses and thus, an unlimited number of modifications.
173 NHEJ is however rather inefficient in pandoravirus and mollivirus and usually generates large deletions.
174 Accordingly, mutant viruses should be screened to isolate viruses with short deletions and avoid the
175 knockout of neighboring genes. Importantly, HR and CRISPR/Cas9 can be combined to generate marker-
176 free viruses with precise modifications, bypassing most of the limitations of the system and might be
177 preferred to NHEJ repair depending on the aims of the researcher. Regardless of the tools used for gene
178 modification, gene knockout of essential genes remains still challenging and the success of trans-
179 complementation should be empirically tested. Future efforts should be directed to generate vectors and
180 protocols to achieve conditional depletion of genes (like Cre recombinase²⁷, auxin-inducible degron²⁸ or
181 mRNA degradation²⁹) in order to attain the full potential of genetic manipulation in GVs.

182
183 **H2 Experimental design.**
184

185 We have currently designed vectors for endogenous tagging (HA and FLAG epitope tags) at the C-terminus
186 of proteins. These vectors can be adapted for N-terminal tagging or internal tagging if necessary (vector
187 maps and sequences are available at Addgene. IDs are provided in Table 1). A schematic representation of
188 the design is shown in Figure 2a. After the identification of a gene of interest, homology arms (250-500
189 bp) should be selected in the area where the tag will be inserted. If the tag is to be introduced at the C-
190 terminal of the protein, the stop codon should be excluded from the 5'-homology arm. Homology arms
191 can be amplified by PCR from genomic DNA (gDNA) using primers bearing homology to the tagging
192 acceptor vector (Table 1). These flanking regions allow the cloning of the homology arms by In-Fusion®
193 seamless cloning (TaKaRa) into the desired vector (Primer templates are shown in Table 2). Other cloning
194 methods can also be utilized to insert the homology arms in the vector.

195 Several critical steps should be considered. First, confirm that the tag is in frame with the open reading
196 frame of the gene of interest (goi) and that the endogenous stop codon was removed. We currently utilize
197 ApE plasmid editing software with this aim (https://jorgensen.biology.utah.edu/wayned/ape/). Second,
198 ensure that the homology arms do not contain the restriction sites used for the linearization of the vector.
199 If that is the case, a second restriction site (absent in the homology arms and the acceptor vector) can be
200 introduced in the primers. Alternatively, PCR amplification can be used as a substitute to plasmid digestion.
160 production and functionality. This allows ectopic expression of viral genes from plasmids localized at the
161 nucleus of the amoeba to produce fully functional proteins. We have experienced that early expressed
162 genes are easier to trans-complement, while genes expressed late in the infectious cycle of the virus tend
163 not to be efficiently complemented in trans. These differences are likely related to protein expression
164 levels and/or timing. Thus, the success of this strategy is gene dependent and should be tested empirically.
165 Importantly, we suggest trans-complementation only as a tool to generate recombinant viruses with
166 modifications that otherwise would impair viral replication. To confirm that the phenotype observed upon
167 gene knockout is specific to the gene modification and not an off-target effect would be accomplished by
168 cis-complementation, by inserting the gene back at the right locus with the endogenous promoter.

169 Finally, CRISPR/Cas9 can also be used for the modification of nuclear GVs¹⁵. Cytoplasmic giant viruses
170 efficiently protect their DNA, rendering the use of Cas9 impossible¹⁵. Cas9 can be used to induce double-
171 strand breaks in the genome of nuclear GVs and allow NHEJ to generate recombinant viruses¹⁵. This
172 technique allows the generation of marker-free viruses and thus, an unlimited number of modifications.
173 NHEJ is however rather inefficient in pandoravirus and mollivirus and usually generates large deletions.
174 Accordingly, mutant viruses should be screened to isolate viruses with short deletions and avoid the
175 knockout of neighboring genes. Importantly, HR and CRISPR/Cas9 can be combined to generate marker-
176 free viruses with precise modifications, bypassing most of the limitations of the system and might be
177 preferred to NHEJ repair depending on the aims of the researcher. Regardless of the tools used for gene
178 modification, gene knockout of essential genes remains still challenging and the success of trans-
179 complementation should be empirically tested. Future efforts should be directed to generate vectors and
180 protocols to achieve conditional depletion of genes (like Cre recombinase²⁷, auxin-inducible degron²⁸ or
181 mRNA degradation²⁹) in order to attain the full potential of genetic manipulation in GVs.

182
183 **H2 Experimental design.**
184

185 We have currently designed vectors for endogenous tagging (HA and FLAG epitope tags) at the C-terminus
186 of proteins. These vectors can be adapted for N-terminal tagging or internal tagging if necessary (vector
187 maps and sequences are available at Addgene. IDs are provided in Table 1). A schematic representation of
188 the design is shown in Figure 2a. After the identification of a gene of interest, homology arms (250-500
189 bp) should be selected in the area where the tag will be inserted. If the tag is to be introduced at the C-
190 terminal of the protein, the stop codon should be excluded from the 5'-homology arm. Homology arms
191 can be amplified by PCR from genomic DNA (gDNA) using primers bearing homology to the tagging
192 acceptor vector (Table 1). These flanking regions allow the cloning of the homology arms by In-Fusion®
193 seamless cloning (TaKaRa) into the desired vector (Primer templates are shown in Table 2). Other cloning
194 methods can also be utilized to insert the homology arms in the vector.

195 Several critical steps should be considered. First, confirm that the tag is in frame with the open reading
196 frame of the gene of interest (goi) and that the endogenous stop codon was removed. We currently utilize
197 ApE plasmid editing software with this aim (https://jorgensen.biology.utah.edu/wayned/ape/). Second,
198 ensure that the homology arms do not contain the restriction sites used for the linearization of the vector.
199 If that is the case, a second restriction site (absent in the homology arms and the acceptor vector) can be
200 introduced in the primers. Alternatively, PCR amplification can be used as a substitute to plasmid digestion.
Finally, in order to efficiently assess integration and clonality of recombinant lines, a negative control must be produced for every independent set of transfections. This negative control is done by incubating amoebas with PolyFect Transfection Reagent but no DNA. Infection and drug selection are performed in parallel with other samples. Differences in viral growth as well as DNA amplification by PCR will be critical to assess if the experiment is successful or not.

## H3 Gene knockout by HR

Vectors for gene knockout using HR in mimivirus/megavirus and pandoravirus/mollivirus are currently available (Table 1). Homology arms of about 250-500 bp should be selected to target a gene of interest as shown in Figure 2b. We recommend designing two homology arms, wherein 5' arm covers the beginning of the gene whereas the 3' arm covers the end of the gene, allowing the deletion of a section of the coding region. Homology arms can be amplified by PCR from gDNA using primers bearing homology to the tagging acceptor vector (Table 1). These flanking regions allow the cloning of the homology arms by In-Fusion® seamless cloning (TaKaRa) into the desired vector (Primer templates are shown in Table 2). Any other cloning method can also be used.

Several critical steps should be considered. First, ensure that the homology arms do not contain the restriction sites used for the linearization of the vector. If that is the case, a second restriction site (absent in the homology arms and the acceptor vector) can be introduced in the primers. Alternatively, PCR amplification can be used as a substitute to plasmid digestion. Finally, negative control as described in *Endogenous tagging* section must be included.

## H3 Gene knock out by CRISPR/Cas9

The basic principles of CRISPR/Cas9 and the rules to select appropriate gRNA sequences can be found in³⁰. gRNA target sequences must include a PAM (protospacer adjacent motif) sequence and be significantly different from other sequences in the genome of *A. castellanii* (if gene knockout will be performed in this organism) or both *A. castellanii* and nuclear GV (if gene knockout will be performed in a nuclear GV). We currently utilize the Eukaryotic Pathogen CRISPR gRNA Design Tool (EuPaGDT)(http://grna.ctegd.uga.edu/), which allows users to rank gRNA target sequences (by analyzing GC content and flanking microhomology pair score) and analyzes potential off-target editing in the genome of *A. castellanii*. Potential off-target sites are manually identified by BLASTn using the reference genome of the GV to be genetically modified. We remind any researcher interested in carrying out gene editing by CRISPR/Cas9 that potential off-target sites have a lower risk of cleavage when mismatches with the 3' (seeding region) of the gRNA or lack of a PAM are observed. Once one or multiple gRNAs are selected, these can be inserted into vector vHB8 by mutagenesis (Figure 3). Primers should be designed containing the new gRNAs and bear homology sequences to clone the gRNAs by In-Fusion® seamless cloning (TaKaRa) into vHB8 (Primer templates are shown in Table 2).

Importantly, a second plasmid encoding for Cas9 and gRNAs which do not target the desired genome should be generated and transfected simultaneously with any set of transfections. Selection and genotyping must be done in parallel with other samples in order to assess if the experiment is successful or not. If amoebas are being genetically modified, this strain will be the baseline control for any phenotypic characterization.

## H3 Complementation in cis or in trans
Finally, in order to efficiently assess integration and clonality of recombinant lines, a negative control must be produced for every independent set of transfections. This negative control is done by incubating amoebas with PolyFect Transfection Reagent but no DNA. Infection and drug selection are performed in parallel with other samples. Differences in viral growth as well as DNA amplification by PCR will be critical to assess if the experiment is successful or not.

## H3 Gene knockout by HR

Vectors for gene knockout using HR in mimivirus/megavirus and pandoravirus/mollivirus are currently available (Table 1). Homology arms of about 250-500 bp should be selected to target a gene of interest as shown in Figure 2b. We recommend designing two homology arms, wherein 5' arm covers the beginning of the gene whereas the 3' arm covers the end of the gene, allowing the deletion of a section of the coding region. Homology arms can be amplified by PCR from gDNA using primers bearing homology to the tagging acceptor vector (Table 1). These flanking regions allow the cloning of the homology arms by In-Fusion® seamless cloning (TaKaRa) into the desired vector (Primer templates are shown in Table 2). Any other cloning method can also be used.

Several critical steps should be considered. First, ensure that the homology arms do not contain the restriction sites used for the linearization of the vector. If that is the case, a second restriction site (absent in the homology arms and the acceptor vector) can be introduced in the primers. Alternatively, PCR amplification can be used as a substitute to plasmid digestion. Finally, negative control as described in *Endogenous tagging* section must be included.

## H3 Gene knock out by CRISPR/Cas9

The basic principles of CRISPR/Cas9 and the rules to select appropriate gRNA sequences can be found in³⁰. gRNA target sequences must include a PAM (protospacer adjacent motif) sequence and be significantly different from other sequences in the genome of *A. castellanii* (if gene knockout will be performed in this organism) or both *A. castellanii* and nuclear GV (if gene knockout will be performed in a nuclear GV). We currently utilize the Eukaryotic Pathogen CRISPR gRNA Design Tool (EuPaGDT)(http://grna.ctegd.uga.edu/), which allows users to rank gRNA target sequences (by analyzing GC content and flanking microhomology pair score) and analyzes potential off-target editing in the genome of *A. castellanii*. Potential off-target sites are manually identified by BLASTn using the reference genome of the GV to be genetically modified. We remind any researcher interested in carrying out gene editing by CRISPR/Cas9 that potential off-target sites have a lower risk of cleavage when mismatches with the 3' (seeding region) of the gRNA or lack of a PAM are observed. Once one or multiple gRNAs are selected, these can be inserted into vector vHB8 by mutagenesis (Figure 3). Primers should be designed containing the new gRNAs and bear homology sequences to clone the gRNAs by In-Fusion® seamless cloning (TaKaRa) into vHB8 (Primer templates are shown in Table 2).

Importantly, a second plasmid encoding for Cas9 and gRNAs which do not target the desired genome should be generated and transfected simultaneously with any set of transfections. Selection and genotyping must be done in parallel with other samples in order to assess if the experiment is successful or not. If amoebas are being genetically modified, this strain will be the baseline control for any phenotypic characterization.

## H3 Complementation in cis or in trans
Each time a gene is knocked out and mutant organisms are generated, a confirmation that the phenotype observed is due to the expected modification and not to any off-target modifications is needed. A traditional way to assess this is by complementation of the gene function through reintroducing the coding sequence of the gene knocked out. If the phenotype is restored, it can be concluded that the phenotype associated with the genome modification is due to the knockout of the gene being studied. Importantly, complementation in cis, at the endogenous locus and utilizing the endogenous promoter, is recommended and can be achieved by re-introducing the coding sequence of the gene by homologous recombination. Genome sequencing might also be explored if gene complementation is not possible. Trans-complementation allows the generation of recombinant viruses lacking essential genes. In order to trans-complement the lack of these genes, we have generated a battery of vectors that allows the expression of genes under the regulation of *Acanthamoeba castellanii* weak, medium, or strong promoters (vectors modified from ³¹); specific expression levels are usually important for efficient complementation of gene function). Moreover, vectors encoding geneticin¹² or nourseothricin¹³ are available (see Table 1). The coding sequence of the gene of interest should be cloned into one (or multiple) of these vectors, which are consequently transfected into amoebas for expression of the recombinant protein.

Gene knockout of essential viral genes can be obtained if recombination/CRISPR is performed in an *A. castellanii* host expressing a trans-complementing copy of the gene (mutagenesis or codon optimization are required in order to avoid cleavage by CRISPR/Cas9). Many companies provide services for gene synthesis (which includes codon optimization upon providing a codon usage table for the organism of interest) including Integrated DNA technologies (https://eu.idtdna.com/pages), Genewiz (https://www.genewiz.com/en-GB/) or GeneScript (https://www.genscript.com/). In this case, all steps of selection and propagation of the recombinant viruses might be performed in trans-complementing amoebas.

Several critical steps should be considered. First, codon optimization for expression in *A. castellanii* is highly recommended to achieve optimal expression levels. Second, if trans-complementation is designed to complement gene knockout by CRISPR/Cas9, silent mutations at the gRNA targeting site are necessary to avoid cleavage of the vector by Cas9. Finally, in order to assess efficiency of drug selection, a negative control must be produced for every independent set of transfections. This negative control is done by incubating amoebas with PolyFect Transfection Reagent but no DNA. After one or two days of drug selection, no amoebas in the trophozoite stage should be detected. No amoebal growth should appear until the end of the experiment.

## H3 Selection of mutant viruses by drug cassettes

Selection of viral mutants using selection cassettes is currently performed by the utilization of drugs targeting the translation of their host *A. castellanii*. Thus, amoebas infected by WT viruses and non-infected amoebas will succumb to the inhibition of protein synthesis. Only amoebas infected by a virus conferring resistance to such drugs will be able to complete the round of infection and release a pool of viruses enriched in recombinant ones. Such a strategy allows a single round of infection per culture, because freshly released viruses will not be able to infect amoebas in their neighborhood for a second round, as the selection killed them. Thus, viral growth in the presence of the drug is slow. In order to speed up the process, we have designed a protocol that alternates rounds of infection under selection pressure and in the absence of the drug, allowing faster amplification of the virus.
Each time a gene is knocked out and mutant organisms are generated, a confirmation that the phenotype observed is due to the expected modification and not to any off-target modifications is needed. A traditional way to assess this is by complementation of the gene function through reintroducing the coding sequence of the gene knocked out. If the phenotype is restored, it can be concluded that the phenotype associated with the genome modification is due to the knockout of the gene being studied. Importantly, complementation in cis, at the endogenous locus and utilizing the endogenous promoter, is recommended and can be achieved by re-introducing the coding sequence of the gene by homologous recombination. Genome sequencing might also be explored if gene complementation is not possible. Trans-complementation allows the generation of recombinant viruses lacking essential genes. In order to trans-complement the lack of these genes, we have generated a battery of vectors that allows the expression of genes under the regulation of *Acanthamoeba castellanii* weak, medium, or strong promoters (vectors modified from ³¹); specific expression levels are usually important for efficient complementation of gene function). Moreover, vectors encoding geneticin¹² or nourseothricin¹³ are available (see Table 1). The coding sequence of the gene of interest should be cloned into one (or multiple) of these vectors, which are consequently transfected into amoebas for expression of the recombinant protein.

Gene knockout of essential viral genes can be obtained if recombination/CRISPR is performed in an *A. castellanii* host expressing a trans-complementing copy of the gene (mutagenesis or codon optimization are required in order to avoid cleavage by CRISPR/Cas9). Many companies provide services for gene synthesis (which includes codon optimization upon providing a codon usage table for the organism of interest) including Integrated DNA technologies (https://eu.idtdna.com/pages), Genewiz (https://www.genewiz.com/en-GB/) or GeneScript (https://www.genscript.com/). In this case, all steps of selection and propagation of the recombinant viruses might be performed in trans-complementing amoebas.

Several critical steps should be considered. First, codon optimization for expression in *A. castellanii* is highly recommended to achieve optimal expression levels. Second, if trans-complementation is designed to complement gene knockout by CRISPR/Cas9, silent mutations at the gRNA targeting site are necessary to avoid cleavage of the vector by Cas9. Finally, in order to assess efficiency of drug selection, a negative control must be produced for every independent set of transfections. This negative control is done by incubating amoebas with PolyFect Transfection Reagent but no DNA. After one or two days of drug selection, no amoebas in the trophozoite stage should be detected. No amoebal growth should appear until the end of the experiment.

## H3 Selection of mutant viruses by drug cassettes

Selection of viral mutants using selection cassettes is currently performed by the utilization of drugs targeting the translation of their host *A. castellanii*. Thus, amoebas infected by WT viruses and non-infected amoebas will succumb to the inhibition of protein synthesis. Only amoebas infected by a virus conferring resistance to such drugs will be able to complete the round of infection and release a pool of viruses enriched in recombinant ones. Such a strategy allows a single round of infection per culture, because freshly released viruses will not be able to infect amoebas in their neighborhood for a second round, as the selection killed them. Thus, viral growth in the presence of the drug is slow. In order to speed up the process, we have designed a protocol that alternates rounds of infection under selection pressure and in the absence of the drug, allowing faster amplification of the virus.
282 Importantly, multiplicity of infection (MOI) must be kept 0.1 to 1 when the selection of recombinant
283 viruses is attempted. If a recombinant virus and a wild-type virus infect the same cell, production of the
284 selection cassette by the recombinant virus will trans-complement the lack of it in the wild-type virus,
285 defeating the purpose of the drug selection.

286 **H3 Selection of amoebas by drug selection cassettes.**

287 *A. castellanii* segregates its genetic material randomly<sup>10,11</sup>. Concordantly, the level of expression of genes
288 from vectors in *A. castellanii* shows intrinsic variation which cannot be corrected by cloning the amoebas.
289 Therefore, to select transfected amoebas, we utilize a protocol increasing the concentration of the
290 selective drug at different passages of the transfected amoebas<sup>32</sup>. This allows an increase in the production
291 of expressed genes, likely by increasing the copy number of the plasmids.

292

293 **H2 Expertise needed to implement the protocol.**

294 The protocols here described are simple and do not require particular expertise in molecular biology to be
295 carried out. *A. castellanii* is however a human pathogen and thus requires L2 biosafety measures.

296

297 **H1 MATERIALS**

298 **H2 BIOLOGICAL MATERIALS**

299 • Acanthamoeba castellanii strain NEFF (ATCC 30010™) (https://www.atcc.org/products/30010)

300 • *Escherichia coli* Stellar™ competent cells (TaKaRa, cat. no. 636763)

301 • Mimivirus reunion (MW004169.1)<sup>18</sup>

302 • Pandoravirus neocaledonia (MG011690.1)/ Mollivirus kamchatka (MN812837.1) <sup>15</sup>

303

304 **H2 REAGENTS**

305 **H3 Growth media and buffers**

306 • Proteose peptone (Merck, cat. no. 82450)

307 • Yeast extract (ThermoFisher Scientific, cat. no. BP1422-500)

308 • MgSO₄ - 7 H₂O (Merck, cat. no. 63138)

309 • CaCl₂ anhydrous (Merck, cat. no. 1.02378)

310 • Fe(NH₄)₂(SO₄)₂ (Merck, cat. no. 215406)

311 • Na₂HPO₄ (Merck, cat. no. S9763)

312 • KH₂PO₄ (Merck, cat. no. P5379)

313 • Glucose (Merck, cat. no. G8270)

314 • NaCl (Euromedex, cat. no. 1112)

315 • KCl (Merck, cat. no. P3911)

316 • NaOH (Merck, cat. no. S8045)

317 • LB (Luria/Miller) (Carl-Roth, cat. no. X968.4)

318 • Agar (ThermoFisher Scientific, cat. no. BP1423-500)

319 • TAE 10X – Tris Acetate EDTA Buffer 10X (Merck, cat. no. T9650-1L)
Importantly, multiplicity of infection (MOI) must be kept 0.1 to 1 when the selection of recombinant viruses is attempted. If a recombinant virus and a wild-type virus infect the same cell, production of the selection cassette by the recombinant virus will trans-complement the lack of it in the wild-type virus, defeating the purpose of the drug selection.

## H3 Selection of amoebas by drug selection cassettes.

A. castellanii segregates its genetic material randomly<sup>10,11</sup>. Concordantly, the level of expression of genes from vectors in A. castellanii shows intrinsic variation which cannot be corrected by cloning the amoebas. Therefore, to select transfected amoebas, we utilize a protocol increasing the concentration of the selective drug at different passages of the transfected amoebas<sup>32</sup>. This allows an increase in the production of expressed genes, likely by increasing the copy number of the plasmids.

The protocols here described are simple and do not require particular expertise in molecular biology to be carried out. A. castellanii is however a human pathogen and thus requires L2 biosafety measures.

## H1 MATERIALS

### H2 BIOLOGICAL MATERIALS

* Acanthamoeba castellanii strain NEFF (ATCC 30010™) (https://www.atcc.org/products/30010)

* Escherichia coli Stellar™ competent cells (TaKaRa, cat. no. 636763)

* Mimivirus reunion (MW004169.1)<sup>18</sup>

* Pandoravirus neocaledonia (MG011690.1)/ Mollivirus kamchatka (MN812837.1)<sup>15</sup>

### H2 REAGENTS

#### H3 Growth media and buffers

* Proteose peptone (Merck, cat. no. 82450)

* Yeast extract (ThermoFisher Scientific, cat. no. BP1422-500)

* MgSO₄ - 7 H₂O (Merck, cat. no. 63138)

* CaCl₂ anhydrous (Merck, cat. no. 1.02378)

* Fe(NH₄)₂(SO₄)₂ (Merck, cat. no. 215406)

* Na₂HPO₄ (Merck, cat. no. S9763)

* KH₂PO₄ (Merck, cat. no. P5379)

* Glucose (Merck, cat. no. G8270)

* NaCl (Euromedex, cat. no. 1112)

* KCl (Merck, cat. no. P3911)

* NaOH (Merck, cat. no. S8045)

* LB (Luria/Miller) (Carl-Roth, cat. no. X968.4)

* Agar (ThermoFisher Scientific, cat. no. BP1423-500)

* TAE 10X – Tris Acetate EDTA Buffer 10X (Merck, cat. no. T9650-1L)
320 **H3 Antibiotics**

321 • Geneticin™ (G418 sulfate, 50 mg / ml) (ThermoFisher Scientific cat. no. 10131035)

322 • Nourseothricin (Jena Biosciences, cat. no. AB-102L)

323 • Ampicillin (Euromedex, cat. no. EU0400)

324 • Kanamycin monosulfate (Euromedex, cat. no. UK0010-D)

325 **H3 PCR and cloning**

326 • SsoFast™ EvaGreen® Supermix for qPCR (Biorad, cat. no. 1725202)

327 • PureLink® Genomic DNA Kit from Invitrogen (ThermoFisher Scientific, cat. no. K182001)

328 • Wizard® Genomic DNA Purification Kit (Promega, cat. no. A1120)

329 • Monarch plasmid miniprep kit (New England Biolabs, cat. no. T1010S)

330 • Monarch PCR & DNA Cleanup Kit (New England Biolabs, cat. no. T1030S)

331 • QIAEX II Gel Extraction Kit (Qiagen, cat. no. 20021)

332 • Phusion™ High-Fidelity DNA Polymerase (2 U/µL) (ThermoFisher Scientific, cat. no. F530L)

333 • Terra™ PCR Direct Red Dye Premix (TaKaRa cat. no. 639286)

334 • GoTaq® DNA polymerase (Promega, cat. no. M3008)

335 • In-Fusion® Snap Assembly Master Mix (TaKaRa cat. no. 638943)

336 • Agarose (Merck, cat. no. A9539-500G)

337 • Nuclease free water (Dutscher, cat. no. 972090)

338 • Ethidium bromide (Merck, cat. no. E1510-10ML) **!CAUTION Ethidium Bromide is a known mutagen. Wear a lab coat, eye protection and gloves when working with this chemical. You may prefer to use safe products as SYBR Safe dye instead of ethidium bromide according to the manufacturer's instructions.**

342 • SYBR™ Safe DNA Gel Stain (ThermoFisher Scientific, cat. no. S33102)

343 • 1kb DNA Ladder Plus (Euromedex, cat. no. 03B-1411)

344 • 6X Loading Dye Solution (Euromedex, cat. no. 10-0111)

345 • All restriction enzymes are ordered from New England Biolabs (https://international.neb.com/)

346 • PCR Primers are ordered from Eurofins Genomics (https://eurofinsgenomics.eu/)

347 **H3 Transfection**

348 • PolyFect Transfection Reagent (Qiagen, cat. no. 301105)

349 **H3 Immunofluorescence**

350 • HA Tag Recombinant Rabbit Monoclonal Antibody (Thermo Fisher Scientific Cat# MA5-27915, RRID:AB_2744968)

351 • Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 594 (Thermo Fisher Scientific Cat# A-21207, RRID:AB_141637)

352 • Triton X-100 (EUROMEDEX, 2001-C)

353 • 37% Formaldehyde (Sigma-Aldrich, 252549-100ml)

354 • Hoechst 33258 Pentahydrate (ThermoFisher Scientific, H1398)

355 • BSA (Euromedex, cat. no. 04-100-812-C)

356 **H3 SDS-PAGE and Western-blot**

357 • Acrylamide:bisacrylamide 40% (37,5:1) (Euromedex, cat. no. EU0062-C)

358 • Tween 20 (Euromedex, cat. no. 2001-C)
320 **H3 Antibiotics**

321 • Geneticin™ (G418 sulfate, 50 mg / ml) (ThermoFisher Scientific cat. no. 10131035)

322 • Nourseothricin (Jena Biosciences, cat. no. AB-102L)

323 • Ampicillin (Euromedex, cat. no. EU0400)

324 • Kanamycin monosulfate (Euromedex, cat. no. UK0010-D)

325 **H3 PCR and cloning**

326 • SsoFast™ EvaGreen® Supermix for qPCR (Biorad, cat. no. 1725202)

327 • PureLink® Genomic DNA Kit from Invitrogen (ThermoFisher Scientific, cat. no. K182001)

328 • Wizard® Genomic DNA Purification Kit (Promega, cat. no. A1120)

329 • Monarch plasmid miniprep kit (New England Biolabs, cat. no. T1010S)

330 • Monarch PCR & DNA Cleanup Kit (New England Biolabs, cat. no. T1030S)

331 • QIAEX II Gel Extraction Kit (Qiagen, cat. no. 20021)

332 • Phusion™ High-Fidelity DNA Polymerase (2 U/µL) (ThermoFisher Scientific, cat. no. F530L)

333 • Terra™ PCR Direct Red Dye Premix (TaKaRa cat. no. 639286)

334 • GoTaq® DNA polymerase (Promega, cat. no. M3008)

335 • In-Fusion® Snap Assembly Master Mix (TaKaRa cat. no. 638943)

336 • Agarose (Merck, cat. no. A9539-500G)

337 • Nuclease free water (Dutscher, cat. no. 972090)

338 • Ethidium bromide (Merck, cat. no. E1510-10ML) **!CAUTION Ethidium Bromide is a known mutagen.**

339 Wear a lab coat, eye protection and gloves when working with this chemical. You may prefer to use
340 safe products as SYBR Safe dye instead of ethidium bromide according to the manufacturer's
341 instructions.

342 • SYBR™ Safe DNA Gel Stain (ThermoFisher Scientific, cat. no. S33102)

343 • 1kb DNA Ladder Plus (Euromedex, cat. no. 03B-1411)

344 • 6X Loading Dye Solution (Euromedex, cat. no. 10-0111)

345 • All restriction enzymes are ordered from New England Biolabs (https://international.neb.com/)

346 • PCR Primers are ordered from Eurofins Genomics (https://eurofinsgenomics.eu/)

347 **H3 Transfection**

348 • PolyFect Transfection Reagent (Qiagen, cat. no. 301105)

349 **H3 Immunofluorescence**

350 • HA Tag Recombinant Rabbit Monoclonal Antibody (Thermo Fisher Scientific Cat# MA5-27915,
351 RRID:AB_2744968)

352 • Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 594 (Thermo
353 Fisher Scientific Cat# A-21207, RRID:AB_141637)

354 • Triton X-100 (EUROMEDEX, 2001-C)

355 • 37% Formaldehyde (Sigma-Aldrich, 252549-100ml)

356 • Hoechst 33258 Pentahydrate (ThermoFisher Scientific, H1398)

357 • BSA (Euromedex, cat. no. 04-100-812-C)

358 **H3 SDS-PAGE and Western-blot**

359 • Acrylamide:bisacrylamide 40% (37,5:1) (Euromedex, cat. no. EU0062-C)

360 • Tween 20 (Euromedex, cat. no. 2001-C)
361 • Tris-Glycine-SDS buffer 10x (Euromedex, cat. no. EU0510-B)

362 • SDS (Merck, cat. no. 436143)

363 • TEMED (N,N,N',N'-Tetramethylethylenediamine) (Merck, cat. no. T9281-25ml)

364 • APS (Ammonium persulfate) (Merck, cat. no. A7460-100G)

365 • 2-Mercaptoethanol (Merck, cat. no. 63689-100ML-F)

366 • Coomassie brilliant blue G250 (ThermoFisher Scientific, cat. no. 20279)

367 • Acetic acid glacial (Carlo Erba, cat. no. 401424)

368 • Ethanol 96° (Carlo Erba, cat. no. 528151)

369 • Ethanol absolute (Carlo Erba, cat. no. 528151)

370 • BSA (Euromedex, cat. no. 04-100-812-C)

371 • cOmplete™, EDTA-free Protease Inhibitor Cocktail (Merck, cat. no. 5056489001)

372 • Prestained Protein Ladder 10-180 kDa (Euromedex, cat. no. 06P-0111)

373 • Unstained Prot MW Marker 14.4-116 kDa (Euromedex, cat. no. 06U-0511)

374 • Western Blotting Filter Papers (ThermoFisher Scientific, cat. no. 88600)

375 • nitro blue tetrazolium / 5-bromo-4-chloro-3-indolyl-1-phosphate (NBT / BCIP) (Sigma-Aldrich, B1911-100ml)

376 • Invitrogen™ Power Blotter 1-Step™ Transfer Buffer (5X) (ThermoFisher Scientific, cat. no. PB7300)

378

379 **H2 REAGENT SETUP**

380 • **PYG** In a 2 l glass bottle, add 40 g of proteose peptone and 2 g of yeast extract. Complete with water to ~1.8 l and autoclave at 121°C for 20 min. Prepare 1 l stock solutions of MgSO₄ (400mM), CaCl₂ (50 mM), Fe(NH₄)₂(SO₄)₂ (5 mM), Na₂HPO₄ (250 mM), KH₂PO₄ (250 mM) and glucose (2 M), sterilized with filtration units Stericups. In the autoclaved solution, add 20 ml of MgSO₄, Fe(NH₄)₂(SO₄)₂, Na₂HPO₄ and KH₂PO₄, 16 ml of CaCl₂, and 50 ml of glucose. Complete to 2 l with distilled water (use the graduations of the bottle). Mix well and sterilize on 2 filtration units of 1 l. Work in a class II BioSafety cabinet to ensure sterility of the medium. Medium and stock solutions can be stored at room temperature (22°C approx.) for several months, as long as they are not contaminated.

388 • **LB-agar** Prepare LB as described by the supplier. In a 500 ml glass bottle, add 4.8 g of agar, and 400 ml of LB. Don't mix. Autoclave at 121°C for 20 min. Mix and use to prepare petri dishes for bacterial culture. Medium can be stored at room temperature for several months, as long as it is not contaminated.

392 • **Tris Acetate EDTA (TAE) 0.5X electrophoresis solution** Dilute TAE buffer 10X in distilled water to 0.5X working solution for casting agarose gels and for use as a buffer for gel electrophoresis. Buffer can be stored at room temperature for several months.

395 • **Tris-Glycine-SDS buffer 1X** Dilute TG-SDS 10X in distilled water to 1X working solution for use as a buffer for SDS-PAGE acrylamide gel electrophoresis. Buffer can be stored at room temperature for several months.

398 • **PBS** For 1 l PBS, in a beaker, mix 900 ml distilled water with 8 g NaCl (136.8 mM final), 0.4 g KCl (5.36 mM final), 1.44 g Na₂HPO₄ (10.1 mM final), 0.24 g KH₂PO₄ (1.76 mM final). Adjust the pH to 7.4 with NaOH 1M. Adjust the volume to 1 l with distilled water. Filter sterilize or autoclave at 121°C for 20 min. Buffer can be stored at room temperature for several months.

401
361 • Tris-Glycine-SDS buffer 10x (Euromedex, cat. no. EU0510-B)

362 • SDS (Merck, cat. no. 436143)

363 • TEMED (N,N,N',N'-Tetramethylethylenediamine) (Merck, cat. no. T9281-25ml)

364 • APS (Ammonium persulfate) (Merck, cat. no. A7460-100G)

365 • 2-Mercaptoethanol (Merck, cat. no. 63689-100ML-F)

366 • Coomassie brilliant blue G250 (ThermoFisher Scientific, cat. no. 20279)

367 • Acetic acid glacial (Carlo Erba, cat. no. 401424)

368 • Ethanol 96° (Carlo Erba, cat. no. 528151)

369 • Ethanol absolute (Carlo Erba, cat. no. 528151)

370 • BSA (Euromedex, cat. no. 04-100-812-C)

371 • cOmplete™, EDTA-free Protease Inhibitor Cocktail (Merck, cat. no. 5056489001)

372 • Prestained Protein Ladder 10-180 kDa (Euromedex, cat. no. 06P-0111)

373 • Unstained Prot MW Marker 14.4-116 kDa (Euromedex, cat. no. 06U-0511)

374 • Western Blotting Filter Papers (ThermoFisher Scientific, cat. no. 88600)

375 • nitro blue tetrazolium / 5-bromo-4-chloro-3-indolyl-1-phosphate (NBT / BCIP) (Sigma-Aldrich, B1911-100ml)

376 • Invitrogen™ Power Blotter 1-Step™ Transfer Buffer (5X) (ThermoFisher Scientific, cat. no. PB7300)

378

379 **H2 REAGENT SETUP**

380 • **PYG** In a 2 l glass bottle, add 40 g of proteose peptone and 2 g of yeast extract. Complete with water to ~1.8 l and autoclave at 121°C for 20 min. Prepare 1 l stock solutions of MgSO₄ (400mM), CaCl₂ (50 mM), Fe(NH₄)₂(SO₄)₂ (5 mM), Na₂HPO₄ (250 mM), KH₂PO₄ (250 mM) and glucose (2 M), sterilized with filtration units Stericups. In the autoclaved solution, add 20 ml of MgSO₄, Fe(NH₄)₂(SO₄)₂, Na₂HPO₄ and KH₂PO₄, 16 ml of CaCl₂, and 50 ml of glucose. Complete to 2 l with distilled water (use the graduations of the bottle). Mix well and sterilize on 2 filtration units of 1 l. Work in a class II BioSafety cabinet to ensure sterility of the medium. Medium and stock solutions can be stored at room temperature (22°C approx.) for several months, as long as they are not contaminated.

388 • **LB-agar** Prepare LB as described by the supplier. In a 500 ml glass bottle, add 4.8 g of agar, and 400 ml of LB. Don't mix. Autoclave at 121°C for 20 min. Mix and use to prepare petri dishes for bacterial culture. Medium can be stored at room temperature for several months, as long as it is not contaminated.

392 • **Tris Acetate EDTA (TAE) 0.5X electrophoresis solution** Dilute TAE buffer 10X in distilled water to 0.5X working solution for casting agarose gels and for use as a buffer for gel electrophoresis. Buffer can be stored at room temperature for several months.

395 • **Tris-Glycine-SDS buffer 1X** Dilute TG-SDS 10X in distilled water to 1X working solution for use as a buffer for SDS-PAGE acrylamide gel electrophoresis. Buffer can be stored at room temperature for several months.

398 • **PBS** For 1 l PBS, in a beaker, mix 900 ml distilled water with 8 g NaCl (136.8 mM final), 0.4 g KCl (5.36 mM final), 1.44 g Na₂HPO₄ (10.1 mM final), 0.24 g KH₂PO₄ (1.76 mM final). Adjust the pH to 7.4 with NaOH 1M. Adjust the volume to 1 l with distilled water. Filter sterilize or autoclave at 121°C for 20 min. Buffer can be stored at room temperature for several months.

401
402 PBS Triton X-100 0.2% (PBST) For 1 l PBST, in a beaker, mix 900 ml distilled water with 8 g NaCl (136.8
403 mM final), 0.4 g KCl (5.36 mM final), 1.44 g Na₂HPO₄ (10.1 mM final), 0.24 g KH₂PO₄ (1.76 mM final)
404 and 2mL of Triton X-100 (final 0.2%). Adjust the pH to 7.6 with NaOH 1M. Adjust the volume to 1 l
405 with distilled water. Buffer can be stored at room temperature for several months.
406

407 H2 EQUIPMENT
408 Motic™ AE31 Main Body, Binocular Microscope (ThermoFisher Scientific, cat. no. 15485342)
409 Epifluorescence microscope AXIO Observer Z1 Zeiss
410 https://www.zeiss.com/corporate/int/home.html

411 Class II Biosafety cabinet for *Acanthamoeba* and virus manipulations. ADS Laminaire, Optimale 12

412 Incubator for amoebas and viruses. Froilabo, Bioconcept https://www.froilabo.com/

413 Incubator for bacteria plates. Froilabo, Bioconcept https://www.froilabo.com/

414 Shaking incubator for bacteria. InFors Multitron Eco https://www.infors-ht.com/fr/

415 Transilluminator for agarose gels and western-blots imaging. Vilber Fusion FX Spectra
416 https://www.vilber.com/fusion-fx-spectra/

417 Electrophoresis system for agarose gels. Mupid-One (Dutscher, cat. no. 088900)

418 Electrophoresis system for acrylamide gels from Biorad (Power supplier : PowerPac Basic,
419 Electrophoresis chambers and accessories : Mini-PROTEAN Tetra Handcast Systems)
420 https://www.bio-rad.com/fr-fr/category/electrophoresis-chambers?ID=N3F2N0E8Z

421 Western-Blot transfer system (ThermoFisher Scientific, Power Blotter Station cat. no. 15896152)

422 Thermocycler (Analytic Jena, Biometra Trio) https://www.analytik-jena.com/products/life-science/pcr-qpcr-thermal-cycler/thermal-cycler-pcr/biometra-trio-series/

423 Water bath (ThermoFisher Scientific, Precision GP 05, cat. no. 15385857)

424 NanoDrop spectrophotometer (ThermoFisher Scientific)

425 Centrifuge 5425R (Eppendorf)

426 Centrifuge 4-16kS (Sigma-Aldrich)

428 High-definition Neubauer counting chamber (hemocytometer) (Dutscher, cat. no. 900504)

429 Cell scrapers 24 cm (Sarstedt, cat. no. 83.3951)

430 Cell culture flask Cellstar 25 cm² filter cap Greiner Bio-One (Dutscher, cat. no. 690175)

431 Plates 6 wells cell culture Cellstar standard Greiner Bio-One (Dutscher, cat. no. 657160)

432 Plates 96 wells flat bottom for cell culture Cellstar standard Greiner Bio-One (Dutscher, cat. no. 020035)

434 Greiner Bio-One 96-well sterile cell culture plate with flat bottom, black with transparent bottom, for fluorescence imaging (Dutscher, cat. no. 655090)

435 Greiner Bio-One EASYseal™ adhesive foil for seal microtiter plate (Dutscher, cat. no. 676001)

436 Parafilm tape (Dutscher, cat. no. 090260)

438 Precision wipes Kimtech Science (Dutscher, cat. no. 002907B)

439 Petri dishes 90mm (Sarstedt, cat. no. 82.1472)

440 Filtration unit Stericup 1L (Dutscher, cat. no. 051248B)

441 Tubes 15 ml (Sarstedt, cat. no. 62.554.502)

442 Microtubes 1.5 ml (Sarstedt, cat. no. 72.690.001)
402 • PBS Triton X-100 0.2% (PBST) For 1 l PBST, in a beaker, mix 900 ml distilled water with 8 g NaCl (136.8
403 mM final), 0.4 g KCl (5.36 mM final), 1.44 g Na₂HPO₄ (10.1 mM final), 0.24 g KH₂PO₄ (1.76 mM final)
404 and 2mL of Triton X-100 (final 0.2%). Adjust the pH to 7.6 with NaOH 1M. Adjust the volume to 1 l
405 with distilled water. Buffer can be stored at room temperature for several months.
406

407 H2 EQUIPMENT
408 • Motic™ AE31 Main Body, Binocular Microscope (ThermoFisher Scientific, cat. no. 15485342)
409 • Epifluorescence microscope AXIO Observer Z1 Zeiss
410 https://www.zeiss.com/corporate/int/home.html

411 • Class II Biosafety cabinet for *Acanthamoeba* and virus manipulations. ADS Laminaire, Optimale 12
412 • Incubator for amoebas and viruses. Froilabo, Bioconcept https://www.froilabo.com/

413 • Incubator for bacteria plates. Froilabo, Bioconcept https://www.froilabo.com/

414 • Shaking incubator for bacteria. InFors Multitron Eco https://www.infors-ht.com/fr/

415 • Transilluminator for agarose gels and western-blots imaging. Vilber Fusion FX Spectra
416 https://www.vilber.com/fusion-fx-spectra/

417 • Electrophoresis system for agarose gels. Mupid-One (Dutscher, cat. no. 088900)

418 • Electrophoresis system for acrylamide gels from Biorad (Power supplier : PowerPac Basic,
419 Electrophoresis chambers and accessories : Mini-PROTEAN Tetra Handcast Systems)
420 https://www.bio-rad.com/fr-fr/category/electrophoresis-chambers?ID=N3F2N0E8Z

421 • Western-Blot transfer system (ThermoFisher Scientific, Power Blotter Station cat. no. 15896152)

422 • Thermocycler (Analytic Jena, Biometra Trio) https://www.analytik-jena.com/products/life-science/pcr-qpcr-thermal-cycler/thermal-cycler-pcr/biometra-trio-series/

424 • Water bath (ThermoFisher Scientific, Precision GP 05, cat. no. 15385857)

425 • NanoDrop spectrophotometer (ThermoFisher Scientific)

426 • Centrifuge 5425R (Eppendorf)

427 • Centrifuge 4-16kS (Sigma-Aldrich)

428 • High-definition Neubauer counting chamber (hemocytometer) (Dutscher, cat. no. 900504)

429 • Cell scrapers 24 cm (Sarstedt, cat. no. 83.3951)

430 • Cell culture flask Cellstar 25 cm² filter cap Greiner Bio-One (Dutscher, cat. no. 690175)

431 • Plates 6 wells cell culture Cellstar standard Greiner Bio-One (Dutscher, cat. no. 657160)

432 • Plates 96 wells flat bottom for cell culture Cellstar standard Greiner Bio-One (Dutscher, cat. no. 020035)
433

434 • Greiner Bio-One 96-well sterile cell culture plate with flat bottom, black with transparent bottom, for
435 fluorescence imaging (Dutscher, cat. no. 655090)

436 • Greiner Bio-One EASYseal™ adhesive foil for seal microtiter plate (Dutscher, cat. no. 676001)

437 • Parafilm tape (Dutscher, cat. no. 090260)

438 • Precision wipes Kimtech Science (Dutscher, cat. no. 002907B)

439 • Petri dishes 90mm (Sarstedt, cat. no. 82.1472)

440 • Filtration unit Stericup 1L (Dutscher, cat. no. 051248B)

441 • Tubes 15 ml (Sarstedt, cat. no. 62.554.502)

442 • Microtubes 1.5 ml (Sarstedt, cat. no. 72.690.001)
443 • Microtube Multiply® Pro 0,2 ml with cap (Sarstedt, cat. no. 72.737.002)

444 • Microtube Multiply® µStrip 0,2 ml (Sarstedt, cat. no. 72.985.002)

445 • Caps for PCR tubes strips (Sarstedt, cat. no. 65.989.002)

446 • Filtration unit Stericup PES 0,2µm 1L (Dutscher, cat. no. 051248B)

447

448 **H2 SOFTWARE**

449 • Primer design and tools for In-Fusion® cloning https://www.takarabio.com/learning-centers/cloning/primer-design-and-other-tools

450 • New England Biolabs Tm calculator https://tm calculator.neb.com/#!/main

451 • New England Biolabs double digestion https://nebcloner.neb.com/#!/redigest

452 • gRNAs design http://grna.ctegd.uga.edu/

453 • Blastn sequence alignment tool https://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastn&BLAST_SPEC=GeoBlast&PAGE_TYPE=BlastSearch

454 • Expasy translate tool https://web.expasy.org/translate/

455 • Clustal omega multiple sequence alignment tool https://www.ebi.ac.uk/Tools/msa/clustalo/

456 • Reverse complement tool https://www.bioinformatics.org/sms/rev_comp.html

457 • ApE plasmid editing software³³ https://jorgensen.biology.utah.edu/wayned/ape/

461

462 **H1 PROCEDURE**

463 **H2 Mutagenesis of nuclear viruses and A. castellanii using CRISPR/Cas9**

464

465 **H3 Vector preparation for Cas9 and gRNAs expression in A. castellanii ● TIMING 5 days (5-6 hs hands on time)**

466 The following protocol describes how to clone 2 gRNAs in vHB8 starting from primers containing the gRNAs and vHB16 as a template (See Figure 3).

467 • 1. Prepare a PCR mix for gRNAs amplification according to *table below* and using the primer templates shown in *Table 2*.

<table><thead><tr><th></th><th>Amount</th><th>Final Concentration</th></tr></thead><tbody><tr><td>Nuclease-free water</td><td>Make up to 50 µl</td><td></td></tr><tr><td>5X Phusion HF or GC Buffer</td><td>10 µl</td><td>1x</td></tr><tr><td>10 mM dNTPs</td><td>1 µl</td><td>200 µM</td></tr><tr><td>10 µM Forward Primer 1</td><td>2.5 µl</td><td>0.5 µM</td></tr><tr><td>10 µM Reverse Primer 2</td><td>2.5 µl</td><td>0.5 µM</td></tr><tr><td>Template vHB16</td><td>20 ng</td><td>0.4 ng/µl</td></tr><tr><td>DMSO (optional)</td><td>(1.5 µl)</td><td>3 %</td></tr><tr><td>Phusion DNA Polymerase</td><td>0.5 µl</td><td>1 unit/50 µl PCR</td></tr></tbody></table>

471 ▲**CRITICAL STEP** DMSO is used to inhibit the formation of secondary structures on the DNA templates and should be considered for GC rich templates or templates with predicted secondary structures.

474
443 • Microtube Multiply® Pro 0,2 ml with cap (Sarstedt, cat. no. 72.737.002)

444 • Microtube Multiply® µStrip 0,2 ml (Sarstedt, cat. no. 72.985.002)

445 • Caps for PCR tubes strips (Sarstedt, cat. no. 65.989.002)

446 • Filtration unit Stericup PES 0,2µm 1L (Dutscher, cat. no. 051248B)

447

448 **H2 SOFTWARE**

449 • Primer design and tools for In-Fusion® cloning https://www.takarabio.com/learning-centers/cloning/primer-design-and-other-tools

450 • New England Biolabs Tm calculator https://tm calculator.neb.com/#!/main

451 • New England Biolabs double digestion https://nebcloner.neb.com/#!/redigest

452 • gRNAs design http://grna.ctegd.uga.edu/

453 • Blastn sequence alignment tool https://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastn&BLAST_SPEC=GeoBlast&PAGE_TYPE=BlastSearch

454 • Expasy translate tool https://web.expasy.org/translate/

455 • Clustal omega multiple sequence alignment tool https://www.ebi.ac.uk/Tools/msa/clustalo/

456 • Reverse complement tool https://www.bioinformatics.org/sms/rev_comp.html

457 • ApE plasmid editing software³³ https://jorgensen.biology.utah.edu/wayned/ape/

461

462 **H1 PROCEDURE**

463 **H2 Mutagenesis of nuclear viruses and A. castellanii using CRISPR/Cas9**

464

465 **H3 Vector preparation for Cas9 and gRNAs expression in A. castellanii ● TIMING 5 days (5-6 hs hands on time)**

466 The following protocol describes how to clone 2 gRNAs in vHB8 starting from primers containing the gRNAs and vHB16 as a template (See Figure 3).

467 • 1. Prepare a PCR mix for gRNAs amplification according to *table below* and using the primer templates shown in *Table 2*.

<table><thead><tr><th></th><th>Amount</th><th>Final Concentration</th></tr></thead><tbody><tr><td>Nuclease-free water</td><td>Make up to 50 µl</td><td></td></tr><tr><td>5X Phusion HF or GC Buffer</td><td>10 µl</td><td>1x</td></tr><tr><td>10 mM dNTPs</td><td>1 µl</td><td>200 µM</td></tr><tr><td>10 µM Forward Primer 1</td><td>2.5 µl</td><td>0.5 µM</td></tr><tr><td>10 µM Reverse Primer 2</td><td>2.5 µl</td><td>0.5 µM</td></tr><tr><td>Template vHB16</td><td>20 ng</td><td>0.4 ng/µl</td></tr><tr><td>DMSO (optional)</td><td>(1.5 µl)</td><td>3 %</td></tr><tr><td>Phusion DNA Polymerase</td><td>0.5 µl</td><td>1 unit/50 µl PCR</td></tr></tbody></table>

471 ▲**CRITICAL STEP** DMSO is used to inhibit the formation of secondary structures on the DNA templates and should be considered for GC rich templates or templates with predicted secondary structures.

474
475

2. Perform PCR following the cycling conditions below.

<table><thead><tr><th>Cycle number</th><th>Denature</th><th>Anneal</th><th>Extend</th><th>Final</th></tr></thead><tbody><tr><td>1</td><td>98°C, 30 sec</td><td></td><td></td><td></td></tr><tr><td>2-30</td><td>98°C, 10 sec</td><td>55°C, 30 sec</td><td>72°C, 15 sec</td><td></td></tr><tr><td>31</td><td></td><td></td><td>72°C, 5 min</td><td></td></tr><tr><td>32</td><td></td><td></td><td></td><td>10°C, Hold</td></tr></tbody></table>

476

477

478

479

480

481

482

483

3. Analyze the product by migrating 1 µl of the PCR reaction on a 2 % agarose gel using standard gel electrophoresis conditions (135V, 20 min). Purify the rest of the PCR reaction with Monarch® PCR & DNA Cleanup Kit (see Supplementary Methods). Elute with nuclease-free water.

■ PAUSE POINT Purified DNA can be stored at -20°C.

?TROUBLESHOOTING

4. Digest the PCR product purified from Step 3 with Dpnl in order to eliminate remaining parental plasmid and 1 µg of vHB8 with Notl in parallel at 37°C for 3 hours following the reactions:

<table><thead><tr><th></th><th>Amount</th><th>Final Concentration</th></tr></thead><tbody><tr><td>PCR product or vHB8</td><td>All purified PCR or 1 µg of vHB8</td><td></td></tr><tr><td>NEB CutSmart buffer</td><td>2 µl</td><td>1x</td></tr><tr><td>Dpnl or Notl (HF)</td><td>20 U</td><td>1 U/µl</td></tr><tr><td>Nuclease-free water</td><td>To 20 µl</td><td></td></tr></tbody></table>

484

■ PAUSE POINT Digestion can be performed overnight.

5. Analyze digestion efficiency of vHB8 by Notl by migrating 1 µl of the mixture using standard gel electrophoresis (135V, 20 min). Purify the rest of digested plasmid with Monarch® PCR & DNA Cleanup Kit according to the manufacturer's instructions (Supplementary Methods) ?TROUBLESHOOTING

485

486

487

488

489

6. Assemble an In-Fusion® seamless cloning reaction as follows:

<table><thead><tr><th></th><th>Amount</th><th>Final Concentration</th></tr></thead><tbody><tr><td>Digested and purified PCR fragment from Step 4</td><td>200 ng</td><td>20 ng/µl</td></tr><tr><td>Linearized vector from Step 5</td><td>50 ng</td><td>5 ng/µl</td></tr><tr><td>5X In-Fusion® HD Enzyme Premix</td><td>2 µl</td><td>1x</td></tr><tr><td>Nuclease-free water</td><td>Up to 10 µl</td><td></td></tr></tbody></table>

490

7. Incubate the reaction for 15 min at 50 °C, then place it on ice.

491

492

493

494

495

496

497

498

8. Transform Stellar™ Competent Cells (according to manufacturer's specifications) using 5 µl of the In-Fusion® reaction and at least 25 µl of chemically competent bacteria.

▲ CRITICAL STEP The incubation allowing the recovery of bacteria after transformation is not necessary when using plasmids containing ampicillin resistance (bacteriostatic).

9. Plate cells on LB agar dishes containing Ampicillin 100 µg.ml⁻¹

10. Incubate plates overnight at 37 °C.

11. Perform a colony screening PCR to identify positive clones. The PCR mixture is assembled as follows (one reaction per colony tested):

<table><thead><tr><th></th><th>Amount</th><th>Final Concentration</th></tr></thead><tbody><tr><td>5X Green GoTaq® Buffer</td><td>3 µl</td><td>1x</td></tr><tr><td>10 mM dNTPs</td><td>0.3 µl</td><td>200 µM</td></tr></tbody></table>
475

2. Perform PCR following the cycling conditions below.

<table><thead><tr><th>Cycle number</th><th>Denature</th><th>Anneal</th><th>Extend</th><th>Final</th></tr></thead><tbody><tr><td>1</td><td>98°C, 30 sec</td><td></td><td></td><td></td></tr><tr><td>2-30</td><td>98°C, 10 sec</td><td>55°C, 30 sec</td><td>72°C, 15 sec</td><td></td></tr><tr><td>31</td><td></td><td></td><td>72°C, 5 min</td><td></td></tr><tr><td>32</td><td></td><td></td><td></td><td>10°C, Hold</td></tr></tbody></table>

476

477

478

479

3. Analyze the product by migrating 1 µl of the PCR reaction on a 2 % agarose gel using standard gel electrophoresis conditions (135V, 20 min). Purify the rest of the PCR reaction with Monarch® PCR & DNA Cleanup Kit (see Supplementary Methods). Elute with nuclease-free water.

■ PAUSE POINT Purified DNA can be stored at -20°C.

480

?TROUBLESHOOTING

481

482

483

4. Digest the PCR product purified from Step 3 with Dpnl in order to eliminate remaining parental plasmid and 1 µg of vHB8 with Notl in parallel at 37°C for 3 hours following the reactions:

<table><thead><tr><th></th><th>Amount</th><th>Final Concentration</th></tr></thead><tbody><tr><td>PCR product or vHB8</td><td>All purified PCR or 1 µg of vHB8</td><td></td></tr><tr><td>NEB CutSmart buffer</td><td>2 µl</td><td>1x</td></tr><tr><td>Dpnl or Notl (HF)</td><td>20 U</td><td>1 U/µl</td></tr><tr><td>Nuclease-free water</td><td>To 20 µl</td><td></td></tr></tbody></table>

484

■ PAUSE POINT Digestion can be performed overnight.

485

486

487

488

5. Analyze digestion efficiency of vHB8 by Notl by migrating 1 µl of the mixture using standard gel electrophoresis (135V, 20 min). Purify the rest of digested plasmid with Monarch® PCR & DNA Cleanup Kit according to the manufacturer's instructions (Supplementary Methods) ?TROUBLESHOOTING

489

6. Assemble an In-Fusion® seamless cloning reaction as follows:

<table><thead><tr><th></th><th>Amount</th><th>Final Concentration</th></tr></thead><tbody><tr><td>Digested and purified PCR fragment from Step 4</td><td>200 ng</td><td>20 ng/µl</td></tr><tr><td>Linearized vector from Step 5</td><td>50 ng</td><td>5 ng/µl</td></tr><tr><td>5X In-Fusion® HD Enzyme Premix</td><td>2 µl</td><td>1x</td></tr><tr><td>Nuclease-free water</td><td>Up to 10 µl</td><td></td></tr></tbody></table>

490

7. Incubate the reaction for 15 min at 50 °C, then place it on ice.

491

492

8. Transform Stellar™ Competent Cells (according to manufacturer's specifications) using 5 µl of the In-Fusion® reaction and at least 25 µl of chemically competent bacteria.

493

494

▲ CRITICAL STEP The incubation allowing the recovery of bacteria after transformation is not necessary when using plasmids containing ampicillin resistance (bacteriostatic).

495

9. Plate cells on LB agar dishes containing Ampicillin 100 µg.ml⁻¹

496

10. Incubate plates overnight at 37 °C.

497

498

11. Perform a colony screening PCR to identify positive clones. The PCR mixture is assembled as follows (one reaction per colony tested):

<table><thead><tr><th></th><th>Amount</th><th>Final Concentration</th></tr></thead><tbody><tr><td>5X Green GoTaq® Buffer</td><td>3 µl</td><td>1x</td></tr><tr><td>10 mM dNTPs</td><td>0.3 µl</td><td>200 µM</td></tr></tbody></table>
<table><tr><td>10 µM Forward Primer from Step 1</td><td>0.75 µl</td><td>0.5 µM</td></tr><tr><td>10 µM Reverse Primer (11, see Table 1)</td><td>0.75 µl</td><td>0.5 µM</td></tr><tr><td>GoTaq® DNA Polymerase</td><td>0.075 µl</td><td>0.375 units</td></tr><tr><td>Nuclease-free water</td><td>Up to 15 µl</td><td></td></tr><tr><td>Template</td><td>See Step 12</td><td></td></tr></table>

▲ CRITICAL STEP Primer 11 was designed next to the insertion site on vHB8 plasmid. Forward primer used in step 1 can be used in combination.

12. To perform the PCR reactions, number each colony to be analyzed by PCR using a marker pen on the petri dish, to later recognize the colonies bearing the desired vector. Using a pipette tip, collect approximately half of each colony and vigorously shake it in the PCR reaction solution.

13. Perform PCR following the cycling conditions below, and analyze the products by running a standard gel electrophoresis (135V, 20 min).

<table><thead><tr><td>Cycle number</td><td>Denature</td><td>Anneal</td><td>Extend</td><td>Final</td></tr></thead><tbody><tr><td>1</td><td>95°C, 2 min</td><td></td><td></td><td></td></tr><tr><td>2-30</td><td>95°C, 45 sec</td><td>42-65°C, 45 sec</td><td>72°C, 1 min/kb</td><td></td></tr><tr><td>31</td><td></td><td></td><td>72°C, 5 min</td><td></td></tr><tr><td>32</td><td></td><td></td><td></td><td>10°C, Hold</td></tr></tbody></table>

▲ CRITICAL STEP Calculate annealing temperature using the NEB Tm Calculator (https://tm calculator.neb.com/#!/main), selecting Taq DNA Polymerase from the given options

14. Pick individual colonies bearing the desired plasmid from the plates in Step 10 and incubate them in 5 ml of LB (Luria-Bertani) medium in presence of 100 µg.ml⁻¹ ampicillin at 37 °C, 200-250 RPM agitation for 12-16 hours (one colony per tube).

15. Centrifuge the tubes at 3000 g for 10 min at 4°C, remove the supernatant and purify the plasmid in the pellet using Monarch® Plasmid DNA Miniprep Kit Protocol, following manufacturer's instructions.

16. Send an aliquot of the plasmid for sequencing and confirm the absence of undesirable mutations.

H3 Transfection of A. castellanii ● TIMING 2 days (2-3 hs hands on time)

▲ CRITICAL This protocol can be used to transfect any kind of DNA into Acanthamoeba castellanii, i.e. linear DNA serving as a template for recombination or circular DNA (plasmid) designed for the stable expression of a transgene like cas9, gRNAs, or any other fusion gene. The vector should also contain a selection marker such as neo (Neomycin phosphotransferase) conferring resistance to geneticin, or NAT (Nourseothricin N-Acetyl Transferase) conferring resistance to nourseothricin (Table 1).

17. In a 25 cm² flask, inoculate 5 x 10⁵ fresh A. castellanii cells in 5 ml PPYG medium (2 x 10⁴ cells / cm²). Incubate at 32 °C for 24 hs.

18. Recover the amoebas into a 15 ml tube after scraping the flask with a cell scraper.

19. Count the cells and determine their concentration with a hemocytometer.
<table><tr><td>10 µM Forward Primer from Step 1</td><td>0.75 µl</td><td>0.5 µM</td></tr><tr><td>10 µM Reverse Primer (11, see Table 1)</td><td>0.75 µl</td><td>0.5 µM</td></tr><tr><td>GoTaq® DNA Polymerase</td><td>0.075 µl</td><td>0.375 units</td></tr><tr><td>Nuclease-free water</td><td>Up to 15 µl</td><td></td></tr><tr><td>Template</td><td>See Step 12</td><td></td></tr></table>

▲ CRITICAL STEP Primer 11 was designed next to the insertion site on vHB8 plasmid. Forward primer used in step 1 can be used in combination.

12. To perform the PCR reactions, number each colony to be analyzed by PCR using a marker pen on the petri dish, to later recognize the colonies bearing the desired vector. Using a pipette tip, collect approximately half of each colony and vigorously shake it in the PCR reaction solution.

13. Perform PCR following the cycling conditions below, and analyze the products by running a standard gel electrophoresis (135V, 20 min).

<table><thead><tr><td>Cycle number</td><td>Denature</td><td>Anneal</td><td>Extend</td><td>Final</td></tr></thead><tbody><tr><td>1</td><td>95°C, 2 min</td><td></td><td></td><td></td></tr><tr><td>2-30</td><td>95°C, 45 sec</td><td>42-65°C, 45 sec</td><td>72°C, 1 min/kb</td><td></td></tr><tr><td>31</td><td></td><td></td><td>72°C, 5 min</td><td></td></tr><tr><td>32</td><td></td><td></td><td></td><td>10°C, Hold</td></tr></tbody></table>

▲ CRITICAL STEP Calculate annealing temperature using the NEB Tm Calculator (https://tm calculator.neb.com/#!/main), selecting Taq DNA Polymerase from the given options

14. Pick individual colonies bearing the desired plasmid from the plates in Step 10 and incubate them in 5 ml of LB (Luria-Bertani) medium in presence of 100 µg.ml⁻¹ ampicillin at 37 °C, 200-250 RPM agitation for 12-16 hours (one colony per tube).

15. Centrifuge the tubes at 3000 g for 10 min at 4°C, remove the supernatant and purify the plasmid in the pellet using Monarch® Plasmid DNA Miniprep Kit Protocol, following manufacturer's instructions.

16. Send an aliquot of the plasmid for sequencing and confirm the absence of undesirable mutations.

## H3 Transfection of A. castellanii ● TIMING 2 days (2-3 hs hands on time)

▲ CRITICAL This protocol can be used to transfect any kind of DNA into *Acanthamoeba castellanii*, i.e. linear DNA serving as a template for recombination or circular DNA (plasmid) designed for the stable expression of a transgene like *cas9*, gRNAs, or any other fusion gene. The vector should also contain a selection marker such as *neo* (Neomycin phosphotransferase) conferring resistance to geneticin, or *NAT* (Nourseothricin N-Acetyl Transferase) conferring resistance to nourseothricin (Table 1).

17. In a 25 cm² flask, inoculate 5 x 10⁵ fresh *A. castellanii* cells in 5 ml PPYG medium (2 x 10⁴ cells / cm²). Incubate at 32 °C for 24 hs.

18. Recover the amoebas into a 15 ml tube after scraping the flask with a cell scraper.

19. Count the cells and determine their concentration with a hemocytometer.
20. In a 6-well dish, add 1.5 x 10⁵ cells in one well per assay containing 2 ml PYG. Incubate for a few minutes to allow the cells to adhere at the bottom (5 minutes is usually enough for healthy cells).

▲ CRITICAL STEP Include an additional well for the negative control which will be transfected with no DNA. Such amoebas will be treated using the same selection procedure as other samples. No trophozoites should be observed after 1 or 2 days of drug selection (refer to ³⁴ for brightfield images of different stages of *Acanthamoeba*). At the end of the experiment, no cells should be observed in the negative control sample.

▲ CRITICAL STEP If CRISPR/Cas9 vectors are transfected, include a vector expressing Cas9 and gRNAs which do not target the organism of interest as negative control for future genotyping or phenotypic characterization.

▲ CRITICAL STEP Before using the culture, with a bench microscope, check that the cells in the flask are in a good shape, i.e. adherent, in trophozoite stage, and in monolayer. This drastically enhances the transfection efficiency.

21. Simultaneously, in a 1.5 ml microtube, mix ~4 µg of DNA diluted in PBS to a final volume of 100 µl, then add 10 µl PolyFect (Qiagen), vortex for 20 sec, and incubate for 5-10 min at room temperature to allow the formation of transfection complexes.

▲ CRITICAL STEP The use of PBS is critical to ensure maximum transfection efficiency. Homogenize the PolyFect solution by tapping before pipetting.

22. During the incubation of the transfection mix, remove the supernatant from the cells from Step 20, carefully wash them by adding 1 ml of PBS, and gently rock the plate.

▲ CRITICAL STEP Add the liquid on the walls of the wells, not directly onto the cells, to avoid their detachment from the plate. The use of PBS is critical to ensure maximum transfection efficiency.

23. Repeat Step 22 and leave the PBS in the wells.

24. Delicately add the transfection mix from Step 21 (110 µl) drop by drop onto the cells. Homogenize by gently rocking the plate.

25. Incubate for 1 h at 32 °C.

26. Gently aspirate the PBS using a pipette tip without disturbing attached cells, and delicately add 2 ml PYG.

▲ CRITICAL STEP Add the liquid on the walls of the wells, not directly onto the cells, to avoid their detachment from the plate.

27. For stable transfection in *A. castellanii*, to express Cas9 and gRNAs for example, continue to the next section.

For genomic manipulations of viruses by homologous recombination, infect the cells directly with the desired virus (Steps 37-75).

**H3 Selection of amoebas stably expressing a transgene such as cas9 and gRNAs ● TIMING 7-10 days (2-3 hs hands on time)**

28. Seal the plate with parafilm to avoid evaporation and incubate overnight at 32 °C. Overnight growth allows the recovery of the cells and the expression of the selection marker.
20. In a 6-well dish, add 1.5 x 10⁵ cells in one well per assay containing 2 ml PYG. Incubate for a few minutes to allow the cells to adhere at the bottom (5 minutes is usually enough for healthy cells).

▲ CRITICAL STEP Include an additional well for the negative control which will be transfected with no DNA. Such amoebas will be treated using the same selection procedure as other samples. No trophozoites should be observed after 1 or 2 days of drug selection (refer to ³⁴ for brightfield images of different stages of *Acanthamoeba*). At the end of the experiment, no cells should be observed in the negative control sample.

▲ CRITICAL STEP If CRISPR/Cas9 vectors are transfected, include a vector expressing Cas9 and gRNAs which do not target the organism of interest as negative control for future genotyping or phenotypic characterization.

▲ CRITICAL STEP Before using the culture, with a bench microscope, check that the cells in the flask are in a good shape, i.e. adherent, in trophozoite stage, and in monolayer. This drastically enhances the transfection efficiency.

21. Simultaneously, in a 1.5 ml microtube, mix ~4 µg of DNA diluted in PBS to a final volume of 100 µl, then add 10 µl PolyFect (Qiagen), vortex for 20 sec, and incubate for 5-10 min at room temperature to allow the formation of transfection complexes.

▲ CRITICAL STEP The use of PBS is critical to ensure maximum transfection efficiency. Homogenize the PolyFect solution by tapping before pipetting.

22. During the incubation of the transfection mix, remove the supernatant from the cells from Step 20, carefully wash them by adding 1 ml of PBS, and gently rock the plate.

▲ CRITICAL STEP Add the liquid on the walls of the wells, not directly onto the cells, to avoid their detachment from the plate. The use of PBS is critical to ensure maximum transfection efficiency.

23. Repeat Step 22 and leave the PBS in the wells.

24. Delicately add the transfection mix from Step 21 (110 µl) drop by drop onto the cells. Homogenize by gently rocking the plate.

25. Incubate for 1 h at 32 °C.

26. Gently aspirate the PBS using a pipette tip without disturbing attached cells, and delicately add 2 ml PYG.

▲ CRITICAL STEP Add the liquid on the walls of the wells, not directly onto the cells, to avoid their detachment from the plate.

27. For stable transfection in *A. castellanii*, to express Cas9 and gRNAs for example, continue to the next section.

For genomic manipulations of viruses by homologous recombination, infect the cells directly with the desired virus (Steps 37-75).

**H3 Selection of amoebas stably expressing a transgene such as cas9 and gRNAs ● TIMING 7-10 days (2-3 hs hands on time)**

28. Seal the plate with parafilm to avoid evaporation and incubate overnight at 32 °C. Overnight growth allows the recovery of the cells and the expression of the selection marker.
570 ▲ CRITICAL STEP Check the appearance of the cells with a microscope: they should be mainly at
571 the trophozoite stage.

572 29. Remove the supernatant by pipetting/aspiration and replace it delicately with 2 ml PYG containing
573 30 µg.ml⁻¹ geneticin or 30 µg.ml⁻¹ nourseothricin (according to the vector selection cassette (see
574 Table 1)).

575 30. Incubate for 2 days at 32 °C.

576 31. Check the appearance of the cells using an inverted microscope magnification x 400.

577 ▲ CRITICAL STEP: As the transfection efficiency is quite low, most cells should appear dead, i.e.
578 round and detached from the bottom of the dish, and a few should remain as healthy trophozoites.
579 These ones are most probably containing the vector and stably expressing the selection marker.

580 ?TROUBLESHOOTING

581 32. Remove dead cells by replacing the culture media every 2-3 days with fresh PYG containing 30 µg.ml⁻¹ of the selection drug until a confluent monolayer of trophozoites is observed. The timing needed to obtain a confluent monolayer is highly variable according to transfection efficiency and the effect of the transgene on the growth of the amoebas. A minimum of one week is needed.

585 33. Recover the cells from the wells either by scraping or by carefully pipetting up and down.

586 34. Prepare two 25 cm² flasks per culture, one containing 5 ml PYG and 30 µg.ml⁻¹ geneticin/nourseothricin, and one with 5 ml PYG and 100 µg.ml⁻¹ geneticin/nourseothricin.

588 35. Distribute 1 ml of cells in each flask. Visualization of the amoebas by fluorescence microscopy should be used to confirm expression of GFP-Cas9. Clear nuclear localization should be observed. Change the medium every 2-3 days until the cultures can be used for further experiments and passage into a new dish whenever cultures reach confluency. Amoebas can be maintained indefinitely in this condition.

593 ▲ CRITICAL STEP: The medium containing 100 µg.ml⁻¹ antibiotic allows increased expression of plasmid-contained genes likely by increasing copy number of the plasmid. The medium containing 30 µg.ml⁻¹ of antibiotic will serve as a backup in case the amoebas do not grow properly in the first one.

597 36. At this point, there are two options according to the aim of the experiment: Option A, Mutagenesis of nuclear viruses with CRISPR/Cas9 or Option B, Mutagenesis of amoebas using CRISPR/Cas9.

599 A. Mutagenesis of nuclear viruses with CRISPR/Cas9 ● TIMING 7-10 days (3-4 hs hands on time)

601 i. In a 25 cm² flask, inoculate 5 x 10⁵ fresh *A. castellanii* cells expressing Cas9 and gRNAs produced during Step 35 in 5 ml PYG medium containing geneticin 100 µg.ml⁻¹ to keep selecting for Cas9 expression (2 x 10⁴ cells / cm²). Incubate at 32 °C for 24 hs.

605 ii. Infect the culture with the virus you wish to mutagenize with an approximate MOI of 5-10 for pandoravirus and mollivirus. Gently rock the plate to homogenize.

607 iii. Incubate 1 h at 32 °C.

608 iv. Remove the supernatant by pipetting/aspiration to remove the viruses that were not internalized and replace with 5 ml PYG containing geneticin 100 µg.ml⁻¹.
570 ▲ CRITICAL STEP Check the appearance of the cells with a microscope: they should be mainly at
571 the trophozoite stage.

572 29. Remove the supernatant by pipetting/aspiration and replace it delicately with 2 ml PYG containing
573 30 µg.ml⁻¹ geneticin or 30 µg.ml⁻¹ nourseothricin (according to the vector selection cassette (see
574 Table 1)).

575 30. Incubate for 2 days at 32 °C.

576 31. Check the appearance of the cells using an inverted microscope magnification x 400.

577 ▲ CRITICAL STEP: As the transfection efficiency is quite low, most cells should appear dead, i.e.
578 round and detached from the bottom of the dish, and a few should remain as healthy trophozoites.
579 These ones are most probably containing the vector and stably expressing the selection marker.

580 ?TROUBLESHOOTING

581 32. Remove dead cells by replacing the culture media every 2-3 days with fresh PYG containing 30 µg.ml⁻¹ of the selection drug until a confluent monolayer of trophozoites is observed. The timing needed to obtain a confluent monolayer is highly variable according to transfection efficiency and the effect of the transgene on the growth of the amoebas. A minimum of one week is needed.

585 33. Recover the cells from the wells either by scraping or by carefully pipetting up and down.

586 34. Prepare two 25 cm² flasks per culture, one containing 5 ml PYG and 30 µg.ml⁻¹ geneticin/nourseothricin, and one with 5 ml PYG and 100 µg.ml⁻¹ geneticin/nourseothricin.

588 35. Distribute 1 ml of cells in each flask. Visualization of the amoebas by fluorescence microscopy should be used to confirm expression of GFP-Cas9. Clear nuclear localization should be observed. Change the medium every 2-3 days until the cultures can be used for further experiments and passage into a new dish whenever cultures reach confluency. Amoebas can be maintained indefinitely in this condition.

593 ▲ CRITICAL STEP: The medium containing 100 µg.ml⁻¹ antibiotic allows increased expression of plasmid-contained genes likely by increasing copy number of the plasmid. The medium containing 30 µg.ml⁻¹ of antibiotic will serve as a backup in case the amoebas do not grow properly in the first one.

597 36. At this point, there are two options according to the aim of the experiment: Option A, Mutagenesis of nuclear viruses with CRISPR/Cas9 or Option B, Mutagenesis of amoebas using CRISPR/Cas9.

599

600 A. Mutagenesis of nuclear viruses with CRISPR/Cas9 ● TIMING 7-10 days (3-4 hs hands on time)

601 i. In a 25 cm² flask, inoculate 5 x 10⁵ fresh A. castellanii cells expressing Cas9 and gRNAs produced during Step 35 in 5 ml PYG medium containing geneticin 100 µg.ml⁻¹ to keep selecting for Cas9 expression (2 x 10⁴ cells / cm²). Incubate at 32 °C for 24 hs.

605 ii. Infect the culture with the virus you wish to mutagenize with an approximate MOI of 5-10 for pandoravirus and mollivirus. Gently rock the plate to homogenize.

607 iii. Incubate 1 h at 32 °C.

608 iv. Remove the supernatant by pipetting/aspiration to remove the viruses that were not internalized and replace with 5 ml PYG containing geneticin 100 µg.ml⁻¹.
610 v. Incubate at 32 °C until you start to observe cell lysis and production of new viruses
611 (approximately 2 to 5 days). Evaluate the production of viruses by observing the flask with
612 an inverted microscope magnification x 400.

613 ▲ CRITICAL STEP Part of the suspension corresponds to mutant viruses.

614 ▲ CRITICAL STEP Infected cells appear lysed or round, and the viruses appear as small dots
615 between the cells. This step is important to evaluate roughly how much suspension is needed for
616 next round of infection.

617 vi. Recover the viruses by pipetting up and down to resuspend viral particles and transfer into
618 a 15 ml tube, transfer 500 µl into two separate 1.5 ml microtubes, and proceed to cloning
619 and genotyping to detect mutant viruses (see Steps 76-91 and 97-102). Keep the
620 remaining suspension at 4 °C. PAUSE POINT: Samples are stable for at least 1 year if there
621 is no bacterial contamination.

622 vii. Prepare a culture of *A. castellanii* expressing Cas9 and gRNAs by repeating Step 36Ai and
623 infect with the viral suspension obtained in Step 36Avi (use 500 µl from one of the
624 microtubes), as described in Step 36Aii. Continue to Step 36Aiii-vi.

625 viii. Repeat step 36Avii two more times.

626 ▲ CRITICAL STEP Due to the low efficiency of NHEJ in nuclear giant viruses and its tendency to
627 generate large deletions, the number of passages needed to increase the proportion of mutants
628 with discrete deletions needs to be analyzed empirically for each gene (usually between 1 and 3
629 passages). Over 5 consecutive passages might increase natural mutations in the gRNA targeting
630 site and thus, should also be avoided.

631 ▲ CRITICAL STEP Cloning (Steps 76 - 91) and genotyping of clones (Steps 97 - 102) should be
632 performed at each passage to maximize the success rate of the experiment. Viral lines should be
633 collected and passaged every day.

634
635 B. Mutagenesis of amoebas using CRISPR/Cas9 ● TIMING 3 weeks (10-15 hs hands on time)

636 i. Prepare a vector designed for CRISPR/Cas9 mutagenesis targeting the gene of interest as
637 described in Steps 1-16

638 ii. Transfect *A. castellanii* with this plasmid as described in Steps 17-27.

639 iii. Select and amplify cells expressing Cas9 and gRNAs as described in Steps 2-8-36.

640 iv. When a confluent population is obtained, proceed to cloning of amoebas (see Steps 92-96). ▲ CRITICAL STEP Modification of the genome of *A. castellanii* occurs during the selection and amplification process.

643 v. Identify mutant strains by genotyping (see Steps 97-102).

644 ▲ CRITICAL STEP Ensure that the mutations obtained create an efficient knock-out. i.e. the indels
645 generate frameshifts or the absence of protein production is confirmed by detection with
646 antibodies (Western blot).

647
648 H2 Mutagenesis of nuclear and cytoplasmic viruses using recombination
649
650 H3 Vector preparation for homologous recombination experiments ● TIMING 10 days (4-6 h hands-on
651 time)

650
651 H3 Vector preparation for homologous recombination experiments ● TIMING 10 days (4-6 h hands-on time)
610 v. Incubate at 32 °C until you start to observe cell lysis and production of new viruses
611 (approximately 2 to 5 days). Evaluate the production of viruses by observing the flask with
612 an inverted microscope magnification x 400.

613 ▲ CRITICAL STEP Part of the suspension corresponds to mutant viruses.

614 ▲ CRITICAL STEP Infected cells appear lysed or round, and the viruses appear as small dots
615 between the cells. This step is important to evaluate roughly how much suspension is needed for
616 next round of infection.

617 vi. Recover the viruses by pipetting up and down to resuspend viral particles and transfer into
618 a 15 ml tube, transfer 500 µl into two separate 1.5 ml microtubes, and proceed to cloning
619 and genotyping to detect mutant viruses (see Steps 76-91 and 97-102). Keep the
620 remaining suspension at 4 °C. PAUSE POINT: Samples are stable for at least 1 year if there
621 is no bacterial contamination.

622 vii. Prepare a culture of *A. castellanii* expressing Cas9 and gRNAs by repeating Step 36Ai and
623 infect with the viral suspension obtained in Step 36Avi (use 500 µl from one of the
624 microtubes), as described in Step 36Aii. Continue to Step 36Aiii-vi.

625 viii. Repeat step 36Avii two more times.

626 ▲ CRITICAL STEP Due to the low efficiency of NHEJ in nuclear giant viruses and its tendency to
627 generate large deletions, the number of passages needed to increase the proportion of mutants
628 with discrete deletions needs to be analyzed empirically for each gene (usually between 1 and 3
629 passages). Over 5 consecutive passages might increase natural mutations in the gRNA targeting
630 site and thus, should also be avoided.

631 ▲ CRITICAL STEP Cloning (Steps 76 - 91) and genotyping of clones (Steps 97 - 102) should be
632 performed at each passage to maximize the success rate of the experiment. Viral lines should be
633 collected and passaged every day.

634
635 B. Mutagenesis of amoebas using CRISPR/Cas9 ● TIMING 3 weeks (10-15 hs hands on time)

636 i. Prepare a vector designed for CRISPR/Cas9 mutagenesis targeting the gene of interest as
637 described in Steps 1-16

638 ii. Transfect *A. castellanii* with this plasmid as described in Steps 17-27.

639 iii. Select and amplify cells expressing Cas9 and gRNAs as described in Steps 2-8-36.

640 iv. When a confluent population is obtained, proceed to cloning of amoebas (see Steps 92-96). ▲ CRITICAL STEP Modification of the genome of *A. castellanii* occurs during the selection and amplification process.

643 v. Identify mutant strains by genotyping (see Steps 97-102).

644 ▲ CRITICAL STEP Ensure that the mutations obtained create an efficient knock-out. i.e. the indels
645 generate frameshifts or the absence of protein production is confirmed by detection with
646 antibodies (Western blot).

647
648 H2 Mutagenesis of nuclear and cytoplasmic viruses using recombination
649
650 H3 Vector preparation for homologous recombination experiments ● TIMING 10 days (4-6 h hands-on
651 time)

650
651 H3 Vector preparation for homologous recombination experiments ● TIMING 10 days (4-6 h hands-on time)
37. Prepare a PCR mix for amplification of each homology arm according to the following **table** and using the primer templates shown in **Table 2**. The genomic DNA of the selected virus must be used as a template for PCR amplification. gDNA is obtained using PureLink® Genomic DNA Kit from Invitrogen by extracting it from purified viral particles or infected cells, following manufacturer's instructions.

<table><thead><tr><td></td><td>Amount</td><td>Final Concentration</td></tr></thead><tbody><tr><td>Nuclease-free water</td><td>Up to 50 µl</td><td></td></tr><tr><td>5X Phusion HF or GC Buffer</td><td>10 µl</td><td>1x</td></tr><tr><td>10 mM dNTPs</td><td>1 µl</td><td>200 µM</td></tr><tr><td>10 µM Forward Primer 1, 3, 5 or 7</td><td>2.5 µl</td><td>0.5 µM</td></tr><tr><td>10 µM Reverse Primer 2, 4, 6 or 8</td><td>2.5 µl</td><td>0.5 µM</td></tr><tr><td>Template gDNA</td><td>100 ng</td><td>2 ng/µl</td></tr><tr><td>DMSO (optional)</td><td>(1.5 µl)</td><td>3%</td></tr><tr><td>Phusion DNA Polymerase</td><td>0.6 µl</td><td>1 unit/50 µl PCR</td></tr></tbody></table>

▲ **CRITICAL STEP** DMSO is used to inhibit the formation of secondary structures on the DNA templates and should be considered for GC rich templates or templates with predicted secondary structures.

38. Perform PCR following the cycling conditions below, and analyze the products by running 1 µl of the PCR reaction using standard agarose gel electrophoresis (135V, 20 min).

<table><thead><tr><td>Cycle number</td><td>Denature</td><td>Anneal</td><td>Extend</td><td>Final</td></tr></thead><tbody><tr><td>1</td><td>98°C, 30 sec</td><td></td><td></td><td></td></tr><tr><td>2-30</td><td>98°C, 10 sec</td><td>45-72°C, 30 sec</td><td>72°C, 15 sec</td><td></td></tr><tr><td>31</td><td></td><td></td><td>72°C, 5 min</td><td></td></tr><tr><td>32</td><td></td><td></td><td></td><td>10°C, Hold</td></tr></tbody></table>

▲ **CRITICAL STEP** Calculate the annealing temperature using the NEB Tm Calculator (https://tm calculator.neb.com/#!/main) without considering the 15 bp corresponding to the receptor vector and used for In-Fusion® recombination

### ?TROUBLESHOOTING

39. Purify the rest of the PCR reaction with Monarch® PCR & DNA Cleanup Kit according to the manufacturer's recommendations (Supplementary Methods). **PAUSE POINT** Purified DNA can be stored at -20°C.

40. In parallel, linearize at least 1 µg of the desired receptor vector (Table 1) by digesting it with HindIII or EcoRI for the cloning of 5' homology arm at 37°C for 3 hours following the reaction:

<table><thead><tr><td></td><td>Amount</td><td>Final Concentration</td></tr></thead><tbody><tr><td>Vector</td><td>1 µg</td><td>50 ng/µl</td></tr><tr><td>NEB 3.1 or NEB CutSmart buffer</td><td>2 µl</td><td>1x</td></tr><tr><td>HindIII or EcoRI</td><td>20 U</td><td>1 U/µl</td></tr><tr><td>Nuclease-free water</td><td>To 20 µl</td><td></td></tr></tbody></table>

**PAUSE POINT** Digestion can be performed overnight.
37. Prepare a PCR mix for amplification of each homology arm according to the following **table** and using the primer templates shown in **Table 2**. The genomic DNA of the selected virus must be used as a template for PCR amplification. gDNA is obtained using PureLink® Genomic DNA Kit from Invitrogen by extracting it from purified viral particles or infected cells, following manufacturer's instructions.

<table><thead><tr><td></td><td>Amount</td><td>Final Concentration</td></tr></thead><tbody><tr><td>Nuclease-free water</td><td>Up to 50 µl</td><td></td></tr><tr><td>5X Phusion HF or GC Buffer</td><td>10 µl</td><td>1x</td></tr><tr><td>10 mM dNTPs</td><td>1 µl</td><td>200 µM</td></tr><tr><td>10 µM Forward Primer 1, 3, 5 or 7</td><td>2.5 µl</td><td>0.5 µM</td></tr><tr><td>10 µM Reverse Primer 2, 4, 6 or 8</td><td>2.5 µl</td><td>0.5 µM</td></tr><tr><td>Template gDNA</td><td>100 ng</td><td>2 ng/µl</td></tr><tr><td>DMSO (optional)</td><td>(1.5 µl)</td><td>3%</td></tr><tr><td>Phusion DNA Polymerase</td><td>0.6 µl</td><td>1 unit/50 µl PCR</td></tr></tbody></table>

▲**CRITICAL STEP** DMSO is used to inhibit the formation of secondary structures on the DNA templates and should be considered for GC rich templates or templates with predicted secondary structures.

38. Perform PCR following the cycling conditions below, and analyze the products by running 1 µl of the PCR reaction using standard agarose gel electrophoresis (135V, 20 min).

<table><thead><tr><td>Cycle number</td><td>Denature</td><td>Anneal</td><td>Extend</td><td>Final</td></tr></thead><tbody><tr><td>1</td><td>98°C, 30 sec</td><td></td><td></td><td></td></tr><tr><td>2-30</td><td>98°C, 10 sec</td><td>45-72°C, 30 sec</td><td>72°C, 15 sec</td><td></td></tr><tr><td>31</td><td></td><td></td><td>72°C, 5 min</td><td></td></tr><tr><td>32</td><td></td><td></td><td></td><td>10°C, Hold</td></tr></tbody></table>

▲**CRITICAL STEP** Calculate the annealing temperature using the NEB Tm Calculator (https://tm calculator.neb.com/#!/main) without considering the 15 bp corresponding to the receptor vector and used for In-Fusion® recombination

## ?TROUBLESHOOTING

39. Purify the rest of the PCR reaction with Monarch® PCR & DNA Cleanup Kit according to the manufacturer's recommendations (Supplementary Methods). **PAUSE POINT** Purified DNA can be stored at -20°C.

40. In parallel, linearize at least 1 µg of the desired receptor vector (Table 1) by digesting it with HindIII or EcoRI for the cloning of 5' homology arm at 37°C for 3 hours following the reaction:

<table><thead><tr><td></td><td>Amount</td><td>Final Concentration</td></tr></thead><tbody><tr><td>Vector</td><td>1 µg</td><td>50 ng/µl</td></tr><tr><td>NEB 3.1 or NEB CutSmart buffer</td><td>2 µl</td><td>1x</td></tr><tr><td>HindIII or EcoRI</td><td>20 U</td><td>1 U/µl</td></tr><tr><td>Nuclease-free water</td><td>To 20 µl</td><td></td></tr></tbody></table>

**PAUSE POINT** Digestion can be performed overnight.
673 41. Analyze digestion efficiency by running 1 µl of the digested plasmid using standard agarose gel electrophoresis (135V, 20 min). Purify the rest of the digested plasmid with Monarch® PCR & DNA Cleanup Kit according to the manufacturer's instructions (Supplementary Methods). ?TROUBLESHOOTING

677 ■ PAUSE POINT Purified DNA can be stored at -20°C.

678 42. Proceed as described in Steps 6-16 to clone the 5' homology arm in the digested vector.

679 43. Digest the vector obtained in the previous step using NotI as described in Step 4 to clone the 3'HR in the previously obtained vector, following Steps 5-16.

681
682 H3 Mutagenesis of viruses by recombination with the insertion of a resistance cassette ● TIMING 5-7 days
683 (8-10 hs hands-on time)

684
685 44. Linearize 6 µg of the vector produced in Step 43 by digesting it overnight with appropriate restriction enzymes (HindIII/EcoRI and NotI, See Figures 1, 2) as follows

686
687

---

---

<table>
   <thead>
    <tr>
     <td>
     </td>
     <td>
      Amount
     </td>
     <td>
      Final Concentration
     </td>
    </tr>
   </thead>
   <tbody>
    <tr>
     <td>
      Vector
     </td>
     <td>
      6 µg
     </td>
     <td>
      120 ng/µl
     </td>
    </tr>
    <tr>
     <td>
      Appropriate NEB buffer
     </td>
     <td>
      5 µl
     </td>
     <td>
      1x
     </td>
    </tr>
    <tr>
     <td>
      Enzyme 1
     </td>
     <td>
      20 U
     </td>
     <td>
      0.4 U/µl
     </td>
    </tr>
    <tr>
     <td>
      Enzyme 2
     </td>
     <td>
      20 U
     </td>
     <td>
      0.4 U/µl
     </td>
    </tr>
    <tr>
     <td>
      Nuclease-free water
     </td>
     <td>
      To 50 µl
     </td>
     <td>
     </td>
    </tr>
   </tbody>
  </table>

688
689 45. Analyze digestion efficiency by running 1 µl of the digested plasmid using standard agarose
gel electrophoresis (135V, 20 min). Purify the rest of the digested plasmid with Monarch®
690
691 PCR & DNA Cleanup Kit according to the manufacturer's instructions (Supplementary
692 Methods).

693 ▲ CRITICAL STEP It is not necessary to separate the two fragments of the plasmid obtained after restriction digestion, for transfection.

695 ▲ CRITICAL STEP Split the sample in two and purify using two columns to avoid saturation and DNA loss during purification.

697 ?TROUBLESHOOTING

698 46. Transfect wt A. castellanii with the linearized vector as described in Steps 17-27.

699 ▲ CRITICAL STEP Include as a negative control a well with cells transfected with no DNA. samples
700 must be collected in parallel in order to assess the specificity of genotyping.

701 47. Infect transfected cells with a high MOI of virus. Gently rock the plate to homogenize.

702 ▲ CRITICAL STEP The aim is to infect every transfected cell. For genetic manipulation of
703 Mimiviridae, a MOI of 10 is sufficient, for pandoravirus or mollivirus, a MOI of 50-100 is preferable.

704 48. Incubate 1 h at 32 °C.

705 49. Remove the supernatant to wash away non-internalized viruses and replace with 2 ml PYG.

707 50. Incubate overnight at 32 °C.
673 41. Analyze digestion efficiency by running 1 µl of the digested plasmid using standard agarose gel electrophoresis (135V, 20 min). Purify the rest of the digested plasmid with Monarch® PCR & DNA Cleanup Kit according to the manufacturer's instructions (Supplementary Methods). ?TROUBLESHOOTING

677 ■ PAUSE POINT Purified DNA can be stored at -20°C.

678 42. Proceed as described in Steps 6-16 to clone the 5' homology arm in the digested vector.

679 43. Digest the vector obtained in the previous step using NotI as described in Step 4 to clone the 3'HR in the previously obtained vector, following Steps 5-16.

681 H3 Mutagenesis of viruses by recombination with the insertion of a resistance cassette ● TIMING 5-7 days

683 (8-10 hs hands-on time)

684 44. Linearize 6 µg of the vector produced in Step 43 by digesting it overnight with appropriate restriction enzymes (HindIII/EcoRI and NotI, See Figures 1, 2) as follows

686

687
<table><thead><tr><td></td><td>Amount</td><td>Final Concentration</td></tr></thead><tbody><tr><td>Vector</td><td>6 µg</td><td>120 ng/µl</td></tr><tr><td>Appropriate NEB buffer</td><td>5 µl</td><td>1x</td></tr><tr><td>Enzyme 1</td><td>20 U</td><td>0.4 U/µl</td></tr><tr><td>Enzyme 2</td><td>20 U</td><td>0.4 U/µl</td></tr><tr><td>Nuclease-free water</td><td>To 50 µl</td><td></td></tr></tbody></table>

688 45. Analyze digestion efficiency by running 1 µl of the digested plasmid using standard agarose gel electrophoresis (135V, 20 min). Purify the rest of the digested plasmid with Monarch® PCR & DNA Cleanup Kit according to the manufacturer's instructions (Supplementary Methods).

693 ▲ CRITICAL STEP It is not necessary to separate the two fragments of the plasmid obtained after restriction digestion, for transfection.

695 ▲ CRITICAL STEP Split the sample in two and purify using two columns to avoid saturation and DNA loss during purification.

697 ?TROUBLESHOOTING

698 46. Transfect wt A. castellanii with the linearized vector as described in Steps 17-27.

699 ▲ CRITICAL STEP Include as a negative control a well with cells transfected with no DNA. samples must be collected in parallel in order to assess the specificity of genotyping.

701 47. Infect transfected cells with a high MOI of virus. Gently rock the plate to homogenize.

702 ▲ CRITICAL STEP The aim is to infect every transfected cell. For genetic manipulation of Mimiviridae, a MOI of 10 is sufficient, for pandoravirus or mollivirus, a MOI of 50-100 is preferable.

704 48. Incubate 1 h at 32 °C.

705 49. Remove the supernatant to wash away non-internalized viruses and replace with 2 ml PYG.

707 50. Incubate overnight at 32 °C.
708 **PAUSE POINT** Viruses can be kept in the incubator for several days (typically 2-3 days), in which
case, seal the plates with parafilm to avoid evaporation of the culture media. If more time is
needed, perform the incubation at room temperature.

711 In 25 cm² flasks, prepare one culture per assay, including the negative control. Inoculate
712 5 x 10⁵ A. castellanii cells in 5 ml PYG (2 x 10⁴ cells / cm²), and incubate 24 hs at 32 °C, to
713 obtain healthy amoebas in a monolayer for the next round of infection.

714 Recover each well into 2 separate 1.5 ml microtubes (1 ml/tube). Centrifuge one of the
715 tubes at maximum speed (~16,000 g) for 20 min at room temperature to pellet the viruses.
716 Remove the supernatant by pipetting/aspiration and keep the pellet for genotyping.
717 Proceed to PCR genotyping to assess the successful integration of the genomic
718 modification (see Steps 97-102).

719 ▲ CRITICAL STEP At this point, recombination events should be independent of the phenotype
720 associated with the modification of the genome and thus, recombinant DNA should be identified
721 by genotyping. Lack of amplification using primers P1+P3 (Figure 4) indicates inefficient
722 transfection or problems with PCR amplification.

723 **PAUSE POINT** Pellets can be stored at -20 °C for at least one month to proceed later to
724 genotyping.

725 ?-TROUBLESHOOTING

726 53. Infect a 25 cm² flask of confluent cells produced in step 51 with the contents of the second
tube obtained during step 52.

728 54. Incubate 1 h at 32 °C.

729 ▲ CRITICAL STEP: Incubation for 1 h without a selection marker allows the expression of the
730 resistance cassette by the virus.

731 55. Wash twice with 1 ml of PYG to eliminate extracellular viruses. Proceed carefully to avoid
detaching cells from the surface.

733 56. Add 5 ml of PYG containing nourseothricin 100 µg.ml⁻¹ or geneticin 100 µg.ml⁻¹ according
to the selection cassette introduced in the viral genome.

735 57. Incubate overnight at 32 °C.

736 **PAUSE POINT** Viruses can be kept in the incubator for several days (typically 2-3 days). If more
time is needed, perform the incubation at room temperature.

738 58. Prepare cultures of amoebas as described in step 51.

739 Recover the contents of the flasks in 15 ml tubes and centrifuge at maximum speed
740 (depending on the centrifuge 6,800 g for 45 min (swinging buckets) or 16,000 g for 20 min
741 (fixed rotor)) at room temperature to pellet the viruses. Discard the supernatants and
742 leave only a small volume to resuspend the pellets.

743 ▲ CRITICAL STEP Centrifugation is necessary to remove the selection marker and allow a new
744 round of viral infection.

745 For each assay, infect one 25 cm² flask of confluent cells produced in step 58 with the
content of a tube obtained during step 59.

747 61. Repeat steps 54-57

748 62. Prepare cultures of amoebas as described in step 53.

749 63. Recover the viruses as described in step 59.

750 64. Repeat steps 54-55

751 65. Replace medium with 5 ml of PYG without selection.
708 **PAUSE POINT** Viruses can be kept in the incubator for several days (typically 2-3 days), in which
case, seal the plates with parafilm to avoid evaporation of the culture media. If more time is
needed, perform the incubation at room temperature.

711 In 25 cm² flasks, prepare one culture per assay, including the negative control. Inoculate
712 5 x 10⁵ A. castellanii cells in 5 ml PYG (2 x 10⁴ cells / cm²), and incubate 24 hs at 32 °C, to
713 obtain healthy amoebas in a monolayer for the next round of infection.

714 Recover each well into 2 separate 1.5 ml microtubes (1 ml/tube). Centrifuge one of the
715 tubes at maximum speed (~16,000 g) for 20 min at room temperature to pellet the viruses.
716 Remove the supernatant by pipetting/aspiration and keep the pellet for genotyping.
717 Proceed to PCR genotyping to assess the successful integration of the genomic
718 modification (see Steps 97-102).

719 ▲ CRITICAL STEP At this point, recombination events should be independent of the phenotype
720 associated with the modification of the genome and thus, recombinant DNA should be identified
721 by genotyping. Lack of amplification using primers P1+P3 (Figure 4) indicates inefficient
722 transfection or problems with PCR amplification.

723 **PAUSE POINT** Pellets can be stored at -20 °C for at least one month to proceed later to
724 genotyping.

725 ?-TROUBLESHOOTING

726 53. Infect a 25 cm² flask of confluent cells produced in step 51 with the contents of the second
tube obtained during step 52.

728 54. Incubate 1 h at 32 °C.

729 ▲ CRITICAL STEP: Incubation for 1 h without a selection marker allows the expression of the
730 resistance cassette by the virus.

731 55. Wash twice with 1 ml of PYG to eliminate extracellular viruses. Proceed carefully to avoid
detaching cells from the surface.

733 56. Add 5 ml of PYG containing nourseothricin 100 µg.ml⁻¹ or geneticin 100 µg.ml⁻¹ according
to the selection cassette introduced in the viral genome.

735 57. Incubate overnight at 32 °C.

736 **PAUSE POINT** Viruses can be kept in the incubator for several days (typically 2-3 days). If more
time is needed, perform the incubation at room temperature.

738 58. Prepare cultures of amoebas as described in step 51.

739 Recover the contents of the flasks in 15 ml tubes and centrifuge at maximum speed
740 (depending on the centrifuge 6,800 g for 45 min (swinging buckets) or 16,000 g for 20 min
741 (fixed rotor)) at room temperature to pellet the viruses. Discard the supernatants and
742 leave only a small volume to resuspend the pellets.

743 ▲ CRITICAL STEP Centrifugation is necessary to remove the selection marker and allow a new
744 round of viral infection.

745 For each assay, infect one 25 cm² flask of confluent cells produced in step 58 with the
content of a tube obtained during step 59.

747 61. Repeat steps 54-57

748 62. Prepare cultures of amoebas as described in step 53.

749 63. Recover the viruses as described in step 59.

750 64. Repeat steps 54-55

751 65. Replace medium with 5 ml of PYG without selection.
752 66. Incubate overnight at 32 °C.

753 ■ **PAUSE POINT** Viruses can be kept in the incubator for several days (typically 2-3 days). If more
754 time is needed, perform the incubation at room temperature.

755 67. Control viral production by observing the culture using an inverted microscope
756 magnification x400.

757 ▲ **CRITICAL STEP** At this step, significant differences between the samples and the negative
758 control are expected. Viruses and cell lysis should be easily recognized in the samples while less
759 abundant in the negative control.

760 ▲ **CRITICAL STEP** Long incubation of the culture will decrease the differences between the samples
761 and the negative control. This will not affect the overall success of the experiment.

762 ?-TROUBLESHOOTING

763 68. Recover the contents of the flasks in 15 ml tubes. Transfer 100 µl in one 1.5 ml microtube
764 for the last round of enrichment in mutant viruses. Keep the remaining suspension at 4 °C.
765 (OPTIONAL) Transfer 1 ml in a 1.5 ml tube and centrifuge at maximum speed and room
766 temperature. Proceed to genotyping (step 97).

767 69. Prepare cultures of amoebas as described in step 51.

768 70. Infect a 25 cm² flask produced in step 68 with 5-100 µl of viruses according to the amount
769 of viral particle present in the culture.

770 71. Repeat steps 54-57

771 ■ **PAUSE POINT** Viruses can be kept in the incubator for several days (typically 2-3 days). If more
772 time is needed, perform the incubation at room temperature.

773 72. Control viral production with an inverted microscope magnification x 400 to evaluate the
774 quantity of produced viruses. Differences between samples and negative control are
775 expected.

776 73. Recover the contents of the flasks in 15 ml tubes.

777 74. Transfer 1 ml in a 1.5 ml tube and centrifuge at maximum speed at room temperature for
778 genotyping of the virus pellet (steps 97-102). Eliminate the supernatant. Keep the
779 remaining suspension at 4 °C.

780 ?-TROUBLESHOOTING

781 75. Proceed to cloning to isolate recombinant viruses (steps 76-91)

782
783 H2 CLONING AND GENOTYPING OF VIRUSES AND AMOEBAS

784
785 H3 Cloning of viruses ● TIMING 5-7 days (3-5 h hands-on time)

786
787 76. In one 25 cm² flask, inoculate 5 x 10⁵ wt amoebas in 5 ml PYG (2 x 10⁴ cells / cm²), and
788 incubate 24 h at 32 °C, to obtain a monolayer of healthy amoebas.

789 77. Recover cells from step 76

790 78. Determine their concentration using a hemocytometer.

791 79. In a 6-well dish, add 1.5 x 10⁵ wt cells per well, one well per assay. Let the amoebas adhere
792 to the bottom, 5 minutes is usually enough with fresh cells.
752 66. Incubate overnight at 32 °C.

753 ■ **PAUSE POINT** Viruses can be kept in the incubator for several days (typically 2-3 days). If more
754 time is needed, perform the incubation at room temperature.

755 67. Control viral production by observing the culture using an inverted microscope
756 magnification x400.

757 ▲ **CRITICAL STEP** At this step, significant differences between the samples and the negative
758 control are expected. Viruses and cell lysis should be easily recognized in the samples while less
759 abundant in the negative control.

760 ▲ **CRITICAL STEP** Long incubation of the culture will decrease the differences between the samples
761 and the negative control. This will not affect the overall success of the experiment.

762 ?-TROUBLESHOOTING

763 68. Recover the contents of the flasks in 15 ml tubes. Transfer 100 µl in one 1.5 ml microtube
764 for the last round of enrichment in mutant viruses. Keep the remaining suspension at 4 °C.
765 (OPTIONAL) Transfer 1 ml in a 1.5 ml tube and centrifuge at maximum speed and room
766 temperature. Proceed to genotyping (step 97).

767 69. Prepare cultures of amoebas as described in step 51.

768 70. Infect a 25 cm² flask produced in step 68 with 5-100 µl of viruses according to the amount
769 of viral particle present in the culture.

770 71. Repeat steps 54-57

771 ■ **PAUSE POINT** Viruses can be kept in the incubator for several days (typically 2-3 days). If more
772 time is needed, perform the incubation at room temperature.

773 72. Control viral production with an inverted microscope magnification x 400 to evaluate the
774 quantity of produced viruses. Differences between samples and negative control are
775 expected.

776 73. Recover the contents of the flasks in 15 ml tubes.

777 74. Transfer 1 ml in a 1.5 ml tube and centrifuge at maximum speed at room temperature for
778 genotyping of the virus pellet (steps 97-102). Eliminate the supernatant. Keep the
779 remaining suspension at 4 °C.

780 ?-TROUBLESHOOTING

781 75. Proceed to cloning to isolate recombinant viruses (steps 76-91)

782
783 H2 CLONING AND GENOTYPING OF VIRUSES AND AMOEBAS

784
785 H3 Cloning of viruses ● TIMING 5-7 days (3-5 h hands-on time)

786
787 76. In one 25 cm² flask, inoculate 5 x 10⁵ wt amoebas in 5 ml PYG (2 x 10⁴ cells / cm²), and
788 incubate 24 h at 32 °C, to obtain a monolayer of healthy amoebas.

789 77. Recover cells from step 76

790 78. Determine their concentration using a hemocytometer.

791 79. In a 6-well dish, add 1.5 x 10⁵ wt cells per well, one well per assay. Let the amoebas adhere
792 to the bottom, 5 minutes is usually enough with fresh cells.
793 80. Infect the cells prepared in step 77 with 2-10 µl of viral suspension, i.e. add 2 µl if the
794 apparent quantity of viruses is high, to 10 µl if few viruses are visible in steps vi and 72.
795 Gently rock the plate. Incubate 1 h at 32 °C.

796 ▲ CRITICAL STEP In order to isolate/clone the viruses, the MOI should be less than 1. This way,
797 each cell will likely be infected by a single virus. Bear in mind that the proportion of infectious
798 particles of mimivirus are higher than pandoravirus or mollivirus. Thus, particle numbers should
799 be adapted for each different virus. Moreover, gene modification might affect infectivity and
800 consequently, the optimal number of viruses must be addressed empirically.

801 81. Wash 5-10 times with 1 ml of PYG to eliminate the maximum of extracellular viruses.

802 82. Collect the viruses from the wells by gently pipetting up and down into a 1.5 ml microtubes.

804 83. Determine the cell concentration with a hemocytometer and dilute the sample with PYG
805 to adjust the concentration to ~1 amoeba/µl of PYG.

806 ▲ CRITICAL STEP Control the concentration by depositing 5 drops of 1 µl in an empty well of the
807 6-well dish and observing with an inverted microscope magnification x100 or less. There should
808 be 0-3 amoeba(s)/drop. Adjust the dilution if necessary.

809 84. Dilute the suspension in PYG 10 times to have 1 amoeba / 10 µl, which is easier to handle
810 with a multichannel pipet.

811 85. Prepare one 96 well dish per viral strain. In each well, add 100-1,000 of wt amoebas in 200 µL PYG. ▲ CRITICAL STEP It is recommended to add 100 amoebas/well for assays with pandoravirus and mollivirus, as their infectious cycle is long and amoebas tend to overgrow the viruses. For experiments with mimivirus or megavirus, 1000 cells per well is appropriate. No control is needed at this point.

816 86. Add 10 µl of diluted infected amoebas from step 84 to each well of the plate prepared in
817 step 85 using a multichannel pipette. Seal the plates to avoid evaporation, with parafilm
818 or adhesive seals.

819 87. Incubate at 32 °C for at least 2 days (generally 3 to 4 days).

820 88. Every well with an infection contains a potential recombinant virus. Observe each well of
821 the 96 well plates with an inverted microscope magnification x 400 to detect the
822 production of viruses.

823 ▲ CRITICAL STEP If all wells appear infected, repeat the procedure from step 80 infecting with a
824 lower quantity of viruses. When all wells are lysed, it usually indicates infection with multiple
825 viruses.

826 ▲ CRITICAL STEP Lysis induction depends not only on the type of virus (pandoravirus and
827 mollivirus tend to induce a lower degree of cell lysis), but also on the gene knocked out (and the
828 phenotype associated with that deletion). Concordantly, the infection should be carefully assessed
829 in each well to identify the presence or absence of viruses.

830 89. Mix the wells by pipetting vigorously up and down and transfer 100 µl of viral/cell
831 suspension from ~8 wells into a 1.5 ml microtube. Keep the plates sealed at 4 °C.

832 ▲ CRITICAL STEP Label tubes and wells picked from the 96 wells plate. Upon identification of
833 recombinant and clonal viruses, the remaining 100 µl left in the wells will be used to amplify the
834 viruses.
793 80. Infect the cells prepared in step 77 with 2-10 µl of viral suspension, i.e. add 2 µl if the
794 apparent quantity of viruses is high, to 10 µl if few viruses are visible in steps vi and 72.
795 Gently rock the plate. Incubate 1 h at 32 °C.

796 ▲ CRITICAL STEP In order to isolate/clone the viruses, the MOI should be less than 1. This way,
797 each cell will likely be infected by a single virus. Bear in mind that the proportion of infectious
798 particles of mimivirus are higher than pandoravirus or mollivirus. Thus, particle numbers should
799 be adapted for each different virus. Moreover, gene modification might affect infectivity and
800 consequently, the optimal number of viruses must be addressed empirically.

801 81. Wash 5-10 times with 1 ml of PYG to eliminate the maximum of extracellular viruses.

802 82. Collect the viruses from the wells by gently pipetting up and down into a 1.5 ml microtubes.

804 83. Determine the cell concentration with a hemocytometer and dilute the sample with PYG
805 to adjust the concentration to ~1 amoeba/µl of PYG.

806 ▲ CRITICAL STEP Control the concentration by depositing 5 drops of 1 µl in an empty well of the
807 6-well dish and observing with an inverted microscope magnification x100 or less. There should
808 be 0-3 amoeba(s)/drop. Adjust the dilution if necessary.

809 84. Dilute the suspension in PYG 10 times to have 1 amoeba / 10 µl, which is easier to handle
810 with a multichannel pipet.

811 85. Prepare one 96 well dish per viral strain. In each well, add 100-1,000 of wt amoebas in 200 µL PYG. ▲ CRITICAL STEP It is recommended to add 100 amoebas/well for assays with pandoravirus and mollivirus, as their infectious cycle is long and amoebas tend to overgrow the viruses. For experiments with mimivirus or megavirus, 1000 cells per well is appropriate. No control is needed at this point.

816 86. Add 10 µl of diluted infected amoebas from step 84 to each well of the plate prepared in
817 step 85 using a multichannel pipette. Seal the plates to avoid evaporation, with parafilm
818 or adhesive seals.

819 87. Incubate at 32 °C for at least 2 days (generally 3 to 4 days).

820 88. Every well with an infection contains a potential recombinant virus. Observe each well of
821 the 96 well plates with an inverted microscope magnification x 400 to detect the
822 production of viruses.

823 ▲ CRITICAL STEP If all wells appear infected, repeat the procedure from step 80 infecting with a
824 lower quantity of viruses. When all wells are lysed, it usually indicates infection with multiple
825 viruses.

826 ▲ CRITICAL STEP Lysis induction depends not only on the type of virus (pandoravirus and
827 mollivirus tend to induce a lower degree of cell lysis), but also on the gene knocked out (and the
828 phenotype associated with that deletion). Concordantly, the infection should be carefully assessed
829 in each well to identify the presence or absence of viruses.

830 89. Mix the wells by pipetting vigorously up and down and transfer 100 µl of viral/cell
831 suspension from ~8 wells into a 1.5 ml microtube. Keep the plates sealed at 4 °C.

832 ▲ CRITICAL STEP Label tubes and wells picked from the 96 wells plate. Upon identification of
833 recombinant and clonal viruses, the remaining 100 µl left in the wells will be used to amplify the
834 viruses.
835 90. Centrifuge the microtubes of step 89 at maximum speed (~16,000 g) for 20 min at room temperature and use the pellets for genotyping (see steps 97-102).

836 837 838 839 840 841 842 91. Once mutant clones are identified, use the remaining content of the wells to infect 25 cm² flasks containing monolayers of amoebas. At this step and the next ones, no selection with nourseothricin is needed. Incubate at 32 °C until an acceptable quantity of viruses and cell lysis are observable. Then, proceed to the purification of viral particles³⁵ and phenotyping (see Supplementary Methods).

843 844 845 846 847 848 849 850 851 852 853 854 855 856 92. Collect the cells prepared in Step 36Biv, either by scraping or by gently pipetting up and down.

847 848 849 850 851 852 853 854 855 856 93. Repeat Steps 83-86.

848 849 850 851 852 853 854 855 856 94. Incubate at 32 °C until growth can be observed in the wells.

851 852 853 854 855 856 95. Repeat Step 89.

857 858 859 860 861 862 863 864 865 866 867 868 869 870 97. Design primers for genotyping according to Figure 4. Different sets of primers should allow the identification of recombinant and wild-type locus. To facilitate PCR amplification, design primers to amplify DNA products no longer than 2 kb in size.

858 859 860 861 862 863 864 865 866 867 868 869 870 98. Resuspend the pellets of cultures in 20 µl to 100 µl DNase-RNase free water depending on the size of the pellet.

862 863 864 865 866 867 868 869 870 91. ▲ CRITICAL STEP Calculate the annealing temperature using the NEB Tm Calculator (https://tm calculator.neb.com/#!/main). Preferentially, design primers with annealing temperatures between 55 and 60°C.

865 866 867 868 869 870 98. Resuspend the pellets of cultures in 20 µl to 100 µl DNase-RNase free water depending on the size of the pellet.

865 866 867 868 869 870 91. ▲ CRITICAL STEP Include a sample of parental amoebas or virus (wild-type) as control for PCR specific amplification.

865 866 867 868 869 870 99. Use samples prepared in step 98 as direct templates for PCR amplification with Terra™ PCR Direct Red Dye Premix (TaKaRa) according to the following reaction mixture:

<table><thead><tr><td></td><td>Amount</td><td>Final Concentration</td></tr></thead><tbody><tr><td>Nuclease-free water</td><td>To 15 µl</td><td></td></tr><tr><td>2X Terra PCR Direct Red Dye Premix</td><td>7.5 µl</td><td>1x</td></tr><tr><td>10 µM Forward Primer</td><td>0.45 µl</td><td>0.3 µM</td></tr><tr><td>10 µM Reverse Primer</td><td>0.45 µl</td><td>0.3 µM</td></tr></tbody></table>
835 90. Centrifuge the microtubes of step 89 at maximum speed (~16,000 g) for 20 min at room temperature and use the pellets for genotyping (see steps 97-102).

836 837 838 839 840 841 842 91. Once mutant clones are identified, use the remaining content of the wells to infect 25 cm² flasks containing monolayers of amoebas. At this step and the next ones, no selection with nourseothricin is needed. Incubate at 32 °C until an acceptable quantity of viruses and cell lysis are observable. Then, proceed to the purification of viral particles³⁵ and phenotyping (see Supplementary Methods).

843 844 845 846 847 848 849 850 851 852 853 854 855 856 92. Collect the cells prepared in Step 36Biv, either by scraping or by gently pipetting up and down.

847 848 849 850 851 852 853 854 855 856 93. Repeat Steps 83-86.

847 848 849 850 851 852 853 854 855 856 94. Incubate at 32 °C until growth can be observed in the wells.

847 848 849 850 851 852 853 854 855 856 95. Repeat Step 89.

857 858 859 860 861 862 863 864 865 866 867 868 869 870 97. Design primers for genotyping according to Figure 4. Different sets of primers should allow the identification of recombinant and wild-type locus. To facilitate PCR amplification, design primers to amplify DNA products no longer than 2 kb in size.

857 858 859 860 861 862 863 864 865 866 867 868 869 870 ▲ CRITICAL STEP Calculate the annealing temperature using the NEB Tm Calculator (https://tm calculator.neb.com/#!/main). Preferentially, design primers with annealing temperatures between 55 and 60°C.

857 858 859 860 861 862 863 864 865 866 867 868 869 870 98. Resuspend the pellets of cultures in 20 µl to 100 µl DNase-RNase free water depending on the size of the pellet.

857 858 859 860 861 862 863 864 865 866 867 868 869 870 ▲ CRITICAL STEP Include a sample of parental amoebas or virus (wild-type) as control for PCR specific amplification.

857 858 859 860 861 862 863 864 865 866 867 868 869 870 99. Use samples prepared in step 98 as direct templates for PCR amplification with Terra™ PCR Direct Red Dye Premix (TaKaRa) according to the following reaction mixture:

<table><thead><tr><td></td><td>Amount</td><td>Final Concentration</td></tr></thead><tbody><tr><td>Nuclease-free water</td><td>To 15 µl</td><td></td></tr><tr><td>2X Terra PCR Direct Red Dye Premix</td><td>7.5 µl</td><td>1x</td></tr><tr><td>10 µM Forward Primer</td><td>0.45 µl</td><td>0.3 µM</td></tr><tr><td>10 µM Reverse Primer</td><td>0.45 µl</td><td>0.3 µM</td></tr></tbody></table>
<table><tr><td>Template</td><td>1 µl</td><td>---</td></tr></table>

100. Perform PCR following the cycling conditions below, and analyze the products by migrating the PCR reaction using standard agarose gel electrophoresis conditions (135V, 20 min).

<table><thead><tr><td>Cycle number</td><td>Denature</td><td>Anneal</td><td>Extend</td><td>Final</td></tr></thead><tbody><tr><td>1</td><td>98°C, 2 min</td><td></td><td></td><td></td></tr><tr><td>2-28</td><td>98°C, 10 sec</td><td>50-60°C, 15 sec</td><td>68°C, 1-2 min</td><td></td></tr><tr><td>29</td><td></td><td></td><td></td><td>10°C, Hold</td></tr></tbody></table>

### ?-TROUBLESHOOTING See Table 3

101. Check the sequence of the mutations with the help of a company performing standard Sanger sequencing, according to their recommendations.

**▲ CRITICAL STEP** Next-Generation Sequencing is an efficient way of analyzing all the alleles of A. castellanii in a single experiment. If primers are barcoded, multiple clones of amoebas can be analyzed in a single run of sequencing.

102. Select the clones carrying the desired genomic modifications (See Figure 4 and Anticipated results section), and proceed to amplification, purification using the protocol previously described in ³⁵ (in case of viruses) and phenotyping (Step 109).

**▲ CRITICAL STEP** In case Terra amplifications do not lead to conclusive results, purify the gDNA of the samples using PureLink® Genomic DNA Kit (Invitrogen) or Wizard® Genomic DNA Purification Kit (Promega) and elute in the minimal possible volume according to manufacturers' instructions. Then, proceed to genotyping using Phusion™ High-Fidelity DNA Polymerase (see steps 1-2 for PCR conditions).

## H3 Vector preparation for trans-complementation ● TIMING 5 days

103. Prepare a PCR mix as described in step 1 using the appropriate template, most probably gDNA (10 to 50 ng per 50-µl reaction volume) from the studied virus or A. castellanii.

104. Perform PCR following the cycling conditions below, and analyze the products by running 1 µl of the PCR reaction using standard gel electrophoresis (135V, 20 min).

<table><thead><tr><td>Cycle number</td><td>Denature</td><td>Anneal</td><td>Extend</td><td>Final</td></tr></thead><tbody><tr><td>1</td><td>98°C, 30 sec</td><td></td><td></td><td></td></tr><tr><td>2-30</td><td>98°C, 10 sec</td><td>45-72°C, 30 sec</td><td>72°C, 30 sec/kb</td><td></td></tr><tr><td>31</td><td></td><td></td><td>72°C, 5 min</td><td></td></tr><tr><td>32</td><td></td><td></td><td></td><td>10°C, Hold</td></tr></tbody></table>

**▲ CRITICAL STEP** Calculate the annealing temperature using the NEB Tm Calculator (https://tm calculator.neb.com/#!/main) without considering the 15 bp corresponding to the receptor vector and used for In-Fusion® recombination

105. Analyze and purify PCR reaction as described in step 3.

### ?TROUBLESHOOTING

106. Linearize at least 1 µg of the desired vector (Table 1) by digesting it with one or two restriction enzymes at 37°C between 3 hours to overnight following the reaction:

<table><tr><td>Amount</td><td>Final Concentration</td></tr></table>
<table><tr><td>Template</td><td>1 µl</td><td>---</td></tr></table>

100. Perform PCR following the cycling conditions below, and analyze the products by migrating the PCR reaction using standard agarose gel electrophoresis conditions (135V, 20 min).

<table><thead><tr><td>Cycle number</td><td>Denature</td><td>Anneal</td><td>Extend</td><td>Final</td></tr></thead><tbody><tr><td>1</td><td>98°C, 2 min</td><td></td><td></td><td></td></tr><tr><td>2-28</td><td>98°C, 10 sec</td><td>50-60°C, 15 sec</td><td>68°C, 1-2 min</td><td></td></tr><tr><td>29</td><td></td><td></td><td></td><td>10°C, Hold</td></tr></tbody></table>

**?TROUBLESHOOTING** See Table 3

101. Check the sequence of the mutations with the help of a company performing standard Sanger sequencing, according to their recommendations.

**▲ CRITICAL STEP** Next-Generation Sequencing is an efficient way of analyzing all the alleles of *A. castellanii* in a single experiment. If primers are barcoded, multiple clones of amoebas can be analyzed in a single run of sequencing.

102. Select the clones carrying the desired genomic modifications (See Figure 4 and Anticipated results section), and proceed to amplification, purification using the protocol previously described in ³⁵ (in case of viruses) and phenotyping (Step 109).

**▲ CRITICAL STEP** In case Terra amplifications do not lead to conclusive results, purify the gDNA of the samples using PureLink® Genomic DNA Kit (Invitrogen) or Wizard® Genomic DNA Purification Kit (Promega) and elute in the minimal possible volume according to manufacturers' instructions. Then, proceed to genotyping using Phusion™ High-Fidelity DNA Polymerase (see steps 1-2 for PCR conditions).

**H3 Vector preparation for trans-complementation ● TIMING 5 days**

103. Prepare a PCR mix as described in step 1 using the appropriate template, most probably gDNA (10 to 50 ng per 50-µl reaction volume) from the studied virus or *A. castellanii*.

104. Perform PCR following the cycling conditions below, and analyze the products by running 1 µl of the PCR reaction using standard gel electrophoresis (135V, 20 min).

<table><thead><tr><td>Cycle number</td><td>Denature</td><td>Anneal</td><td>Extend</td><td>Final</td></tr></thead><tbody><tr><td>1</td><td>98°C, 30 sec</td><td></td><td></td><td></td></tr><tr><td>2-30</td><td>98°C, 10 sec</td><td>45-72°C, 30 sec</td><td>72°C, 30 sec/kb</td><td></td></tr><tr><td>31</td><td></td><td></td><td>72°C, 5 min</td><td></td></tr><tr><td>32</td><td></td><td></td><td></td><td>10°C, Hold</td></tr></tbody></table>

**▲ CRITICAL STEP** Calculate the annealing temperature using the NEB Tm Calculator (https://tm calculator.neb.com/#!/main) without considering the 15 bp corresponding to the receptor vector and used for In-Fusion® recombination

105. Analyze and purify PCR reaction as described in step 3.

**?TROUBLESHOOTING**

106. Linearize at least 1 µg of the desired vector (Table 1) by digesting it with one or two restriction enzymes at 37°C between 3 hours to overnight following the reaction:

<table><tr><td>Amount</td><td>Final Concentration</td></tr></table>
<table><tr><td>Vector</td><td>1 µg</td><td>50 ng/µl</td></tr><tr><td>NEB 3.1 or NEB CutSmart buffer</td><td>2 µl</td><td>1x</td></tr><tr><td>Restriction enzyme(s)</td><td>20 U (or 10U + 10U)</td><td>1 U/µl (or 0.5 U/µl of each)</td></tr><tr><td>Nuclease-free water</td><td>To 20 µl</td><td></td></tr></table>

107. Repeat steps 5-16. ?TROUBLESHOOTING

108. Proceed to transfection and selection of amoebas as described in steps 17-37

## H3 Viral phenotyping TIMING: variable

109. Refer to Supplementary Methods for viral particle quantification¹⁵, infectious dose determination¹⁵,³⁶, DNA replication measurements¹⁵, Immunofluorescence assays (adapted from³⁷) or western blotting (adapted from³⁸).

## H1 ANTICIPATED RESULTS

Successful examples of genome manipulation are shown in Figure 4. Genotyping and sequencing of PCR products must be performed in parallel with a negative control (amplified from parental virus/amoebas) to demonstrate specificity of the PCR reactions. Other examples of gene manipulation can be found in our publications¹⁵,¹⁸. We typically obtain recombinant viruses or amoebas in 3 weeks or one month, respectively), with low rates of unsuccessful attempts (less than 5%). Importantly, in our experience, essential genes are still difficult to modify and further effort to develop conditional systems for gene depletion will be essential to efficiently target the variety of genes encoded by both *A. castellanii* and *GVs*.

## H1 TIMING

Steps 1-16, Vector preparation for CRISPR/Cas9 expression: 5 days.

Steps 17-27, Transfection in *A. castellanii*: 2 days.

Steps 28-35, Selection of amoebas stably expressing a transgene such as cas9 and gRNAs: 7-10 days.

Step 36A, Mutagenesis of viruses with CRISPR/Cas9: 7-10 days.

Step 36B, Mutagenesis of amoebas using CRISPR/Cas9: 3 weeks.

Steps 37-43, Vector preparation for homologous recombination experiments: 10 days.

Steps 44-75, Mutagenesis of viruses by recombination with the insertion of a resistance cassette: 5-7 days.

Steps 76-91, Cloning of viruses: 5-7 days.

Steps 92-96, Cloning of amoebas: 5-7 days.

Steps 97-102, Genotyping of viruses and amoebas: 4 days

Steps 103-108, Vector preparation for trans-complementation: 5 days

Step 109, Viral phenotyping: variable
<table><tr><td>Vector</td><td>1 µg</td><td>50 ng/µl</td></tr><tr><td>NEB 3.1 or NEB CutSmart buffer</td><td>2 µl</td><td>1x</td></tr><tr><td>Restriction enzyme(s)</td><td>20 U (or 10U + 10U)</td><td>1 U/µl (or 0.5 U/µl of each)</td></tr><tr><td>Nuclease-free water</td><td>To 20 µl</td><td></td></tr></table>

107. Repeat steps 5-16. ?TROUBLESHOOTING

108. Proceed to transfection and selection of amoebas as described in steps 17-37

### H3 Viral phenotyping TIMING: variable

109. Refer to Supplementary Methods for viral particle quantification¹⁵, infectious dose determination¹⁵,³⁶, DNA replication measurements¹⁵, Immunofluorescence assays (adapted from³⁷) or western blotting (adapted from³⁸).

### H1 ANTICIPATED RESULTS

Successful examples of genome manipulation are shown in Figure 4. Genotyping and sequencing of PCR products must be performed in parallel with a negative control (amplified from parental virus/amoebas) to demonstrate specificity of the PCR reactions. Other examples of gene manipulation can be found in our publications¹⁵,¹⁸. We typically obtain recombinant viruses or amoebas in 3 weeks or one month, respectively), with low rates of unsuccessful attempts (less than 5%). Importantly, in our experience, essential genes are still difficult to modify and further effort to develop conditional systems for gene depletion will be essential to efficiently target the variety of genes encoded by both *A. castellanii* and *GVs*.

### H1 TIMING

Steps 1-16, Vector preparation for CRISPR/Cas9 expression: 5 days.

Steps 17-27, Transfection in *A. castellanii*: 2 days.

Steps 28-35, Selection of amoebas stably expressing a transgene such as cas9 and gRNAs: 7-10 days.

Step 36A, Mutagenesis of viruses with CRISPR/Cas9: 7-10 days.

Step 36B, Mutagenesis of amoebas using CRISPR/Cas9: 3 weeks.

Steps 37-43, Vector preparation for homologous recombination experiments: 10 days.

Steps 44-75, Mutagenesis of viruses by recombination with the insertion of a resistance cassette: 5-7 days.

Steps 76-91, Cloning of viruses: 5-7 days.

Steps 92-96, Cloning of amoebas: 5-7 days.

Steps 97-102, Genotyping of viruses and amoebas: 4 days

Steps 103-108, Vector preparation for trans-complementation: 5 days

Step 109, Viral phenotyping: variable
933 **H1 TROUBLESHOOTING**

934 Troubleshooting advice can be found in Table 3.

935

936 **H1 REFERENCES**

937 1 Rayamajhee, B., Willcox, M. D., Henriquez, F. L., Petsoglou, C. & Carnt, N. Acanthamoeba keratitis: an increasingly common infectious disease of the cornea. *Lancet Microbe* **2**, e345-e346, doi:10.1016/S2666-5247(21)00093-8 (2021).

938 2 Guimaraes, A. J., Gomes, K. X., Cortines, J. R., Peralta, J. M. & Peralta, R. H. Acanthamoeba spp. as a universal host for pathogenic microorganisms: One bridge from environment to host virulence. *Microbiol Res* **193**, 30-38, doi:10.1016/j.micres.2016.08.001 (2016).

943 3 Raoult, D. et al. The 1.2-megabase genome sequence of Mimivirus. *Science* **306**, 1344-1350, doi:10.1126/science.1101485 (2004).

945 4 Philippe, N. et al. Pandoraviruses: amoeba viruses with genomes up to 2.5 Mb reaching that of parasitic eukaryotes. *Science* **341**, 281-286, doi:10.1126/science.1239181 (2013).

947 5 Legendre, M. et al. Thirty-thousand-year-old distant relative of giant icosahedral DNA viruses with a pandoravirus morphology. *Proc Natl Acad Sci U S A* **111**, 4274-4279, doi:10.1073/pnas.1320670111 (2014).

950 6 Legendre, M. et al. In-depth study of Mollivirus sibericum, a new 30,000-y-old giant virus infecting Acanthamoeba. *Proc Natl Acad Sci U S A* **112**, E5327-5335, doi:10.1073/pnas.1510795112 (2015).

952 7 Schulz, F., Abergel, C. & Woyke, T. Giant virus biology and diversity in the era of genome-resolved metagenomics. *Nat Rev Microbiol*, **20**, 721-736, doi:10.1038/s41579-022-00754-5 (2022).

954 8 de Oliveira, E. G. et al. Giant Viruses as a Source of Novel Enzymes for Biotechnological Application. *Pathogens* **11**, 1453, doi:10.3390/pathogens11121453 (2022).

956 9 Byers, T. J. Molecular biology of DNA in Acanthamoeba, Amoeba, Entamoeba, and Naegleria. *Int Rev Cyto* **99**, 311-341, doi:10.1016/s0074-7696(08)61430-8 (1986).

958 10 Gicquaud, C. & Tremblay, N. Observations with Hoechst Staining of Amitosis in Acanthamoeba-Castellanii. *J Protozool* **38**, 221-224, doi:DOI 10.1111/j.1550-7408.1991.tb04432.x (1991).

960 11 Matthey-Doret, C. et al. Chromosome-scale assemblies of Acanthamoeba castellanii genomes provide insights into Legionella pneumophila infection-related chromatin reorganization. *Genome Res* **32**, 1698-1710, doi:10.1101/gr.276375.121 (2022).

963 12 Peng, Z., Omaruddin, R. & Bateman, E. Stable transfection of Acanthamoeba castellanii. *Biochim Biophys Acta* **1743**, 93-100, doi:10.1016/j.bbamcr.2004.08.014 (2005).

965 13 Liu, Y. et al. Virus-encoded histone doublets are essential and form nucleosome-like structures. *Cell* **184**, 4237-4250 e4219, doi:10.1016/j.cell.2021.06.032 (2021).

967 14 Fridmann-Sirkis, Y. et al. Efficiency in Complexity: Composition and Dynamic Nature of Mimivirus Replication Factories. *J Virol* **90**, 10039-10047, doi:10.1128/JVI.01319-16 (2016).

969 15 Bisio, H. et al. Evolution of giant pandoravirus revealed by CRISPR/Cas9. *Nat Commun* **14**, 428, doi:10.1038/s41467-023-36145-4 (2023).

971 16 Aqeel, Y., Siddiqui, R. & Khan, N. A. Silencing of xylose isomerase and cellulose synthase by siRNA inhibits encystation in Acanthamoeba castellanii. *Parasitol Res* **112**, 1221-1227, doi:10.1007/s00436-012-3254-6 (2013).

974 17 Sobhy, H., Scola, B. L., Pagnier, I., Raoult, D. & Colson, P. Identification of giant Mimivirus protein functions using RNA interference. *Front Microbiol* **6**, 345, doi:10.3389/fmicb.2015.00345 (2015).

976 18 Alempic, J.-M. et al. Knockout of GMC-oxidoreductase genes reveals functional redundancy in mimivirus. *bioRxiv*, 2023.2004.2028.538727, doi:10.1101/2023.04.28.538727 (2023).
933 **H1 TROUBLESHOOTING**

934 Troubleshooting advice can be found in Table 3.

935

936 **H1 REFERENCES**

937 1 Rayamajhee, B., Willcox, M. D., Henriquez, F. L., Petsoglou, C. & Carnt, N. Acanthamoeba keratitis: an increasingly common infectious disease of the cornea. *Lancet Microbe* **2**, e345-e346, doi:10.1016/S2666-5247(21)00093-8 (2021).

938 2 Guimaraes, A. J., Gomes, K. X., Cortines, J. R., Peralta, J. M. & Peralta, R. H. Acanthamoeba spp. as a universal host for pathogenic microorganisms: One bridge from environment to host virulence. *Microbiol Res* **193**, 30-38, doi:10.1016/j.micres.2016.08.001 (2016).

943 3 Raoult, D. et al. The 1.2-megabase genome sequence of Mimivirus. *Science* **306**, 1344-1350, doi:10.1126/science.1101485 (2004).

945 4 Philippe, N. et al. Pandoraviruses: amoeba viruses with genomes up to 2.5 Mb reaching that of parasitic eukaryotes. *Science* **341**, 281-286, doi:10.1126/science.1239181 (2013).

947 5 Legendre, M. et al. Thirty-thousand-year-old distant relative of giant icosahedral DNA viruses with a pandoravirus morphology. *Proc Natl Acad Sci U S A* **111**, 4274-4279, doi:10.1073/pnas.1320670111 (2014).

950 6 Legendre, M. et al. In-depth study of Mollivirus sibericum, a new 30,000-y-old giant virus infecting Acanthamoeba. *Proc Natl Acad Sci U S A* **112**, E5327-5335, doi:10.1073/pnas.1510795112 (2015).

952 7 Schulz, F., Abergel, C. & Woyke, T. Giant virus biology and diversity in the era of genome-resolved metagenomics. *Nat Rev Microbiol*, **20**, 721-736, doi:10.1038/s41579-022-00754-5 (2022).

954 8 de Oliveira, E. G. et al. Giant Viruses as a Source of Novel Enzymes for Biotechnological Application. *Pathogens* **11**, 1453, doi:10.3390/pathogens11121453 (2022).

956 9 Byers, T. J. Molecular biology of DNA in Acanthamoeba, Amoeba, Entamoeba, and Naegleria. *Int Rev Cyto* **99**, 311-341, doi:10.1016/s0074-7696(08)61430-8 (1986).

958 10 Gicquaud, C. & Tremblay, N. Observations with Hoechst Staining of Amitosis in Acanthamoeba-Castellanii. *J Protozool* **38**, 221-224, doi:DOI 10.1111/j.1550-7408.1991.tb04432.x (1991).

960 11 Matthey-Doret, C. et al. Chromosome-scale assemblies of Acanthamoeba castellanii genomes provide insights into Legionella pneumophila infection-related chromatin reorganization. *Genome Res* **32**, 1698-1710, doi:10.1101/gr.276375.121 (2022).

963 12 Peng, Z., Omaruddin, R. & Bateman, E. Stable transfection of Acanthamoeba castellanii. *Biochim Biophys Acta* **1743**, 93-100, doi:10.1016/j.bbamcr.2004.08.014 (2005).

965 13 Liu, Y. et al. Virus-encoded histone doublets are essential and form nucleosome-like structures. *Cell* **184**, 4237-4250 e4219, doi:10.1016/j.cell.2021.06.032 (2021).

967 14 Fridmann-Sirkis, Y. et al. Efficiency in Complexity: Composition and Dynamic Nature of Mimivirus Replication Factories. *J Virol* **90**, 10039-10047, doi:10.1128/JVI.01319-16 (2016).

969 15 Bisio, H. et al. Evolution of giant pandoravirus revealed by CRISPR/Cas9. *Nat Commun* **14**, 428, doi:10.1038/s41467-023-36145-4 (2023).

971 16 Aqeel, Y., Siddiqui, R. & Khan, N. A. Silencing of xylose isomerase and cellulose synthase by siRNA inhibits encystation in Acanthamoeba castellanii. *Parasitol Res* **112**, 1221-1227, doi:10.1007/s00436-012-3254-6 (2013).

974 17 Sobhy, H., Scola, B. L., Pagnier, I., Raoult, D. & Colson, P. Identification of giant Mimivirus protein functions using RNA interference. *Front Microbiol* **6**, 345, doi:10.3389/fmicb.2015.00345 (2015).

976 18 Alempic, J.-M. et al. Knockout of GMC-oxidoreductase genes reveals functional redundancy in mimivirus. *bioRxiv*, 2023.2004.2028.538727, doi:10.1101/2023.04.28.538727 (2023).
978 19 Jiang, F. & Doudna, J. A. CRISPR-Cas9 Structures and Mechanisms. *Annu Rev Biophys* **46**, 505-529, doi:10.1146/annurev-biophys-062215-010822 (2017).

980 20 Moon, E. K., Hong, Y., Chung, D. I., Goo, Y. K. & Kong, H. H. Down-regulation of cellulose synthase inhibits the formation of endocysts in Acanthamoeba. *Korean J Parasitol* **52**, 131-135, doi:10.3347/kjp.2014.52.2.131 (2014).

983 21 Groenenboom, M. A., Maree, A. F. & Hogeweg, P. The RNA silencing pathway: the bits and pieces that matter. *PLoS Comput Biol* **1**, 155-165, doi:10.1371/journal.pcbi.0010021 (2005).

985 22 Wei, J. R. et al. Depletion of antibiotic targets has widely varying effects on growth. *Proc Natl Acad Sci U S A* **108**, 4176-4181, doi:10.1073/pnas.1018301108 (2011).

987 23 Curtis, C. D. & Nardulli, A. M. Using RNA interference to study protein function. *Methods Mol Biol* **505**, 187-204, doi:10.1007/978-1-60327-575-0_11 (2009).

989 24 Levasseur, A. et al. MIMIVIRE is a defence system in mimivirus that confers resistance to virophage. *Nature* **531**, 249-252, doi:10.1038/nature17146 (2016).

991 25 Bekliz, M. et al. Experimental Analysis of Mimivirus Translation Initiation Factor 4a Reveals Its Importance in Viral Protein Translation during Infection of Acanthamoeba polyphaga. *J Virol* **92**, doi:10.1128/JVI.00337-18 (2018).

994 26 Mougari, S., Abrahao, J., Oliveira, G. P., Bou Khalil, J. Y. & La Scola, B. Role of the R349 Gene and Its Repeats in the MIMIVIRE Defense System. *Front Microbiol* **10**, 1147, doi:10.3389/fmicb.2019.01147 (2019).

997 27 Bisio, H. et al. The ZIP Code of Vesicle Trafficking in Apicomplexa: SEC1/Munc18 and SNARE Proteins. *mBio* **11**, e02092-20, doi:10.1128/mBio.02092-20 (2020).

999 28 Bisio, H., Krishnan, A., Marq, J. B. & Soldati-Favre, D. Toxoplasma gondii phosphatidylserine flippase complex ATP2B-CDC50.4 critically participates in microneme exocytosis. *PLoS Pathog* **18**, e1010438, doi:10.1371/journal.ppat.1010438 (2022).

1002 29 Ali, Z., Mahas, A. & Mahfouz, M. CRISPR/Cas13 as a Tool for RNA Interference. *Trends Plant Sci* **23**, 374-378, doi:10.1016/j.tplants.2018.03.003 (2018).

1004 30 Pallares Masmitja, M., Knodlseder, N. & Guell, M. CRISPR-gRNA Design. *Methods Mol Biol* **1961**, 3-11, doi:10.1007/978-1-4939-9170-9_1 (2019).

1006 31 Bateman, E. Expression plasmids and production of EGFP in stably transfected Acanthamoeba. *Protein Expr Purif* **70**, 95-100, doi:10.1016/j.pep.2009.10.008 (2010).

1008 32 Fabre, E. et al. Noumeavirus replication relies on a transient remote control of the host nucleus. *Nat Commun* **8**, 15087, doi:10.1038/ncomms15087 (2017).

1010 33 Davis, M. W. & Jorgensen, E. M. ApE, A Plasmid Editor: A Freely Available DNA Manipulation and Visualization Program. *Front Bioinform* **2**, 818619, doi:10.3389/fbinf.2022.818619 (2022).

1012 34 Dos Santos, D. L. et al. Clinical and molecular diagnosis of Acanthamoeba keratitis in contact lens wearers in southern Brazil reveals the presence of an endosymbiont. *Parasitol Res* **121**, 1447-1454, doi:10.1007/s00436-022-07474-y (2022).

1015 35 Bertaux, L., Lartigue, A. & Jeudy, S. Giant Mimiviridae CsCl Purification Protocol. *Bio Protoc* **10**, e3827, doi:10.21769/BioProtoc.3827 (2020).

1017 36 Lei, C., Yang, J., Hu, J. & Sun, X. On the Calculation of TCID(50) for Quantitation of Virus Infectivity. *Virol Sin* **36**, 141-144, doi:10.1007/s12250-020-00230-5 (2021).

1019 37 Burda, P. C., Bisio, H., Marq, J. B., Soldati-Favre, D. & Heussler, V. T. CRISPR/Cas9-Based Knockout of GNAQ Reveals Differences in Host Cell Signaling Necessary for Egress of Apicomplexan Parasites. *mSphere* **5**, e01001-20, doi:10.1128/mSphere.01001-20 (2020).

1022 38 Bisio, H., Lunghi, M., Brochet, M. & Soldati-Favre, D. Phosphatidic acid governs natural egress in Toxoplasma gondii via a guanylate cyclase receptor platform. *Nat Microbiol* **4**, 420-428, doi:10.1038/s41564-018-0339-8 (2019).
978 19 Jiang, F. & Doudna, J. A. CRISPR-Cas9 Structures and Mechanisms. *Annu Rev Biophys* **46**, 505-529, doi:10.1146/annurev-biophys-062215-010822 (2017).

980 20 Moon, E. K., Hong, Y., Chung, D. I., Goo, Y. K. & Kong, H. H. Down-regulation of cellulose synthase inhibits the formation of endocysts in Acanthamoeba. *Korean J Parasitol* **52**, 131-135, doi:10.3347/kjp.2014.52.2.131 (2014).

983 21 Groenenboom, M. A., Maree, A. F. & Hogeweg, P. The RNA silencing pathway: the bits and pieces that matter. *PLoS Comput Biol* **1**, 155-165, doi:10.1371/journal.pcbi.0010021 (2005).

985 22 Wei, J. R. et al. Depletion of antibiotic targets has widely varying effects on growth. *Proc Natl Acad Sci U S A* **108**, 4176-4181, doi:10.1073/pnas.1018301108 (2011).

987 23 Curtis, C. D. & Nardulli, A. M. Using RNA interference to study protein function. *Methods Mol Biol* **505**, 187-204, doi:10.1007/978-1-60327-575-0_11 (2009).

989 24 Levasseur, A. et al. MIMIVIRE is a defence system in mimivirus that confers resistance to virophage. *Nature* **531**, 249-252, doi:10.1038/nature17146 (2016).

991 25 Bekliz, M. et al. Experimental Analysis of Mimivirus Translation Initiation Factor 4a Reveals Its Importance in Viral Protein Translation during Infection of Acanthamoeba polyphaga. *J Virol* **92**, doi:10.1128/JVI.00337-18 (2018).

994 26 Mougari, S., Abrahao, J., Oliveira, G. P., Bou Khalil, J. Y. & La Scola, B. Role of the R349 Gene and Its Repeats in the MIMIVIRE Defense System. *Front Microbiol* **10**, 1147, doi:10.3389/fmicb.2019.01147 (2019).

997 27 Bisio, H. et al. The ZIP Code of Vesicle Trafficking in Apicomplexa: SEC1/Munc18 and SNARE Proteins. *mBio* **11**, e02092-20, doi:10.1128/mBio.02092-20 (2020).

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1008 32 Fabre, E. et al. Noumeavirus replication relies on a transient remote control of the host nucleus. *Nat Commun* **8**, 15087, doi:10.1038/ncomms15087 (2017).

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1012 34 Dos Santos, D. L. et al. Clinical and molecular diagnosis of Acanthamoeba keratitis in contact lens wearers in southern Brazil reveals the presence of an endosymbiont. *Parasitol Res* **121**, 1447-1454, doi:10.1007/s00436-022-07474-y (2022).

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1019 37 Burda, P. C., Bisio, H., Marq, J. B., Soldati-Favre, D. & Heussler, V. T. CRISPR/Cas9-Based Knockout of GNAQ Reveals Differences in Host Cell Signaling Necessary for Egress of Apicomplexan Parasites. *mSphere* **5**, e01001-20, doi:10.1128/mSphere.01001-20 (2020).

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1025 **H1 Acknowledgments**

1026 We thank Sandra Jeudy for helpful advice during the development of the protocol. This study was founded
1027 by the European Research Council (ERC) under the European Union's Horizon 2020 research and
1028 innovation program (grant agreement No 832601; C.A.). H.B. is the recipient of an EMBO Long-Term
1029 Fellowship (ALTF 979-2019).

1030 **H1 Data availability**

1031 All data supporting this paper are included within the article in the form of figures and tables. Raw data
1032 associated with Figure 4 are provided in the primary articles¹⁵,¹⁸. All plasmids were deposited in Addgene
1033 (reference number indicated in Table 1).

1034 **H1 Author Contributions Statement**

1035 H.B. Writing of the original draft. N.P. Writing of the original draft. A.S. Writing of the original draft. C.A.
1036 Project administration, funding acquisition and writing of the original draft.

1037 **H1 Competing interests**

1038 The authors declare no competing interests as defined by Nature Research, or other interests that might
1039 be perceived to influence the interpretation of the article
1025 **H1 Acknowledgments**

1026 We thank Sandra Jeudy for helpful advice during the development of the protocol. This study was founded
1027 by the European Research Council (ERC) under the European Union's Horizon 2020 research and
1028 innovation program (grant agreement No 832601; C.A.). H.B. is the recipient of an EMBO Long-Term
1029 Fellowship (ALTF 979-2019).

1030 **H1 Data availability**

1031 All data supporting this paper are included within the article in the form of figures and tables. Raw data
1032 associated with Figure 4 are provided in the primary articles¹⁵,¹⁸. All plasmids were deposited in Addgene
1033 (reference number indicated in Table 1).

1034 **H1 Author Contributions Statement**

1035 H.B. Writing of the original draft. N.P. Writing of the original draft. A.S. Writing of the original draft. C.A.
1036 Project administration, funding acquisition and writing of the original draft.

1037 **H1 Competing interests**

1038 The authors declare no competing interests as defined by Nature Research, or other interests that might
1039 be perceived to influence the interpretation of the article
<figure><img src="image_3.png" /></figure>
<figure><img src="image_2.png" /></figure>
1043 A general workflow of the Procedure is shown. Genome modification by recombination or CRISPR/Cas9
1044 are depicted, including vector preparation, transfection, selection, cloning, and phenotyping. The nature
1045 of the modification expected on the genomes of A. castellanii or GV's is also indicated. vAS1, vHB46, and
1046 vH47 were developed for gene editing in cytoplasmic GV's (mimivirus, megavirus). vHB178, vHB179, and
1047 vHB63 were developed for gene editing of nuclear GV's (pandoravirus, mollivirus) (Table 1). pmg741,
1048 pmg153 are the promoters from the *Megavirus chilensis* early genes *mg741* and *mg153*. They are efficient
1049 in Mimivirus as well. pneo650 is the promoter from *Pandoravirus neocaledonia pneo_cds_650* early gene.
1050 NAT: Nourseothricin N-Acetyl Transferase conferring resistance to nourseothricin. NEO: Neomycin
1051 phosphotransferase conferring resistance to geneticin. GOI: Gene Of Interest. N: nucleus of A. castellanii.
1052 pU6: type III promoter from U6 gene of A. castellanii driving the transcription of small RNAs such as tRNAs.
1053 Green ellipses in vectors vAS1, vHB178, and 179: HA or FLAG tags. The yellow box in vAS1, green box in
1054 vHB178 and 179, and boxes in 3' position of NAT: 3' UTRs. vHB16 template to produce a DNA sequence
1055 containing 2 consecutive gRNAs that will be cloned into vHB8 digested with NotI. NLS: nuclear localization
1056 signal. GFP: green fluorescent protein. HR: homologous recombination. NHEJ: Non-homologous end
1057 joining. Restriction enzymes are shown in purple with purple arrowheads indicating the cut site.

<figure><img src="image_4.png" /></figure>

1058

1059

<caption>Figure 2. Design of homology arms for gene modification by homologous recombination.</caption>

1060

1061

1062

1063

1064

1065

1066

1067

1068

1069

a. Schematic representation of the strategy to endogenously tag a gene using recombination. Example primer sequences, labelled 1-4, are shown in Table 2. 500bp at the end of the gene of interest (goi), excluding the stop codon, and 500bp at the 3' of the gene are amplified by PCR and used as homology arms for recombination. Homology arms are cloned into a vector containing the epitope tag and a drug-resistance cassette for the selection of recombinant viruses. Homologous recombination allows the insertion of the linearized cassette into the locus of the goi. Asterisks denotate stop codons and dashed line delimitate areas of homologous recombination.

b. Schematic representation of the strategy for gene knockout using recombination as an example. Example primer sequences, labeled 5-8, are shown in Table 2. 500bp at each extremity of the goi are amplified by PCR and cloned into a vector containing a drug resistance cassette. Homologous
1043 A general workflow of the Procedure is shown. Genome modification by recombination or CRISPR/Cas9
1044 are depicted, including vector preparation, transfection, selection, cloning, and phenotyping. The nature
1045 of the modification expected on the genomes of A. castellanii or GV's is also indicated. vAS1, vHB46, and
1046 vH47 were developed for gene editing in cytoplasmic GV's (mimivirus, megavirus). vHB178, vHB179, and
1047 vHB63 were developed for gene editing of nuclear GV's (pandoravirus, mollivirus) (Table 1). pmg741,
1048 pmg153 are the promoters from the *Megavirus chilensis* early genes *mg741* and *mg153*. They are efficient
1049 in Mimivirus as well. pneo650 is the promoter from *Pandoravirus neocaledonia pneo_cds_650* early gene.
1050 NAT: Nourseothricin N-Acetyl Transferase conferring resistance to nourseothricin. NEO: Neomycin
1051 phosphotransferase conferring resistance to geneticin. GOI: Gene Of Interest. N: nucleus of A. castellanii.
1052 pU6: type III promoter from U6 gene of A. castellanii driving the transcription of small RNAs such as tRNAs.
1053 Green ellipses in vectors vAS1, vHB178, and 179: HA or FLAG tags. The yellow box in vAS1, green box in
1054 vHB178 and 179, and boxes in 3' position of NAT: 3' UTRs. vHB16 template to produce a DNA sequence
1055 containing 2 consecutive gRNAs that will be cloned into vHB8 digested with NotI. NLS: nuclear localization
1056 signal. GFP: green fluorescent protein. HR: homologous recombination. NHEJ: Non-homologous end
1057 joining. Restriction enzymes are shown in purple with purple arrowheads indicating the cut site.

<figure><img src="image_3.png" /><figcaption>1058
1059 Figure 2. Design of homology arms for gene modification by homologous recombination.</figcaption></figure>

1060 a. Schematic representation of the strategy to endogenously tag a gene using recombination. 1061 1062 Example primer sequences, labelled 1-4, are shown in Table 2. 500bp at the end of the gene of 1063 interest (goi), excluding the stop codon, and 500bp at the 3' of the gene are amplified by PCR and 1064 used as homology arms for recombination. Homology arms are cloned into a vector containing the 1065 epitope tag and a drug-resistance cassette for the selection of recombinant viruses. Homologous 1066 recombination allows the insertion of the linearized cassette into the locus of the goi. Asterisks 1067 denotate stop codons and dashed line delimitate areas of homologous recombination. 1068

1069 b. Schematic representation of the strategy for gene knockout using recombination as an example. 1070 Example primer sequences, labeled 5-8, are shown in Table 2. 500bp at each extremity of the goi 1071 are amplified by PCR and cloned into a vector containing a drug resistance cassette. Homologous
1070 recombination allows the insertion of the linearized cassette into the targeted locus. Asterisks
1071 denotate stop codons and dashed line delimitate areas of homologous recombination.

1072

<figure><img src="image_5.png" /></figure>

1073

<caption>1074 Figure 3. Cloning of polycistronic DNA sequences for gRNAs expression.</caption>

1075 Schematic representation of the strategy to clone 2 gRNA sequences in the plasmid vHB8 encoding
1076 CRISPR/Cas9. Primers I and J containing the new gRNA sequences (coloured diamonds) are used for PCR
1077 amplification of a tracrRNA and tRNA, using vHB16 as a template (upper panel). The resulting product is
1078 then cloned into vHB8 digested with NotI which encodes a single tRNA and tracrRNA (lower panel). vHB8
1079 does not contain any gRNA but rather a NotI site instead, allowing the insertion of the PCR product
1080 between the tRNA and tracrRNA.
1070 recombination allows the insertion of the linearized cassette into the targeted locus. Asterisks
1071 denotate stop codons and dashed line delimitate areas of homologous recombination.

1072

<figure><img src="image_4.png" /></figure>

1073

<caption>1074 Figure 3. Cloning of polycistronic DNA sequences for gRNAs expression.</caption>

1075 Schematic representation of the strategy to clone 2 gRNA sequences in the plasmid vHB8 encoding
1076 CRISPR/Cas9. Primers I and J containing the new gRNA sequences (coloured diamonds) are used for PCR
1077 amplification of a tracrRNA and tRNA, using vHB16 as a template (upper panel). The resulting product is
1078 then cloned into vHB8 digested with NotI which encodes a single tRNA and tracrRNA (lower panel). vHB8
1079 does not contain any gRNA but rather a NotI site instead, allowing the insertion of the PCR product
1080 between the tRNA and tracrRNA.
<figure><img src="image_6.png" /><figcaption>Figure 4. Design of primers for genotyping.</figcaption></figure>

A. Design of primers and genotyping for modification of GVs by homologous recombination. Primers should be designed outside of the homology arms to detect recombination at the expected locus. 'Pool' indicates suspension of viruses after drug selection but prior to cloning (Step 75). 'Clone'

1081

1082

1083

1084

1085
<figure><img src="image_5.png" /><figcaption>Figure 4. Design of primers for genotyping.</figcaption></figure>

A. Design of primers and genotyping for modification of GVs by homologous recombination. Primers should be designed outside of the homology arms to detect recombination at the expected locus. 'Pool' indicates suspension of viruses after drug selection but prior to cloning (Step 75). 'Clone'

1081

1082

1083

1084

1085
1086 indicates a homogeneous population after successful cloning (Step 91). Primers P1 and P2 allow to detect unmodified locus while primers P1 and P3 would allow amplifying the recombinant locus.
1087 Analyzing the presence of recombinant viruses right after transfection (Step 66) is highly recommended and both P1+P2 and P1+P3 are expected to display positive amplification. As an example, genotyping of the insertion of a second copy of *mcp* into the genome of *Mollivirus sibericum* is shown¹⁵.

1092 B. Design of primers and genotyping for modification of nuclear GVs by CRISPR/Cas9. Primers should be designed flanking the regions targeted by gRNAs (black arrows). Clonal viruses that display a smaller PCR amplification product should be preferentially selected. The absence of PCR product indicates larger deletions including one or both of the primer annealing sequences. As an example, genotyping of the knockout of *pneo_254* in *Pandoravirus neocaledonia* is shown¹⁵

1097 C. Design of primers and genotyping for modification of *A. castellanii* by CRISPR/Cas9. Primers should be designed flanking the regions targeted by gRNAs (black arrows). *A. castellanii* tends to repair double-strand breaks by small indels and thus, shifts in PCR product size are rarely observed. Sequencing of PCR products is necessary for the analysis of recombinant cells. Importantly, since *A. castellanii* is a polyploid organism, multiple recombinant alleles are expected for each PCR amplification. As an example, genotyping of the knockout of the *cellulose synthase 1* of *A. castellanii* is shown¹⁵

1104
1105 Table 1. Vectors

<table><thead><tr><td>Identifier</td><td>Genotype</td><td>Resistance cassette</td><td>Comments</td><td>Addgene ID</td></tr></thead><tbody><tr><td>PAM1</td><td>pTFIID-GFP</td><td>Geneticin</td><td>Expression in A. castellanii (weak promoter)</td><td>198740</td></tr><tr><td>PAM2</td><td>pGAPDH-GFP</td><td>Geneticin</td><td>Expression in A. castellanii (strong promoter)</td><td>198741</td></tr><tr><td>PAM3</td><td>pTPBF-GFP</td><td>Geneticin</td><td>Expression in A. castellanii (medium promoter)</td><td>198742</td></tr><tr><td>PAM10</td><td>pEF1-GFP</td><td>Geneticin</td><td>Expression in A. castellanii (very strong promoter)</td><td>198743</td></tr><tr><td>PAM22 (vc241)</td><td>pEF1-GFP</td><td>Nourseothricin</td><td>Expression in A. castellanii (very strong promoter)</td><td>193453</td></tr><tr><td>PAM24</td><td>pGAPDH-GFP</td><td>Nourseothricin</td><td>Expression in A. castellanii (strong promoter)</td><td>198744</td></tr><tr><td>vAS1</td><td>3xHA-3'UTRmg105 -pmg153-NAT</td><td>Nourseothricin</td><td>3xHA endogenous tagging in mimivirus and megavirus</td><td>198745</td></tr><tr><td>vHB8</td><td>pEFI-Cas9-NLS, pU6-tRNA-NotI-tracrRNA</td><td>Geneticin</td><td>Expression of nuclear Cas9 and gRNAs in amoebas (for cloning)</td><td>193454</td></tr><tr><td>vHB16</td><td>pEFI-Cas9-NLS, pU6-2x(tRNA-gRNA-tracrRNA)</td><td>Geneticin</td><td>Expression of nuclear Cas9 and gRNAs in amoebas (template for PCR)</td><td>193455</td></tr><tr><td>vHB46</td><td>pmg153-NAT</td><td>Nourseothricin</td><td>Vector for knockout in mimivirus/megavirus</td><td>198746</td></tr></tbody></table>
1086 indicates a homogeneous population after successful cloning (Step 91). Primers P1 and P2 allow to detect unmodified locus while primers P1 and P3 would allow amplifying the recombinant locus.
1087
1088 Analyzing the presence of recombinant viruses right after transfection (Step 66) is highly recommended and both P1+P2 and P1+P3 are expected to display positive amplification. As an example, genotyping of the insertion of a second copy of *mcp* into the genome of *Mollivirus sibericum* is shown¹⁵.
1089
1090
1091

B. Design of primers and genotyping for modification of nuclear GVs by CRISPR/Cas9. Primers should be designed flanking the regions targeted by gRNAs (black arrows). Clonal viruses that display a smaller PCR amplification product should be preferentially selected. The absence of PCR product indicates larger deletions including one or both of the primer annealing sequences. As an example, genotyping of the knockout of *pneo_254* in *Pandoravirus neocaledonia* is shown¹⁵

1092
1093
1094
1095
1096
1097
1098
1099
1100
1101
1102
1103
1104
1105

C. Design of primers and genotyping for modification of *A. castellanii* by CRISPR/Cas9. Primers should be designed flanking the regions targeted by gRNAs (black arrows). *A. castellanii* tends to repair double-strand breaks by small indels and thus, shifts in PCR product size are rarely observed. Sequencing of PCR products is necessary for the analysis of recombinant cells. Importantly, since *A. castellanii* is a polyploid organism, multiple recombinant alleles are expected for each PCR amplification. As an example, genotyping of the knockout of the *cellulose synthase 1* of *A. castellanii* is shown¹⁵

**Table 1. Vectors**

<table><thead><tr><td>Identifier</td><td>Genotype</td><td>Resistance cassette</td><td>Comments</td><td>Addgene ID</td></tr></thead><tbody><tr><td>PAM1</td><td>pTFIID-GFP</td><td>Geneticin</td><td>Expression in A. castellanii (weak promoter)</td><td>198740</td></tr><tr><td>PAM2</td><td>pGAPDH-GFP</td><td>Geneticin</td><td>Expression in A. castellanii (strong promoter)</td><td>198741</td></tr><tr><td>PAM3</td><td>pTPBF-GFP</td><td>Geneticin</td><td>Expression in A. castellanii (medium promoter)</td><td>198742</td></tr><tr><td>PAM10</td><td>pEF1-GFP</td><td>Geneticin</td><td>Expression in A. castellanii (very strong promoter)</td><td>198743</td></tr><tr><td>PAM22 (vc241)</td><td>pEF1-GFP</td><td>Nourseothricin</td><td>Expression in A. castellanii (very strong promoter)</td><td>193453</td></tr><tr><td>PAM24</td><td>pGAPDH-GFP</td><td>Nourseothricin</td><td>Expression in A. castellanii (strong promoter)</td><td>198744</td></tr><tr><td>vAS1</td><td>3xHA-3'UTRmg105 -pmg153-NAT</td><td>Nourseothricin</td><td>3xHA endogenous tagging in mimivirus and megavirus</td><td>198745</td></tr><tr><td>vHB8</td><td>pEFI-Cas9-NLS, pU6-tRNA-NotI-tracrRNA</td><td>Geneticin</td><td>Expression of nuclear Cas9 and gRNAs in amoebas (for cloning)</td><td>193454</td></tr><tr><td>vHB16</td><td>pEFI-Cas9-NLS, pU6-2x(tRNA-gRNA-tracrRNA)</td><td>Geneticin</td><td>Expression of nuclear Cas9 and gRNAs in amoebas (template for PCR)</td><td>193455</td></tr><tr><td>vHB46</td><td>pmg153-NAT</td><td>Nourseothricin</td><td>Vector for knockout in mimivirus/megavirus</td><td>198746</td></tr></tbody></table>
<table><tr><td>vHB47</td><td>pmg741-NAT</td><td>Nourseothricin</td><td>Vector for knockout in mimivirus/megavirus</td><td>198747</td></tr><tr><td>vHB63</td><td>pneo650-NAT</td><td>Nourseothricin</td><td>Vector for knockout in pandoravirus/mollivirus</td><td>198748</td></tr><tr><td>vHB178</td><td>3xHA-pneo650-NAT</td><td>Nourseothricin</td><td>3xHA endogenous tagging in pandoravirus and mollivirus</td><td>193457</td></tr><tr><td>vHB179</td><td>3xFLAG-pneo650-NAT</td><td>Nourseothricin</td><td>3xFLAG endogenous tagging in pandoravirus and mollivirus</td><td>193458</td></tr></table>

1106

<table><caption>Table 2. Primers</caption><thead><tr><td>Primer</td><td>Sequence</td><td>Comments</td></tr></thead><tbody><tr><td>1</td><td>CTTTTGCAAAAAGCTTNNNNNNNNNNNNNNNNNNN</td><td>Contains the 5' homology arm for cloning in vector vAS1 HindIII. Forward</td></tr><tr><td>2</td><td>GAACATCGTATGGGTANNNNNNNNNNNNNNNNNNNN</td><td>Contains the 5' homology arm for cloning in vector vAS1 HindIII. Reverse</td></tr><tr><td>3</td><td>AAATAGTCCTTTAGANNNNNNNNNNNNNNNNNNNN</td><td>Contains the 3' homology arm for cloning in vector vAS1 NotI. Forward</td></tr><tr><td>4</td><td>CTTATCGCTGCGGCCGCNNNNNNNNNNNNNNNNNNN</td><td>Contains the 3' homology arm for cloning in vector vAS1 NotI. Reverse</td></tr><tr><td>5</td><td>CTTTTGCAAAAAGCTTNNNNNNNNNNNNNNNNNNN</td><td>Contains the 5' homology arm for cloning in vector vHB47 HindIII. Forward</td></tr><tr><td>6</td><td>CTAATATTTTAAGCTNNNNNNNNNNNNNNNNNNN</td><td>Contains the 5' homology arm for cloning in vector vHB47 HindIII. Reverse</td></tr><tr><td>7</td><td>TCCTTTAGAGCGGCCNNNNNNNNNNNNNNNNNNN</td><td>Contains the 3' homology arm for cloning in vector vHB47 NotI. Forward</td></tr><tr><td>8</td><td>CTTATCGCTGCGGCCGCNNNNNNNNNNNNNNNNNNN</td><td>Contains the homology arm for cloning in vector vHB47 NotI. Reverse</td></tr><tr><td>9</td><td>TCCCCATACTGGCCANNNNNNNNNNNNNNNNNNNNGT TTTAGAGCTAGAAATAGC</td><td>Contains homology arms to insert into vHB8 NotI. N region depicts the sequence of the gRNA #1. Forward</td></tr><tr><td>10</td><td>TTCTAGCTCTAAAACNNNNNNNNNNNNNNNNNNNTG GCCAGTATGGGG</td><td>Contains homology arms to insert into vHB8 NotI. N region depicts the sequence of gRNA #2. Reverse</td></tr><tr><td>11</td><td>GTTGTTCCGGGTCGACTCTAG</td><td>Primer used for colony screening of vHB8 plasmids</td></tr></tbody></table>

1108 Restriction sites are indicated with underlined text.

1109

<table><caption>Table 3. Troubleshooting Table</caption><thead><tr><td>Step</td><td>Problem</td><td>Possible reason</td><td>Solution</td></tr></thead></table>
<table><tr><td>vHB47</td><td>pmg741-NAT</td><td>Nourseothricin</td><td>Vector for knockout in mimivirus/megavirus</td><td>198747</td></tr><tr><td>vHB63</td><td>pneo650-NAT</td><td>Nourseothricin</td><td>Vector for knockout in pandoravirus/mollivirus</td><td>198748</td></tr><tr><td>vHB178</td><td>3xHA-pneo650-NAT</td><td>Nourseothricin</td><td>3xHA endogenous tagging in pandoravirus and mollivirus</td><td>193457</td></tr><tr><td>vHB179</td><td>3xFLAG-pneo650-NAT</td><td>Nourseothricin</td><td>3xFLAG endogenous tagging in pandoravirus and mollivirus</td><td>193458</td></tr></table>

1106

<table><caption>Table 2. Primers</caption><thead><tr><td>Primer</td><td>Sequence</td><td>Comments</td></tr></thead><tbody><tr><td>1</td><td>CTTTTGCAAAAAGCTTNNNNNNNNNNNNNNNNNNN</td><td>Contains the 5' homology arm for cloning in vector vAS1 HindIII. Forward</td></tr><tr><td>2</td><td>GAACATCGTATGGGTANNNNNNNNNNNNNNNNNNNN</td><td>Contains the 5' homology arm for cloning in vector vAS1 HindIII. Reverse</td></tr><tr><td>3</td><td>AAATAGTCCTTTAGANNNNNNNNNNNNNNNNNNNN</td><td>Contains the 3' homology arm for cloning in vector vAS1 NotI. Forward</td></tr><tr><td>4</td><td>CTTATCGCTGCGGCCGCNNNNNNNNNNNNNNNNNNN</td><td>Contains the 3' homology arm for cloning in vector vAS1 NotI. Reverse</td></tr><tr><td>5</td><td>CTTTTGCAAAAAGCTTNNNNNNNNNNNNNNNNNNN</td><td>Contains the 5' homology arm for cloning in vector vHB47 HindIII. Forward</td></tr><tr><td>6</td><td>CTAATATTTTAAGCTNNNNNNNNNNNNNNNNNNN</td><td>Contains the 5' homology arm for cloning in vector vHB47 HindIII. Reverse</td></tr><tr><td>7</td><td>TCCTTTAGAGCGGCCNNNNNNNNNNNNNNNNNNN</td><td>Contains the 3' homology arm for cloning in vector vHB47 NotI. Forward</td></tr><tr><td>8</td><td>CTTATCGCTGCGGCCGCNNNNNNNNNNNNNNNNNNN</td><td>Contains the homology arm for cloning in vector vHB47 NotI. Reverse</td></tr><tr><td>9</td><td>TCCCCATACTGGCCANNNNNNNNNNNNNNNNNNNNGT TTTAGAGCTAGAAATAGC</td><td>Contains homology arms to insert into vHB8 NotI. N region depicts the sequence of the gRNA #1. Forward</td></tr><tr><td>10</td><td>TTCTAGCTCTAAAACNNNNNNNNNNNNNNNNNNNNNTG GCCAGTATGGGG</td><td>Contains homology arms to insert into vHB8 NotI. N region depicts the sequence of gRNA #2. Reverse</td></tr><tr><td>11</td><td>GTTGTTCCGGGTCGACTCTAG</td><td>Primer used for colony screening of vHB8 plasmids</td></tr></tbody></table>

1108 Restriction sites are indicated with underlined text.

1109

<table><caption>Table 3. Troubleshooting Table</caption><thead><tr><td>Step</td><td>Problem</td><td>Possible reason</td><td>Solution</td></tr></thead></table>
<table><tr><td>5, 41,107</td><td>Multiple bands are visible on the electrophoresis gel</td><td>Incomplete digestion of the vector</td><td>Perform digestion for a longer period of time or slice a single band of interest from an agarose gel and purify it with a DNA gel extraction kit (e.g. QIAEX II Gel Extraction Kit from Qiagen)</td></tr><tr><td>3, 38, 105</td><td>Multiple bands are visible on the electrophoresis gel</td><td>PCR conditions or primers do not allow specific amplification</td><td>Modify the PCR conditions or slice the band from the gel with the correct size and purify with a DNA gel extraction kit (e.g. QIAEX II Gel Extraction Kit from Qiagen)</td></tr><tr><td>45</td><td>More than two bands are visible on the electrophoresis gel.</td><td>Incomplete digestion of the vector</td><td>Purify the plasmid as described in Step 45 and repeat digestion from Step 44.</td></tr><tr><td>321</td><td>Low transfection efficiency</td><td>i) Cells are not in good enough condition<br/>ii) PolyFect was not homogenized before transfection and its concentration is not sufficient<br/>iii) PolyFect efficiency has decreased due to poor conservation, or expiration date</td><td>i) Make sure to use cells that are healthy, adherent, preferentially with low passage rate.<br/>ii) Make sure to homogenize the PolyFect solution by inverting or gently tapping the tube (no vortexing).<br/>iii) Try a new batch of PolyFect.</td></tr><tr><td>52</td><td>Absence of amplification using primers P1+P3 (Figure 4)</td><td>i) Problems of amplification with Terra PCR<br/>ii) Absence of recombinant viruses in the population</td><td>i) Proceed to troubleshooting for step 100<br/>ii) Repeat transfection. If the lack of recombinant viruses persists, new vectors should be prepared with different homology arms.</td></tr><tr><td>67</td><td>Similar high production of viruses in samples and negative control</td><td>i) Too long incubation time of viruses<br/>ii) Inefficient drug selection</td><td>i) Use low MOI for Step 84<br/>ii) Replace the stock of the selection drug or increase its concentration. Confirm the lack of contamination of wild-type viruses (parental) and negative control by PCR amplification with primers</td></tr></table>