# Developing Methods to Circumvent the Conundrum of Chromosomal Rearrangements Occurring in Multiplex Gene Edition

Vinciane Borsenberger, Christian Croux, Fayza Daboussi, Cécile Neuvéglise, Florence Bordes

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Vinciane Borsenberger, Christian Croux, Fayza Daboussi, Cécile Neuvéglise, Florence Bordes. Developing Methods to Circumvent the Conundrum of Chromosomal Rearrangements Occurring in Multiplex Gene Edition. ACS Synthetic Biology, 2020, 9 (9), pp.2562-2575. 10.1021/acssynbio.0c00325. hal-02960618

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# Developing Methods to Circumvent the Conundrum of Chromosomal Rearrangements Occurring in Multiplex Gene Edition

Vinciane Borsenberger,* Christian Croux, Fayza Daboussi, Cécile Neuvéglise, and Florence Bordes

**ABSTRACT:** CRISPR/Cas9 is a powerful tool to edit the genome of the yeast *Yarrowia lipolytica*. Here, we design a simple and robust method to knockout multiple gene families based on the construction of plasmids enabling the simultaneous expression of several sgRNAs. We exemplify the potency of this approach by targeting the well-characterized acyl-CoA oxidase family (POX) and the uncharacterized SPS19 family. We establish a correlation between the high lethality observed upon editing multiple loci and chromosomal translocations resulting from the simultaneous generation of several double-strand breaks (DSBs) and develop multiplex gene editing strategies. Using homologous directed recombination to reduce chromosomal translocations, we demonstrated that simultaneous editing of four genes can be achieved and constructed a strain carrying a sextuple deletion of POX genes. We explore an "excision approach" by simultaneously performing two DSBs in genes and reached 73 to 100% editing efficiency in double disruptions and 41.7% in a triple disruption. This work led to identifying SPS193 as a gene encoding a 2-4 dienoyl-CoA reductase, demonstrating the potential of this method to accelerate knowledge on gene function in expanded gene families.

<figure><img src="image_2.png" /></figure>

**KEYWORDS:** multiplex gene editing, chromosomal rearrangements, CRISPR/Cas9, *Yarrowia lipolytica*

As synthetic biology strives to engineer microbial cell factories into alternatives to oil-based chemical processes such as oleochemicals production, oleaginous yeasts attract more interest because of their appealing aptitudes at handling and stocking fatty acids. Among them, the "generally recognized as safe" (GRAS) unconventional oleaginous yeast *Yarrowia lipolytica*,¹ emerges as a remarkable model organism. This growing interest is fueled by decades of scientific studies,²,³ the yeast's amenability to a variety of growth conditions,²,⁴ the increasing knowledge of its metabolism,⁵⁻⁷ the availability of annotated genomic data on several different strains,⁸⁻¹¹ and a number of efficient genetic tools.²,¹²⁻¹⁴ It has already been modified to accumulate a high quantity of fatty acids,¹⁵ to produce alkanes, alcohols,⁵ medium-chain length fatty acids,¹⁶ ricinoleic acid,¹⁷ PHA,¹⁸,¹⁹ and omega-3-eicosapentaneic acid,²⁰ among many other products.³

Understanding the function of genes implicated in fatty acid metabolism is the key point to metabolic pathways manipulation. Yet, while a number of genes involved in the *de novo* synthesis of fatty acids and their degradation via the beta-oxidation pathways are well characterized,²¹,²² many auxiliary enzymes of these pathways remain to be identified in *Y. lipolytica*. The task is rendered difficult by the important number of paralogues⁸ present in the genome, which means that several candidates per gene family can be considered for undertaking one particular function.

Today screening through gene families has become faster and simpler with the development of CRISPR/Cas9 molecular scissors. The CRISPR/Cas9 system from *Streptococcus pyogenes* has been engineered to become an easily reprogrammable endonuclease. It is constituted by the Cas9 protein in complex with a single guide RNA (sgRNA) which carries a ~20 nt sequence at its 5' end matching a targeted sequence.²,³ The system is highly flexible as the only strong requirement is that the target has to be next to a protospacer adjacent motif (PAM) in the genome to allow the protein to bind DNA, which then generates a double strand break (DSB) in the target site. In *Y. lipolytica*, DSB repair is normally undertaken by the nonhomologous end-joining (NHEJ) machinery that directly ligates DNA broken-ends without the use of extensive homology. NHEJ can be error-prone, so small insertions or deletions (indels) can occur at the cutting site, which can lead to gene disruption and can allow the growth of edited cell lines, if the deletion is not lethal. The second double-strand break repair mechanism is the homology directed recombination
<figure><img src="image_3.png" /><figcaption>**Figure 1.** (A) General principle of golden gate assembly of CRISPR/Cas9 multiplex plasmids for *Y. lipolytica*. Amplicons carrying the expression cassettes for sgRNA are generated from single sgRNA expression plasmids using generic primers. Each primer introduces a *Sap*I site at the amplicons ends represented in red (*Sap*I recognition sequence) and yellow (overhang generated by *Sap*I). They are then assembled into a backbone plasmid using a golden gate strategy with the type IIS restriction enzyme *Sap*I and T4 ligase. (B) Each amplicon contains the promoter for polymerase III (marked in green boxes) and the sgRNA sequence (gray boxes) complete with its target sequence (colored squares). The *Sap*I-generated 5' overhangs direct the assembly by Watson-Crick base-pairing. The terminator sequences for polymerase III (blue boxes), simply composed of 6 thymidines, were introduced by the generic primers. (C) Verification of correct assembly by restriction enzyme digestion. After transformation of the golden gate mix into *E. coli*, 24 colonies were picked at random, cultivated, and submitted to plasmid extraction. The purified plasmids were digested by two enzymes straddling the assembly site, yielding the vector band at 8918 bp and the assembly zone band at 1427 bp (2-amplicon plasmids, gels on the left), or 1962 bp (3-amplicon plasmids, gels on the right). The backbone plasmid pCg69 without insert yields a faint band at 359 bp (last well on the right). Colors were inverted for clarity purposes.</figcaption></figure>

(HDR) that provides high-fidelity template-dependent repair. NHEJ and HDR enhanced by CRISPR/Cas9 allow the simultaneous targeting of several genes and have been extensively applied to a variety of organisms.

CRISPR/Cas9 has been harnessed by several groups for *Y. lipolytica* genome editing<sup>14,24-27</sup> and is rapidly becoming the method of choice due to the simplicity of its implementation. For this purpose, molecular biology toolboxes have been successfully developed. They rely on various methods of delivery for Cas9 protein, either provided on replicative plasmids or inserted in the strain genome. Similarly, the RNA component has been supplied through various methods: self-processing ribozyme on mRNA,<sup>27</sup> artificial RNA III polymerase promoters,<sup>14</sup> or T7 transcription.<sup>26</sup> Genes have successfully been disabled through NHEJ-based mechanisms, such as the introduction of indels at one DSB<sup>14,28</sup> or the excision of a sequence between two DSBs.<sup>29</sup> CRISPR/Cas9 was also successfully used to carry out HDR-based editing with small DNA oligomers<sup>25,28</sup> or matrixes with a large homology arms.<sup>14</sup> Yet, despite the development of many approaches, multiplexing in *Y. lipolytica* has met with limited success. In 2016,

Gao et al.<sup>24</sup> reported the simultaneous disruption of two genes with 36.7 ± 8.5% efficiency and of three genes with 19.3 ± 9.2%. Likewise, Holkenbrink et al.<sup>25</sup> reported editing efficiencies between 6 and 66% for double-disruption and failure at obtaining triple disruptants. Generally, successful multiplex editing by CRISPR/Cas9 is deeply host-dependent. For instance, a report recently demonstrated its ability to target up to 16 loci simultaneously in human cells, (12 EGFP copies and two diploid genes),<sup>30</sup> while other endeavors in different species displayed much more modest achievements. The highest number of loci simultaneously targeted is three in microalgae,<sup>31</sup> four in *Escherichia coli*,<sup>32</sup> and eight in *Saccharomyces cerevisiae*.<sup>33</sup> Obviously, the extent of the research effort deployed for each of these organisms differs greatly, but the contrast in these achievements might also reflect on the intrinsic feature of their respective DNA repair mechanism among other limitations. *S. cerevisiae* is famously renowned for its ability to carry out HDR, even in the absence of a DSB and, as a result, is more readily amenable to gene editing than an NHEJ-prone organism such as *Y. lipolytica*.<sup>34</sup> In this respect,
<figure><img src="image_4.png" /><figcaption>**Figure 2.** (A) Four guides were identified to target the six acyl-CoA oxidases of *Y. lipolytica* (POX1 to POX6), each sgRNA was designed to undertake two genes at the time (POX1 and POX5, POX4 and POX6, POX2 and POX3). Guides were inserted into multiplex plasmids pCg69 (URA3 selection marker and Cas9 expression cassette included) and pg10 (LEU2 selection marker, without Cas9). (B) Transformation efficiency of various combinations of CRISPR/Cas9 plasmids for the disruption of POX genes. Transformations using single plasmids and URA3 selection alone are in black, while cotransformations with LEU2 and URA3 selections are in gray. In both cases, the colony count per quantity of plasmid ratios are given relative to femtomole of plasmids rather than micrograms in order to take into account their size difference. When two plasmids were involved, cfu/fmol was calculated relative to only one of the plasmids provided in equimolar amount. The total count for sgRNA, DSB, and locus implicated is summarized for each transformation. The data reflect the results of three independent experiments.</figcaption></figure>

the latter represents a better model for organisms with a strong NHEJ pathway.

Part of this inability to produce the desired mutants in multiplex gene editing can be linked to the decrease of

transformant numbers observed upon increasing the number of CRISPR-generated DSB in the genome, a recurring observation in multiplexing approaches.³³ Such limitation on the number of targeted loci do not occur when implementing
CRISPR interference strategies that use an inactivated Cas9 to act on gene regulation,³⁵ which indicates that this lethality is likely due to the coexistence of multiple DSBs in the genome during the editing process. Another aspect of the issue, adding to the complexity of the task, is the possible translocation events taking place at the DNA ends during the DSB repairs. Although such events can sometimes be predicted and engineered to produce particular cell lines for the study of chromosomal structural variations, in S. cerevisiae for instance,³⁶ or the investigation of oncogenic chromosomal rearrangements in mouse cell lines,³⁷ they can also occur more randomly.³⁸

In this paper, we develop CRISPR/Cas9 multiplexing approaches which allow targeting up to six genes simultaneously in the well-characterized Acyl-CoA oxidase family (POX). Our study uncovered a high lethality due to a high rate of chromosomal translocations, most likely due to an overburden on the repair system caused by the multiplication of DSBs inside the genome. Nevertheless, this insight allowed us to define general guidelines with which we achieved the simultaneous editing of four genes by HDR. We demonstrated the robustness of a multiple excision approach by simultaneously targeting all three members of the SPS19 family. This allowed us to identify rapidly which gene harbors the 2,4-dienoyl reductase activity in *Y. lipolytica*.

## ■ RESULTS

**Easy Assembly of Multiple sgRNA into a Backbone Plasmid by Golden Gate.** To target multiple sites in *Y. lipolytica* genome, one must be able to simultaneously express several guides in an easy and reliable manner. One approach is to cotransform several plasmids bearing different guides with different selection markers (Supporting Information, SI Figure S1). Another approach, which can also be combined with the first one, is to put several guides on one plasmid. We opted for a systematic method, using a golden gate assembly system,³⁹ through which we could use a generic set of primer to generate the amplicons used in the assembly (Figure 1A and SI Figure S2). The amplicons themselves are amplified from plasmids bearing the expression cassettes for one sgRNA, and thus contain the RNA polymerase III promoter ScR1-tRNA<sub>gly</sub>¹⁴ followed by the sgRNA with the intended target sequence. The primers were designed to introduce a small terminator sequence (six T)<sup>40</sup> at the junction of amplicons during the assembly (Figure 1B). The system was used to assemble plasmids containing either two or three sgRNA expression cassettes. After transformation of the Golden Gate reactions, *E. coli* colonies were picked at random and analyzed by restriction enzyme digestion. In both types of assembly, using optimized conditions, 22/24 plasmids presented the desired restriction profile (Figure 1C), and all the sequenced colonies with correct patterns had the expected sequence (20 in total across a variety of 9 combinations). We established that the key element to achieve this satisfying success rate resided in the primer purity requirements to be around 95%, using a double purification by the manufacturer (reverse phase and ion exchange).

**CRISPR/Cas9 Multiplex Targeting of the POX Family.** The POX family composed of the six acyl CoA oxidases, responsible for undertaking the first step of fatty acid β-oxidation was chosen to investigate multiplexing gene disruptions by CRISPR/Cas9 in *Y. lipolytica*, since it met several criteria. First, the family can be entirely deleted without

compromising strain survival as long as another carbon source other than fatty acids is available.¹⁹ Second, with six loci to target, it allows for an extensive analysis of simultaneous multiple disruption consequences, and finally, as the genes in the family present enough identity (SI Table S2.1) to bear identical sequences next to a NGG PAM sequence, several sites can be targeted with a single guide, resulting in fewer sgRNAs to express. Consequently, four guide sequences were identified that targeted two genes simultaneously: POX1/5 targeting POX1 and POX5, POX4/6 targeting POX4 and POX6, and POX23a and POX23b targeting both POX2 and POX3 (Figure 2A and SI Figure S1A and Table S2.2 for sequences). Each one was incorporated in pCg58 plasmids,²⁸ carrying a URA3 selection marker, as well as Cas9 and sgRNA expression cassettes. From them, several multiplex plasmids were generated carrying either two guides (pCg69-POX2/3 and pg10-POX1456), or three guides (pCg69-allPOX), allowing for the simultaneous disruption of two, four, and six genes (SI Figure S1). Additionally, we designed four 90 nt single-stranded DNA oligomers to perform HDR at each of the DSB sites of POX1, POX4, POX5, and POX6 to insert several stop codons and a frameshift to the gene sequences (SI Table S1 and Figure S3).

The plasmids were inserted into *Y. lipolytica* Po 1d, using either a single plasmid with the URA3 selection marker, or two plasmids, selecting for uracil and leucine prototrophy (Figure 2B). Each transformation was performed in at least three replicates. The quantity of cells was kept constant to minimize the impact of factors such as oxygenation, which we found to be critical for reproducibility. The amount of plasmid provided was adjusted to produce a manageable number of colonies. The total amount of plasmid DNA never exceeded 0.8 µg, which is below the amount of DNA carrier that we use concomitantly, which ensured that the genetic information they carry is the main factor in impacting transformation efficiency rather than their DNA load. Transformations with single plasmids were inherently more efficient than cotransformations as the pg11 and pCg69 URA3 control plasmids on their own generated 13 to 23 times more colonies than in cotransformation with the pg10 LEU2 control (Figure 2B and SI Figure S4). When no sgRNA is expressed, the transformation efficiencies are of the same order of magnitude with (pCg69), or without Cas9 (pg10), which shows that the expression of Cas9 on its own has no detrimental effect. More importantly, colony numbers dropped drastically as the number of CRISPR/Cas9 targeted loci increased. When we compared transformation efficiencies obtained when using Cas9 alone (pCg69) with using Cas9 in combination with sgRNA, we observed a steep decrease in the number of survivors: targeting two loci at once produced a thirty-fold drop on average (from 3848 colonies/fmol in pCg69 transformation to 157 colonies/fmol in pCg58-POX4/6 and 86.7 in pCg69-POX2/3, for instance). Conversely, targeting all six POX genes at once resulted in more than a thousand-fold drop (only 2.2 colonies/fmol of plasmid in the pCg69-allPOX transformation). Furthermore, many colonies in the transformations, which implicated more than two loci, appeared four or five days after plating (66% of pCg69-POX23 + pg10-POX1456 transformation and 38% of pCg69 + pg10-POX1456), when most control colonies usually came up on the second and third day (only 11% of tardy colonies in pCg69 + pg10). Modified strains were found in both “early” and “late” colonies and their delayed appearance notwithstanding, the
<table><caption>Table 1. Strains Constructed in This Study</caption><thead><tr><th>strain</th><th>genotype</th><th>parent strain</th><th>genetic modification</th></tr></thead><tbody><tr><td>CyPo1</td><td>pox2-, pox3-, ura3-, leu2-</td><td>Pold</td><td>double-excision CRISPR</td></tr><tr><td>CyPo2</td><td>pox1-, pox5-, ura3-, leu2-</td><td>Pold</td><td>double NHEJ</td></tr><tr><td>CyPo3</td><td>pox4-, pox6-, ura3-, leu2-</td><td>Pold</td><td>double NHEJ</td></tr><tr><td>CyPo6</td><td>pox1-, pox2-, pox3-, pox4-, pox5-, pox6-, ura3-, leu2-</td><td>Cypo1</td><td>quadruple HDR</td></tr><tr><td>CyPo7</td><td>URA3, LEU2</td><td>Pold</td><td>random integration LEU2 + URA3</td></tr><tr><td>CyPo8</td><td>pox2-, pox3-, URA3, LEU2</td><td>CyPo1</td><td>random integration LEU2 + URA3</td></tr><tr><td>CyPo9</td><td>pox1-, pox5-, URA3, LEU2</td><td>CyPo2</td><td>random integration LEU2 + URA3</td></tr><tr><td>CyPo10</td><td>pox4-, pox6-, URA3, LEU2</td><td>CyPo3</td><td>random integration LEU2 + URA3</td></tr><tr><td>CyPo13</td><td>pox1-, pox2-, pox3-, pox4-, pox5-, pox6-, URA3, LEU2</td><td>CyPo6</td><td>random integration LEU2 + URA3</td></tr><tr><td>CyPo14</td><td>sps191-, ura3-, leu2-</td><td>Pold</td><td>excision CRISPR</td></tr><tr><td>CyPo15</td><td>sps192-, ura3-, leu2-</td><td>Pold</td><td>excision CRISPR</td></tr><tr><td>CyPo16</td><td>sps193-, ura3-, leu2-</td><td>Pold</td><td>excision CRISPR</td></tr><tr><td>CyPo17</td><td>sps192-, sps193-, ura3-, leu2-</td><td>Pold</td><td>double excision CRISPR</td></tr><tr><td>CyPo18</td><td>sps191-, sps193-, ura3-, leu2-</td><td>Pold</td><td>double excision CRISPR</td></tr><tr><td>CyPo19</td><td>sps191-, sps192-, ura3-, leu2-</td><td>Pold</td><td>double-excision CRISPR</td></tr><tr><td>CyPo20</td><td>sps191-, sps192-, sps193-, ura3-, leu2-</td><td>Pold</td><td>double-excision and one flip CRISPR</td></tr><tr><td>CyPo21</td><td>sps191-, URA3, LEU2</td><td>CyPo14</td><td>random integration LEU2 + URA3</td></tr><tr><td>CyPo22</td><td>sps192-, URA3, LEU2</td><td>CyPo15</td><td>random integration LEU2 + URA3</td></tr><tr><td>CyPo23</td><td>sps193-, URA3, LEU2</td><td>CyPo16</td><td>random integration LEU2 + URA3</td></tr><tr><td>CyPo24</td><td>sps192-, sps193-, URA3, LEU2</td><td>CyPo17</td><td>random integration LEU2 + URA3</td></tr><tr><td>CyPo25</td><td>sps191-, sps193-, URA3, LEU2</td><td>CyPo18</td><td>random integration LEU2 + URA3</td></tr><tr><td>CyPo26</td><td>sps191-, sps192-, URA3, LEU2</td><td>CyPo19</td><td>random integration LEU2 + URA3</td></tr><tr><td>CyPo27</td><td>sps191-, sps192-, sps193-, URA3, LEU2</td><td>CyPo20</td><td>random integration LEU2 + URA3</td></tr></tbody></table>

latter grew at the same rate as the former ones (data not shown), which hints that the generation of viable modified strains can be a protracted process in multidisruptions, even when the modified strain fitness is not impaired. Finally, we transformed the CyPo6 strain (Table 1) with the pCg69-POX23 + pg10-POX145 combination. In this strain all the plasmid genomic targets had previously been removed by CRISPR editing, and this transformation had the same efficiency as the control transformation without sgRNA, which established that the high lethality of multiplex transformation is not linked to sgRNA expression *per se*, but rather to their ability to generate DSB (SI Figure S5).

Colonies were picked and grown on rich medium to remove selective pressure for CRISPR/Cas9 plasmids and analyzed by PCR and sequencing at the targeted loci. We observed six types of repair (Figure 3A), depending on the conditions applied in the transformation. Three could be categorized as “punctual modifications” at a DSB (indels, HDR, and exogenous DNA insertions usually from the DNA carrier or HDR donor template). The three other types of modifications implicated several DSB, such as excisions or flipped sequences between two neighboring sites and chromosomal translocations involving sometimes three or four loci (SI Figure S6.1 and Table S5.1). A large proportion of the sequenced translocations (59% of the POX1, POX4, POX5, and POX6 targeting experiments) appeared to have occurred by a direct ligation of DNA ends from different cut sites, without any base deletion or insertion (SI Figure S6.3). Some colonies still had WT genotypes. The proportions of modified strains in each type of transformation are described in Figure 3B. As a general observation, most of the colonies with a modified sequence at one targeted locus were also edited at all the other targeted loci. None of the analyzed colonies transformed with one sgRNA (either POX4/6 or POX 1/5 alone) had a mixture of WT and modified loci: 91.7% were modified at both cut sites and the remaining ones were fully WT. Similarly, when we

used POX4/6 with POX1/5 sgRNAs concomitantly with and without the repair template, only 13.6% and 8.7% of the colonies, respectively, harbored a mixture of WT and modified genotypes, while 33% and 39% were modified at all four targeted sites (SI Table S3). This demonstrates a high propensity of *Y. lipolytica* for the CRISPR/Cas9 coedition of genes.

The preferred repair events for the isolated DSBs produced by POX1/5 and POX4/6 were indels generated through the NHEJ mechanism. They represented 83% of all repair events for pCg58-POX1/5 and pCg58-POX4/6 (Figure 3C and SI Tables S3.1 and S3.2). When a second cut site was introduced in the vicinity of the first cut site, such as when POX2/3a and POX2/3b were paired up, the dominating repair mechanism was the excision of the fragment between the two cut sites, that is, 8/15 colonies at both loci (SI Table S5.3). Other frequently observed outcomes were the inversion of the fragment between the DSBs or chromosomal translocations, and thus indel repair events became the exception (only 1/15 colonies). This constitutes a handy trait, as it allows for a simple screening of modified strain by colony PCR (SI Figure S7.1). When four sites were targeted simultaneously (in pg10-POX1456 colonies), the two main modifications observed were indels (62.5% of all repair events all loci considered) and translocations (32.5%). This transformation with pg10-POX1456 and pCg69 was replicated adding the 90 nt oligomers homologous to the four targets (SI Table S1). The transformation rate did not significantly improve (Figure 3B). In the 22 colonies that were analyzed, HDR was the most favored type of modification at all loci except POX6 (Figure 3C) where the oligonucleotide more often inserted itself (sometimes more than once!) than recombined at the cut site. Within the 11 non-WT colonies, only three did not harbor an HDR edited locus. One had undergone HDR at a single locus, four at two loci, two at three loci, and one was carrying all four correctly edited sequences (SI Table S3.5).
<figure><img src="image_5.png" /></figure>

<figure><img src="image_6.png" /></figure>

<figure><img src="image_7.png" /><figcaption>**Figure 3.** (A) Typical genetic events following CRISPR/Cas9 generated DSB in *Y. lipolytica*. Possible repair outcomes include (a) introduction of small indels at the DSB site, 3 bp away from the PAM, (b) exogenous DNA insertion; if DNA oligomers are provided, then (c) gene edition by HDR; if two DSBs occur in close proximity, either (d) excision of the fragment in between the cut sites (truncation) or (e) excision and reinsertion of the fragment in the other direction (flip); if multiple loci are targeted at the same time (f) reciprocal translocation between the chromosome arms carrying POX5 and POX6 genes and inversion of the DNA region between POX1 and POX4. (B) Proportions of genetic outcomes in colonies transformed with CRISPR/Cas9 plasmids targeting POX genes, depending on the sgRNA provided. The percentage indicated on top of each column represents the proportion of strains harboring translocations in non-WT colonies. (C). Repartition of genetic events locus by locus in these colonies. Data are provided according to the dominant genotype for each locus based on the analysis of 22 to 24 colonies (POX1/5 and POX4/6 transformations) or 15 colonies (POX2/3 transformations).</figcaption></figure>
<table><caption>Table 2. Transcriptomic Data of *Y. lipolytica* SPS19 Genes on Three Different Carbon Sources<sup>a</sup></caption><thead><tr><th>systematic name</th><th>gene</th><th>size (nt)</th><th>no. AO</th><th>no. GU</th><th>no. TB</th><th>RPKM OA</th><th>RPKM GU</th><th>RPKM TB</th><th>rank OA</th><th>rank GU</th><th>rank TB</th></tr></thead><tbody><tr><td>YALI0D1694g</td><td>SPS191</td><td>870</td><td>72</td><td>32</td><td>181</td><td>6</td><td>3</td><td>18</td><td>5359</td><td>5621</td><td>4381</td></tr><tr><td>YALI0E14322g</td><td>SPS192</td><td>867</td><td>716</td><td>481</td><td>1131</td><td>60</td><td>53</td><td>112</td><td>2167</td><td>2450</td><td>1465</td></tr><tr><td>YALI0C03003g</td><td>SPS193</td><td>879</td><td>19308</td><td>2640</td><td>2681</td><td>1593</td><td>285</td><td>262</td><td>101</td><td>574</td><td>697</td></tr></tbody></table>

<sup>a</sup>Notation: AO, oleic acid, GU, glucose, TB, tributyrine; RPKM, reads per kb and per million reads. Rank is based on RPKM values (6210 genes are considered). Bioproject accession number for RNaseq data: PRJEB6632.<sup>43</sup>

Finally, when all six genes were concomitantly targeted using pCg69-POX2/3 with pg10-POX1456, PCR analysis of eight colonies showed that they were either identical to the WT strain (6/8), or had undergone complex chromosomal rearrangements (2/8), which complicated locus analysis because of the numerous possible combinations to screen through (data not shown).

Various POX disruptants were isolated from colonies identified as positive for successful CRISPR/Cas9 editing according to colony PCR. The mutants were grown in rich medium for plasmid curing and isolated on solid medium. Auxotrophic cell lines grown from isolated cells were picked and verified for gene edition and marker loss. A clone deleted of POX2 and POX3 by two excisions was resubmitted to another round of gene edition to add the quadruple disruption of POX1, POX4, POX5, and POX6. Finally, positive clones that were successfully cured of CRISPR/Cas9 plasmids were transformed with LEU2 and URA3 markers by random genomic integration (Table 1). They were grown in synthetic medium supplemented with C12:0 or C18:0 (SI Figure S8). Growth of POX1/POX5, or POX4/POX6 disruptants CyPo9 and CyPo10 appeared to be unaffected compared to the control strain derived directly from Po1d (CyPo7), in line with previous data, which showed POX1, POX5, POX4, and POX6 either had no affinity for the Acyl-CoA of these fatty acids, or a redundant role in their degradation.<sup>21</sup> The POX2/POX3 disruptants (CyPo8) were able to grow on C18 but not on C12, which again is in line with previous data showing that POX3 is the sole gene responsible for the consumption of medium chain fatty acids, and finally the fully disrupted mutant (CyPo13) was unable to grow on both types of fatty acid. Its growth behavior matched that of the strain JMY1233,<sup>18</sup> deleted for the six POX genes.

**SPS19 Family Investigation.** To further exemplify the potency of the multiple sgRNA coexpression system, we sought to apply this strategy to a lesser studied β-oxidation auxiliary enzyme family. A report by Miguzaki et al.<sup>41</sup> described the isolation, purification, and characterization of a 33 kDa 2,4-dienoyl-CoA reductase from *Y. lipolytica* and concluded, through immunogenic precipitation of the protein in the crude extract, that it constituted the only one of this yeast. We performed a phylogenetic analysis and identified three potential candidates for its coding sequence (SI Figures S9.1 and S9.2 and Table S2.3). The family comprises three genes (SPS191-YALI0D1694g, SPS192-YALI0E14322g, and SPS193-YALI0C03003g). The three protein sequences closely resemble SPS19<sup>42</sup> of S. cerevisiae (54.5% to 59.5% identity), which encodes its only known functional 2,4-dienoyl CoA reductase, an auxiliary enzyme in the β-oxidation of linoleic acid. In the *Yarrowia* clade, the gene appears to have undergone two ancestral duplications and many species happen to have kept the three copies of the gene. They retained their C-term peroxysomal targeting signal (SI Figure S6.3), in line with a β-oxidation auxiliary enzyme function.

Comparison of the transcription levels of the three members of the family in *Y. lipolytica* under three different growth conditions showed that only SPS192 and SPS193 appear to be transcribed (Table 2).<sup>43</sup> Furthermore, a strong activation of SPS193 is observed when the yeast is grown on oleic acid, and since enzymes connected to the β-oxidation pathway are expected to undergo an increase in their expression under fatty acid growth conditions, we inferred that this gene was the one most likely responsible for 2,4-dienoyl-CoA reductase in *Y. lipolytica*.

To confirm the validity of this hypothesis, we adopted a similar strategy to the one used with the POX family, so as to produce all possible combinations of SPS19 family disruptants in a short time. We found one sequence of 19 bp identical in SPS191 and SPS192 (SPSm), with a single mismatch 5 nt away from the PAM in SPS193. In addition, we designed six single-target guides to specifically aim at each gene with two DSBs in order to allow for rapid screening by colony PCR of excision events (Figure 4A and SI Table S2.4). The guide sequences were incorporated in pCg58 plasmids (with Cas9 and the URA3 marker), or pg7 (with the LEU2 marker). From them, several multiplex plasmids were generated carrying either two guides (pCg69-SPS191, pCg69-SPS192, and pg10-SPS193), or three guides (pCg69-SPS191/192), (SI Figure S1). The plasmids were inserted into the Po1d strain. Interestingly, when SPSm was used on its own to target SPS191 and SPS192 the outcome was very different depending on locus (Figure 4B and SI Table S4.1). The sequencing of 15 colony PCRs, followed by TIDE analysis,<sup>44</sup> showed that each colony contained a mixture of genotypes predominantly WT at SPS191, while the SPS192 locus was on the contrary always dominated by a unique indel-modified genotype. Additionally, we did not observe any off-target modification at SPS193 locus on any of the twenty-three colonies we analyzed at this locus. When we added the other guide SPS1b, this time, the mixture of WT and indels around the SPSm cut site in SPS191 became less frequent as 50% of colonies had excisions between SPSm and SPS1b.

Interestingly, using pCg69-SPS191 and pCg69-SPS192 on their own led approximately to a 7-fold improvement in transformation efficiency (295 and 378 colonies/fmol, respectively) over using pCg58 and pg7 plasmids concomitantly to express the same sgRNAs (44.1 and 52.9 colonies/fmol, respectively), which confirmed that cotransformations were less efficient than the corresponding transformations using single plasmids (SI Figure S4). As observed with the POX family disruptions, efficiencies were found to be highly influenced by the number of targeted genes (Figure 4C). The lowest survival rate was reached for the six simultaneous DSBs generated by the cotransformation of pCg69-SPS191/192 with pg10-SPS193 (4.0 colony/fmol), which resulted in a thousand-fold drop compared to the transformation of pCg69 and pg10 control (3848 colony/fmol). Colony PCR analysis showed an excellent CRISPR/Cas9 efficiency for all the single-target
<figure><img src="image_8.png" /><figcaption>**Figure 4.** (A) Targeting of SPS19 family in *Y. lipolytica* using two guides 500 to 700 bp apart for each gene. One guide, SPSm, matches the sequence of both SPS191 and SPS192. (B) Efficiency of SPSm guide depending on locus and configuration. The most predominant genotype within CRISPR/Cas9 transformant colonies were determined by colony PCR and sequencing (15 for SPSm only, 16 for SPSm + SPS1b + SPS2b). (C) Transformation efficiency of various combinations of CRISPR/Cas9 plasmids for the disruption of SPS19 genes. Transformations using a single plasmid and URA3 selection alone are in black, while cotransformations with LEU2 and URA3 selections are in gray. In both cases, cfu is given relative to fmol of plasmids. When two plasmids were involved, cfu is calculated relative to only one of the plasmids provided in equimolar amount. The data reflect the results of three independent experiments. The control experiments without guides are the same as in Figure 2B. As all transformations were performed at the same time, only one of them (pCg69) is reported here. (D) Proportions of genetic outcomes in colonies transformed with CRISPR/Cas9 plasmids targeting SPS19 genes, depending on the sgRNA provided. The percentage of strains harboring translocations in non-WT colonies is indicated on top of each column.</figcaption></figure>
<figure><img src="image_9.png" /><figcaption>**Figure 5.** Growth of CRISPR modified strains grown in either YPD medium (light gray) after 22 h, or YNB supplemented with petroselenic acid (dark gray) after 70 h. Error bars reflect the standard deviation of three independent clones.</figcaption></figure>

guides. In the three transformations leading to monodisrup-
tants and the two leading to double disruptants SPS191/193
and SPS192/193, clones were successfully edited with 95 to
100% efficiency (Figure 4D and SI Tables S4.2 and S4.3).
Excisions were conveniently identifiable by PCR (SI Figure
S5.2) and were also often the most favored repair events (69%
of all ΔSPS193 clones), with the exception of the SPS192
locus targeted with SPS2a and SPS2b (only 42%), as a lot of
“flips” also occurred (52%). Remarkably, 41.7% of the colonies
from the cotransformation of pCg69-SPS191/192 with pg10-
SPS193 were correctly disrupted for all three genes and
without chromosomal translocation. Only 4/24 colonies
presented chromosomal translocations (SI Figures S6.2 and
S6.3 and Table S5.2). Of note, as the number of excisions rose,
the number of occurrences where the excised DNA fragment
was recaptured at another DSB site also increased: 7/24
colonies obtained for the triple mutants presented such
insertions, when only 4/48 obtained for the double mutants
had them (SI Table 6). Finally, we generated the plasmid
pCg69-SPS1b-SPS2b and cotransformed it with pg10-SPS3b.
We analyzed the transformants and found that 3/36 colonies
had undergone translocations, a slightly lower rate than the
one obtained with excisions.

Clones produced by CRISPR/Cas9 editing were cured of
their plasmids as described for POX mutants, made competent,
and cotransformed with LEU2 and URA3 markers for random
genomic integration. We selected three different isolated
prototrophic clones per type of mutant and grew them in rich
medium. Every mutant grew similarly to the control
prototrophic transformants made from the original Po1d
strain. The cultures were then used to inoculate cultures where
petroselenic acid (cis-Δ⁶ 18:1) was the sole carbon source
provided. Petroselenic acid is an isomer of oleic acid (cis-Δ⁹
18:1), differing from it by the sole position of its double bond.
WT cells can perform nine rounds of β-oxidation on this fatty
acid, entirely consuming it in the process. In contrast, without
2,4-dienoyl CoA activity in the cell, it can only perform one
round, before being stopped by the double-bond position,
which is insufficient to support growth. This is plainly apparent
in the results (Figure 5), as all ΔSPS193 mutants could not
grow while cell lines with only ΔSPS191 and ΔSPS192
mutations displayed normal growth compared to the control.
From these results we can confirm that SPS193 is the protein

with the 2,4-dienoyl CoA activity in *Y. lipolytica*. Enzymes
involved in the central β-oxidation pathway of *Y. lipolytica* have
been extensively studied, and yet none of the auxiliary enzymes
associated with this pathway had been identified to this day.
Thanks to the development of CRISPR/Cas9 methodology,
we expect the fundamental knowledge on these genes will be
rapidly expanding in the next few years paving the way for
more inventive and efficient metabolic engineering of *Y.
lipolytica*.

■ DISCUSSION

In this study, we explored the practical limits of CRISPR
multiplexing in *Y. lipolytica* and targeted two multigenic
families involved in the processing of fatty acids. For this
purpose, we established a simple and reliable method to
construct plasmids able to express up to three sgRNA per
plasmid simultaneously in *Y. lipolytica* in a robust manner. Its
main advantage is that it relies on a standard set of genetic
material comprising six generic primers and four plasmids in
total (two for single sgRNA expression and two backbones for
Golden Gate assemblies for multiple sgRNA expression) and
thus offers maximum flexibility from a minimal toolbox. The
use of two plasmids permitted the coexpression of five sgRNAs
generating up to eight DSBs in the *Y. lipolytica* genome in one
transformation. On the other hand, combining multiple guides
in a single plasmid enabled simple transformation instead of
cotransformation, which increased the number of trans-
formants by at least 5-fold.

This study gave us the opportunity to test a number of
sgRNA with multiple targets, with a great overall success rate,
since most of the colonies transformed with one of these
guides on their own appeared to be constituted of unique
CRISPR-modified genome at all the intended targets, with the
notable exception of SPSm. It is already well established that
vicinity to a suiting PAM does not guarantee that a target
sequence will be effectively cut, as several parameters, not all of
them well-understood, are likely to affect CRISPR efficiency.
These include, but are not limited to, deleterious sequence
motifs,⁴⁵ GC ratio influence on base pairing strength, sgRNA
secondary structure stability, and genomic target accessibility.
In fact, designing a “good guide” for *Y. lipolytica* is still a rather
fickle process, as algorithms to predict CRISPR/Cas9
efficiencies were built on data sets obtained on organisms
very different from yeast.⁴⁶,⁴⁷ In our experience, SPSm demonstrated very weak efficiency at SPS191 locus, while working perfectly well at SPS192, despite an identical target and PAM sequence. Chromatin accessibility has been shown to be one of the many parameters influencing CRISPR/Cas9 activity in yeast,⁴⁸ and this was confirmed by a recent study in *Y. lipolytica* in which the activity of 47 124 sgRNA was evaluated, establishing a weak correlation (Pearson's *r* = 0.223) between their efficiency and nucleosome occupancy.⁴⁹ We reasoned that the difference in efficiency between SPS191 and SPS192 probably stems from a hindered access to SPS191 locus. It is worth noting that transcriptomics data demonstrate that this gene is not transcribed in contrast to SPS192. Knowing that gene transcription requires an opening of the chromatin, we may hypothesize that it allows better accessibility of the targeted sequence by CRISPR/Cas9 scissors and thus explains the difference in efficiency between SPS191 and SPS192.

Importantly, we observed that we could “rescue” the low efficiency of SPSm at SPS191 by using a sgRNA displaying better efficiency in its vicinity. SPSm leads preferentially to a WT repair by NHEJ when used alone but results preferentially in excisions when SPS1b is added to the mix and generates a DSB 543 bp away from the SPSm cut site. This is in line with the more general observation that repairs by flip or excision are the most favored events by far upon generation of two proximal DSBs, which is in accordance with the findings of a previous study.²⁹ The most frequent type of repair at the connection is a simple ligation of both DNA ends directly at the spot where Cas9 cut them. The majority of the sequenced junctions harbored the three bases of the guide sequence on the PAM sides (SI Table S3.3 for POX2 and POX3), and the 16–17 bp of the other side (SPS genes) which shows that these DNA ends were adequately protected before being ligated. It is likely that the proximity of the two MRX complexes responsible for NHEJ-type repair at the two proximal cuts prompted these small rearrangements. Their overwhelming preponderance implies that these phenomena tend to happen faster than indel creations, which in turn suggests that the introduction of indels is a tricky and lengthy process in *Y. lipolytica*.

Similarly, we repeatedly detected “clean” ligations at the junctions of translocations (SI Figure S6.3). This indicates that these large rearrangements arose through the same mechanism as excisions and flips. Likewise, these events do not require the introduction of indels to destroy the targeted sequence unless the same sgRNA is used to target both genes. It seems that upon the multiplication of DSB, the repair system is still able to provide efficient protection of the DNA ends by ku70/ku80, the first operators in the NHEJ repair, but nevertheless let the DNA sections drift apart in the nucleus, allowing for a junction to another small excised fragment (SI Table S6), chromosome, or chromosome side where other MRX complexes have formed. It is easy to speculate that factors such as accessibility, fragment mobility, and proximity of two cut sites can facilitate these events. There is a question as to whether sequence homology between the different loci can also have a determining influence. Regarding this, we did not find a statistically significant preponderance of “chimera” assemblies that would have resulted of a homology-driven repair where the beginning of one gene is attached to the end of a homologous one (SI Tables S5). Moreover, some simultaneous disruption of similar sequences did not lead to a high

ratio of translocation (the SPS191 and SPS192 disruption experiment yielded none). Likewise, systematic loss of small stretches of DNA that would have been indicative of microhomology driven repair were not observed at any CRISPR Cas9 targeted site (single locus modification or translocation junction). On the contrary, we found that the CRISPR modified strain often harbored a small repeated sequence near the cut site that would have been removed by such a mechanism (SI Figure S6.1.5 shows an example with a 6 bp repeat at the junction site). Taken together these observations indicate that the susceptibility of specific loci to translocation is likely highly influenced by factors linked to chromosome topology, such as the proximity of DSBs and chromatin openness, and not by sequence homology. This is consistent with the hypothesis that the loci were created by a ligation resulting from the encounter of two MRX complexes drifting in the nucleus.

We established that increasing the number of targeted loci increased the proportions of strains bearing chromosomal translocations in the CRISPR-active transformants as well as the number of WT clones. Targeting two POX genes simultaneously yielded 0 to 12.5% of WT colony and 4 to 25% of non-WT colonies harboring translocations, while when the four genes POX1, POX4, POX5, and POX6 were simultaneously targeted in the absence of HDR repair mediating DNA oligomers, the proportion of WT colonies rose to 52% and colonies harboring translocations accounted for 45% of non-WT colonies. This correlation is easily rationalized when we take into account that we only observed the products of translocations that produced viable strains, as any repair event that left a chromosome part disconnected from a centromere would have been lethal, since essential genes would have been lost during cell replication. Therefore, as we increased the number of targeted loci, we increased the number of potential translocations and, similarly, the number of lethal rearrangements. As transformation efficiency dropped, transformants in which for some reason CRISPR did not work (plasmid mutation, random integration of the marker in the genome allowing for the loss of it, etc.) became a significant proportion of the survivors, which resulted in an overall decrease of CRISPR efficiency. Accordingly, generating six DSBs to target six POX genes produced a high proportion of strains with fully WT genotypes or chromosomal rearrangements that prevented their full characterization. On the other hand, generating six DSBs to target three genes (SPS experiments) produced the desired modified strain without translocation with 41.7% overall efficiency, and only 21% of the non-WT strains were harboring chromosomal translocations. These results tend to show that CRISPR/Cas9 multiplexing lethality is more tightly bound to the targeted loci number, their accessibility, and their topology, than to the number of actual DSB sites. We saw that double-targeting of the SPS three genes increased slightly the translocation rate compared to single-targeting (likely because of the increased burden on the repair system) but still produced the triple disruptants in acceptable proportions. Indeed, as repair through excision or flip is the result of a NHEJ type repair, it presents an efficient editing solution for the host, likely to be preferred over a HDR-based approach in NHEJ-prone organisms. Thus, targeting a single gene with multiple sgRNAs presents a number of advantages, first, unlike indels, which leave the sequence in place, excisions or flips more readily remove the possibility of revertants, and second, we saw that it can increase the
efficiency of a weakly active guide. Moreover, it allows for easy
detection of excisions and flips in successful transformants by
colony PCR, enabling high-throughput screening for strains
carrying multiple deletions. With this method, excision-based
PCR screening offers a simple way to robustly isolate triple
knockout, which can be generalized to other hosts.⁵⁰⁻⁵²

We saw that targeting simultaneously four different genes generated modified strains and CRISPR-deficient survivors in similar proportion, which seems to indicate a form of limit to multiplexing CRISPR in *Y. lipolytica* in the absence of positive screening for successfully edited transformants. When DNA oligomers were added in the mix, the transformation efficiency stayed in the same order of magnitude and the proportion of WT transformants was still high. Indeed, *Y. lipolytica* has a strong NHEJ pathway and a very ineffective HDR pathway in comparison to *S. cerevisiae*, which undermines the utilization of repair templates in the occurrence of multiple DSBs. However, we demonstrated that translocation events for the POX quadruple disruptants were lowered in CRISPR-active colonies, presumably because the HDR repair mechanism was managing to compete to some extent with this chromosome shuffling. In this respect, the predisposition for coediting observed in these colonies indicates that a preliminary screening could be set on one single locus (possibly using a selectable marker) to filter out WT false positives, yielding mainly fully edited strains.

■ CONCLUSION

We demonstrated the high potential of multiplexing CRISPR
in *Y. lipolytica* to scan rapidly through gene families using
combined approaches of double gene targeting enabling
excisions, multisite targeting guide, and cotransformation of
multiplex plasmids. Our study underlines the necessity for a
clear appraisal of the impact of translocation events in
CRISPR/Cas9 multiplexing. Translocation ratios are likely to
be highly influenced by chromosome topology, chromatin
accessibility, DSBs proximity, or the size of the detached
fragments. Therefore, they are not easily predictable, but they
can be managed by, for example here, identifying loci that are
the most susceptible to it and editing them separately. We
propose that targeted gene editing may be facilitated by
screening on a single gene, as coediting rates are very high in
HDR-based protocols. On the other hand, we explored an
approach that relies on favoring excision strategies for gene
deletions to circumvent weak HDR pathways in an NHEJ-
prone organism. All these are generalizable approaches to
handle the high lethality associated with multiple disruptions.
As CRISPR/Cas9 technology is deployed in the genome
editing of an expanding multiplicity of hosts, translocation
events are likely to become a recurring issue and will require
specific adaptations. The effectiveness of future editing
strategies will be highly dependent on the DNA repair
pathways available to the targeted cell, and these strategies
should be designed accordingly.

■ MATERIAL AND METHODS

Strains, Plasmids, and Media. *Escherichia coli* strain XL1 blue (Agilent) was used for cloning and plasmid production, the cells were rendered competent using the Mix & Go Competent cells kit (Zymo Research) and transformed following the kit manufacturer instructions. Unless otherwise stated, the Po1d strain was used in all yeast transformations.

The yeast cells were made competent using the Frozen-EZ
Yeast Transformation II Kit (Zymo Research). Successful
transformants were selected on agar plates containing the
appropriate selection medium (YNB + casamino acids for
selection on URA3 only or YNB for simultaneous selection on
LEU2 and URA3). The yeast was typically grown at 28-30 °C
in YPD medium (1% w/v yeast extract, 1% w/v bactopeptone,
1% w/v glucose) or minimum medium YNB containing yeast
nitrogen base (1.7 g/L, without amino acids and ammonium
sulfate), NH4Cl (5 g/L), glucose (10 g/L), and phosphate
buffer (50 mM, pH 6.8), complemented when necessary with
casamino acids (2 g/L) or uracil (1 g/L). Solid medium
contains 1.5% agar. Fatty acids were provided in 20%
emulsions with 2% pluronic detergent. Backbone plasmids
generated for this study were deposited in the Addgene
repository (Addgene ID for pCg58:153304, pCg69:153305,
pg7:153306, and pg10:153307).

**Target Design.** The whole sequences of POX1-YA-
LI0E32835g, POX2-YALI0F10857g, POX3-YALI0D24750g,
POX4-YALI0E27654g, POX5-YALI0C23859g, and POX6-
YALI0E06567g were analyzed for potential spCas9 target
sites using the online tool CRISPOR-TEFOR.⁵³ A total of four
sequences were singled out as they happened to be present in
two proteins at the same time without a single mismatch in the
19 bp (POX2/3b and POX4/6) or the 20 bp next to PAM
(POX 1/5 and POX2/3a); see SI Table S4.1 for the target
sequences. A similar investigation was conducted with SPS191-
YALI0D1694g, SPS192-YALI0E14322g, and SPS193-YA-
LI0C03003g, which returned only one 19 bp sequence
(SPSm) next to an NGG without mismatch between two of
the proteins. Moreover, in the SPS family, two individual target
sites per protein with no significant predictable off-target were
selected in order to obtain monodisruptants (see SI Table S4.2
for the target sequences).

**Optimized Method for Golden Gate Assembly of Multiplex Plasmid.** Short ~500 bp amplicons were generated from pCg58 plasmids containing the sgRNA with its promoter and target sequence using appropriately matched generic primers (see SI Table S1 for sequences). All amplicons as well as the backbone plasmids contain Sapi restriction sites at their ends. In the presence of the restriction enzyme and T4 ligase, 5' overhangs are released, and are ligated in a directed fashion by the joining of 3-base matching sequences. The order of sgRNAs in the final assembly is determined by the primers used in the PCRs. For two-guide assemblies, for instance, the first amplicon is generated using GG_For_S matched with either GG_Rev_GGT (or GG_Rev_TTC), and the last amplicon is generated using GG_For_GGT (or GG_For_TTC if GG_Rev_TTC was used in the first amplicon) and GG_Rev_E. Similarly, for three-guide assemblies the first amplicon is generated using GG_For_S and GG_Rev_GGT, the second using GG_For_GGT and GG_Rev_TTC, and the last amplicon with GG_Rev_TTC and GG_Rev_E (SI Protocols for PCR conditions). The PCRs were digested 1 h at 37 °C with DpnI (NEB), diluted with 200 µL of deionized water, and purified by spin column purification (PCR purification kit from Macherey Nagel). The fragments were assembled by a golden gate reaction with T4 ligase and Sapi (NEB) in Cutsmart buffer supplemented with ATP (1 mM) (SI protocols for conditions). The reactions were directly inserted into XL1 blue competent cells. Colonies were picked at random and cultivated for plasmid propagation and extraction. Correct clones were assessed by digestion with
KpnI and AvrII in Cutsmart buffer, which yields a band at 1427 bp for a successful two-guide assembly and a band at 1962 bp for a three-guide assembly (vectors bands are at 4730 bp and 8918 bp for pg10 and pCg69, respectively), and then ultimately confirmed by sequencing with Seq_guide and/or GG For S primers.

**CRISPR/Cas9 Transformations.** All transformation efficiency evaluation experiments were performed in three separate biological replicates using the same batch of competent cells. Typically, 12.5 µL of cells was transformed with the appropriate amounts of plasmid, gently mixed with 125 µL of EZ3 buffer (Zymo Research) and incubated 1 h at 28 °C, 140 rpm in 1.8 mL tubes. The transformations with single plasmids were plated on YNB medium supplemented with casamino acids; the transformations with two plasmids were plated on YNB plates. The cfu numbers are given relative to fmoI of plasmids. When two plasmids were involved, both were provided in equimolar quantities, and the cfu numbers are given relative to that quantity. For the assessments of genetic events, transformants colonies were picked and transferred on a YPD plate after 3 days at 28 °C. The YPD plate was then incubated at 28 °C, and samples of the outgrown colonies could be taken after a day to be lysed in alkaline solution (NaOH 20 mM, 50 µL, 10 min at 95 °C). The samples were centrifuged, and the supernatants were used directly in PCRs at the targeted loci (SI Protocols). All transformation efficiency data were evaluated on Po 1d competent cells. All the genetic events were evaluated on transformed Po1d cells, except for the tetramutations using pg10-POX1456 and pCg69, with or without oligonucleotides, which were performed on the strain previously deleted of POX2 and POX3 by CRISPR/Cas9 excisions.

**Curing CRISPR Plasmids.** Colonies with satisfying PCR results were grown in 100 mL shake flasks at 160 rpm, 28 °C in 20 mL of YPD medium, for 24 to 48 h, after which, a 1 µL sample was spread out for clone isolation on a YPD agar plate. After another 24–48 h outgrowth, single colonies were picked and transferred in replicate plates of YNB + ura, YNB + casamino acids, and YPD. Clones that were unable to grow on the two selective media and yet fine on YPD medium were analyzed by colony PCR to confirm the intended genetic modification and archived.

**Prototrophy and Differential Growth on Petroselenic Acid of Various Disruptants.** Clones of interest were made competent and 50 µL of cells were transformed simultaneously with PCR amplicons of LEU2 and URA3 with their respective native promoters and terminators (~350 ng each) (see SI Protocols for PCR conditions). Transformations were plated on YNB selective media agar plates. After 3 days at 28 °C, transformants were picked and transferred to YPD medium. Three isolated transformants from each transformation were grown in culture tubes containing 5 mL of YPD, shaken at 28 °C, 140 rpm, for 22 h. The cultures were diluted 10-fold with physiological water and used to inoculate 400 µL of YNB supplemented with 10 g/L of petroselenic acid medium in deep well plates. The deep well plates were sealed with a microporous membrane (Corning) and shaken in an incubator at 28 °C, 600 rpm (Infors) for 71 h. At 21 and 71 h, aliquots of cultures were taken and diluted with a water/IPA 1:3 mixture to clear the emulsion, and their absorbance at 600 nm were measured.

**Identification of SPS19 Homologues in *Saccharomy-cotina* and Phylogeny.** The three SPS genes of the Y.

lipolytica strain E150 were used as queries to search for putative homologues first on GRYC (http://gryc.inra.fr) and the nr database at NCBI (https://blast.ncbi.nlm.nih.gov/Blast.cgi) using blastp, and then in the genome sequence of the 12 species of the *Yarrowia* clade previously published.⁵⁴ Candidates were confirmed by reciprocal blast search. Protein sequences were then aligned with Clustal Omega using Seaview Interface version 4.6.2.⁵⁵ Alignments were cleaned with GBlocks, and the phylogenetic trees were reconstructed with PhyML with a LG substitution model and a Γ-law distribution using four categories of evolution rate.⁵⁶ Confidence was assessed in 100 bootstrap replicates.

**Transcriptome Analysis.** The transcriptomic data deposited at the European Nucleotide Archive (ENA-EMBL) under the project number PRJEB6632 (http://www.ebi.ac.uk/ena/data/view/ERP006181) were used for CLIB122 grown in glucose, oleic acid, and tributyrin as carbon sources. Read trimming, mapping, and counting were performed as previously.⁴³ Expression level is estimated either by the number of mapped reads per kb and per millions reads (RPKM) or per RPKM rank based on 6210 genes.

<figure><img src="image_10.png" /></figure>

## ASSOCIATED CONTENT

### Supporting Information

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acssynbio.0c00325.

Molecular biology protocols for plasmid constructions and PCRs, plasmid maps, additional experiments on transformation efficiencies and multiple DSBs toxicity, translocation sequencing alignments, general summary of genetic repair events by subtype, PCR screening of excisions, growth curves of various POX disruptants with fatty acids, phylogenetic tree of *Saccharomycotina* and SPS19 genes in the *Yarrowia* clade, alignments of the SPS19 gene in the *Yarrowia* clade, a list of oligonucleotide used in this study, identity matrixes for the POX family genes and the SPS19 genes, and tables of sequenced genetic repair events observed in this study (PDF)

<figure><img src="image_11.png" /></figure>

## AUTHOR INFORMATION

### Corresponding Author

Vinciane Borsenberger – Toulouse Biotechnology Institute, Université de Toulouse, INSA, INRAE, CNRS, Toulouse 31077, France; ● orcid.org/0000-0002-6982-4919; Phone: +33 (0) 5 61 55 94 55; Email: vinciane.borsenberger@insa-toulouse.fr

### Authors

Christian Croux – Toulouse Biotechnology Institute, Université de Toulouse, INSA, INRAE, CNRS, Toulouse 31077, France

Fayza Daboussi – Toulouse Biotechnology Institute, Université de Toulouse, INSA, INRAE, CNRS, Toulouse 31077, France; Toulouse White Biotechnology, Ramonville-Saint-Agne 31520, France

Cécile Neuvéglise – AgroParisTech, Micalis Institute, Université Paris-Saclay, INRAE, Paris 78350, France

Florence Bordes – Toulouse Biotechnology Institute, Université de Toulouse, INSA, INRAE, CNRS, Toulouse 31077, France; ● orcid.org/0000-0002-6078-8616

Complete contact information is available at: https://pubs.acs.org/doi/10.1021/acssynbio.0c00325
## Author Contributions

V.B., C.N., F.D., and F.B. conceived and designed the experiments. V.B., C.C., and C.N. performed the experiments. V.B. wrote the original draft. V.B., F.D., C.N., and F.B. reviewed and edited the manuscript.

## Notes

The authors declare no competing financial interest.

## ACKNOWLEDGMENTS

The authors would like thank Dr. Coraline Rigouin for helpful discussions on the genome editing of *Y. lipolytica* and the ICEO facility part of the Integrated Screening Platform of Toulouse (PICT, IBiSA) for providing access to its screening and analytical facilities. This project was funded in the context of the call for precompetitive projects of Toulouse White Biotechnology and was cofunded by Toulouse White Biotechnology (2017/059), Région Occitanie/Pyrénées-Méditerranée (No. 5066717) and the FEDER 2014-2020 program (No. 16005006).

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