DOI: 10.5061/dryad.r4xgxd2q0
title: Data from: Orthogonal transcriptional modulation and gene editing using multiple CRISPR/Cas systems

description: CRISPR/Cas-based transcriptional activation (CRISPRa) and interference
 (CRISPRi) enable transient programmable gene regulation by recruitment or
 fusion of transcriptional regulators to nuclease-deficient Cas (dCas).
 Here we expand on the emerging area of transcriptional engineering and RNA
 delivery by benchmarking combinations of RNA-delivered dCas and
 transcriptional modulators. We utilize dCas9 from Staphylococcus aureus
 and Streptococcus pyogenes for orthogonal transcriptional modulation to
 upregulate one set of genes while downregulating another. We also
 establish trimodal genetic engineering by combining orthogonal
 transcriptional regulation with gene knockout by Cas12a (Acidaminococcus;
 AsCas12a) ribonucleoprotein (RNP) delivery. To simplify trimodal
 engineering, we explore optimal parameters for implementing truncated
 sgRNAs to make use of SpCas9 for knockout and CRISPRa. We find the Cas9
 protein:sgRNA ratio to be crucial for avoiding sgRNA cross-complexation
 and for balancing knockout and activation efficiencies. We demonstrate
 high efficiencies of trimodal genetic engineering in primary human T cells
 while preserving basic T cell health and functionality. This study
 highlights the versatility and potential of complex genetic engineering
 using multiple CRISPR/Cas systems in a simple, one-step process yielding
 transient transcriptome modulation and permanent DNA changes. We believe
 such elaborate engineering can be implemented in regenerative medicine and
 therapies to facilitate more sophisticated treatments.
description: <strong>Plasmid cloning  </strong>
 All plasmids were designed based on a backbone suitable for in
 vitro mRNA transcription previously described in Jensen <em>et
 al</em>. (2021).<sup>22</sup> This plasmid contains a T7
 promotor followed by a 45bp 5’UTR ending with a Kozak sequence after which
 the GOI is inserted. The GOI is followed by a 93bp 3′ UTR of the murine
 Hba-a1 gene and a 50nt poly A tail and a unique restriction site for
 run-off IVT. The GOIs for CRISPRa experiments were as follows: dSpCas9-VPR
 from Jensen <em>et al.</em> (2021), dSpCas9 for
 dSpCas9-VPRmini and dSpCas9-p65p3-HSF1 was amplified from the dSpCas9-VPR
 plasmid.<sup>22</sup> VPRmini was amplified from Addgene
 plasmid 99698. pAAV-SCP1-dSa VPR mini.-2X snRP-1 BsaI gRNA was a gift from
 George Church (Addgene plasmid # 99698 ; http://n2t.net/addgene:99698 ;
 RRID:Addgene_99698). sp65p3-HSF1 was amplified from Addgene plasmid
 129136. pCE059-SiT-Cas12a-[Activ] was a gift from Randall Platt (Addgene
 plasmid # 128136 ; http://n2t.net/addgene:128136 ; RRID:Addgene_128136).
 dSpCas9-SunTag_10x amplified from Addgene plasmid 107310.
 dSV40-NLS-dCas9-HA-NLS-NLS-10xGCN4 was a gift from Hui Yang (Addgene
 plasmid # 107310 ; http://n2t.net/addgene:107310 ; RRID:Addgene_107310).
 scFv-GCN4-VPR, scFv-GCN4-VPRmini, and scFv-GCN4-sp65p3-HSF1 were
 synthesized by Twist Bioscience. scFv-GCN4-VP64 was amplified from Addgene
 plasmid 60904. pHRdSV40-scFv-GCN4-sfGFP-VP64-GB1-NLS was a gift from Ron
 Vale (Addgene plasmid # 60904 ; http://n2t.net/addgene:60904 ;
 RRID:Addgene_60904). CRISPRi IVT template plasmid were produced as
 follows: KOX1-dSpCas9 was from Jensen <em>et al.</em> (2021)
 (as KRAB-dSpCas9) (Addgene plasmid # 205248 ;
 http://n2t.net/addgene:205248 ; RRID:Addgene_205248). dSpCas9-KOX1-MeCP2
 was amplified from Addgene plasmid 110821<em> </em>(as
 dCas9-KRAB-MeCP2). dCas9-KRAB-MeCP2 was a gift from Alejandro Chavez
 & George Church (Addgene plasmid # 110821 ;
 http://n2t.net/addgene:110821 ; RRID:Addgene_110821). ZIM3-dSpCas9 was
 amplified from Addgene plasmid 154472. pLX303-ZIM3-KRAB-dCas9 was a gift
 from Mikko Taipale (Addgene plasmid # 154472 ;
 http://n2t.net/addgene:154472 ; RRID:Addgene_154472). dSaCas9-KOX1 was
 amplified from Addgene plasmid 106219. AAV CMV-dSaCas9-KRAB-bGHpA was a
 gift from Charles Gersbach (Addgene plasmid # 106219 ;
 http://n2t.net/addgene:106219 ; RRID:Addgene_106219). dSaCas9 for
 dSaCas9-KOX1-MeCP2 and dSaCas9-ZIM3 was also amplified from Addgene
 plasmid 106219. dSpCas9 without effector fusion was amplified from the
 dSpCas9-VPR plasmid and dSaCas9 without effector fusion was amplified from
 Addgene plasmid 106219. All plasmids and their respective sequences are
 listed in Table S1. Primers contained at least 30 bp compatible overhangs
 with the backbone for Gibson cloning. All primers were ordered from Sigma
 Aldrich (Merck) and are listed in Table S2. Amplification of GOIs were
 performed using the Phusion Green Hot Start II High-Fidelity PCR Master
 Mix (Thermo Fisher Scientific) according to the manufacturer’s
 instructions. Non-template controls were included for all reactions. GOIs
 were purified from a 1% agarose gel using the GeneJet Gel Extraction Kit
 (Thermo Fisher Scientific) following the supplier’s instructions. The
 assembly of PCR fragments and linearized IVT vector were conducted using
 the NEBuilder HiFi DNA Assembly (New England BioLabs) using recommended
 ratios of insert to vector as well as incubation times provided by the
 supplier. The assembled products were then transformed into 10-beta
 Competent <em>E. coli</em> (New England BioLabs), and finally,
 purified using NucleoBond Extra Midi EF (MACHERY-NAGEL). All plasmid
 constructs were confirmed by Sanger sequencing (Eurofins Genomics and
 Macrogen) or whole-plasmid sequencing using the Oxford Nanophore
 technology. <strong>In vitro
 transcription</strong> IVT mRNA was generated as
 previously described by Jensen <em>et al.</em> (2021), with
 the following modifications: For the IVT reaction, uridine was substituted
 with N1-methyl-pseudouridine (Jena Bioscience) and with a CleanCap:GTP
 ratio of 0.8:1. The mRNA was purified and concentrated using the RNA Clean
 & Concentrator kit (Zymo Research) according to the manufacturer’s
 manual. The mRNA quantity was verified using the DeNovix DS-11 Series
 spectrophotometer, and the RNA quality was verified on an Agilent 2100
 Bioanalyzer (Agilent Technologies) using the RNA 6000 Nano Kit (Agilent
 Technologies) and accompanying protocol.
 <strong>sgRNAs</strong> The sgRNAs
 for the <em>s. pyogenes</em> Cas9 system were ordered from
 Synthego as chemically modified sgRNAs containing 2’-O-methyl on the three
 terminal nucleotides at both ends and 3’phosporothioate between the first
 three and last two bases.<sup>21</sup> sgRNAs for the
 <em>s. aureus </em>Cas9 system were synthesized by either
 Synthego or SBS Genetech Co. with 2’-O-methyl and 3’ phosphorothioate on
 the three terminal nucleotides at both ends. Spacer sequences are listed
 in Table S3. The sgRNA design for CRISPRa and CRISPRi experiments were
 based on guidelines previously devised by Gilbert <em>et
 al.</em> (2014) and Sanson <em>et al.</em> (2018), and
 with at least 30 bp between adjacent protospacer sequences wherever
 possible due to PAM abundance.<sup>16,49</sup>
 <em>TRAC</em> sgRNAs for SpCas9 is from Wieking <em>et
 al.</em> (2020) and <em>TRAC</em> crRNA for AsCas12a is
 from Kath <em>et al.</em>
 (2022).<sup>29,30</sup> <strong>Cell
 culture</strong> Jurkat cells were cultured in
 RPMI-1640 medium supplemented with 5% heat-inactivated FCS, 2mM
 L-glutamine, 100 U/mL penicillin, and 100 ug/mL streptomycin.
 Peripheral-blood mononuclear cells (PBMCs) were isolated from
 de-identified buffy coats obtained from healthy adult donors from the
 Aarhus University Hospital Blood Bank by Ficoll-Plaque plus density
 gradient and from these, primary human T cells were purified by negative
 selection with the EasySep human T cell isolation kit (StemCell
 Technologies). The primary human T cells were cultured in X-VIVO 15 media
 (Lonza) supplemented with 5% human albumin serum (merck), and 10 ng/mL IL7
 and IL2 (Peprotech). The cells were activated for 3 days with Dynabeads
 human T-activator CD3/CD28 (Thermo Fisher Scientific) at a 1:1 cell to
 bead ratio. All cells were counted on a Bio-Rad TC20 automated cell
 counter using tryphan blue to exclude dead cells. 
 <strong>Electroporation</strong>
 All cells were electroporated using the 4D-nucleofector device
 from Lonza (X unit) in 20-µL-format Nucleocuvette strips. Cells were
 electroporated in the following electroporation buffers and programs:
 Jurkat cells: Opti-MEM (Thermo Fisher Scientific), CM138-P3; primary human
 T cells: solution 1 M, EO115-P3.<sup>50</sup> For CRISPRa and
 CRISPRi singleplex RNA-based delivery experiments, unless otherwise
 specified, cells were electroporated with 0.095 µg/µL mRNA + 0.05 µg/µL of
 each of the sgRNAs. For orthogonal CRISPRa and CRISPRi experiments, cells
 were electroporated with 0.095 µg/µL dSpCas9-VPR mRNA along with 0.0125
 µg/µL of each sgRNA for <em>CD123</em> (#1-4) and
 <em>NGFR</em> (#1-4), and 0.095 µg/µL dSaCas9-KOX1 mRNA along
 with 0.0167 µg/µL of each sgRNA for <em>CD5</em> (#1-3) and
 <em>CD3E</em> (#1-3). In primary human T cells for CRISPRa and
 CRISPRi experiments for trimodal engineering, at the optimized condition
 cells were electroporated with 0.095 µg/µL dSpCas9-VPR mRNA+0.0125 µg/µL
 of each sgRNA for <em>CD123 </em>(#1-4) and 0.095 µg/µL
 dSaCas9-KOX1+0.05 µg/µL of each sgRNA for <em>CD5</em>
 (#1-3). For gene editing of <em>TRAC
 </em>with nuclease-active Cas9 protein (IDT. Alt-R S.p. Cas9
 Nuclease V3), Cas9 and sgRNAs were incubated for 15 min at room
 temperature, and later stored at 4°C prior to electroporation. RNP
 complexes were mixed with cells resuspended in 1 M electroporation
 buffer.<sup>51</sup> Cas9 protein and sgRNAs were at a final
 concentration of respectively, 0.320 µg/µL and 0.160 µg/µL. Four days
 post-electroporation, genomic DNA was extracted for analysis of insertions
 or deletions (indels) using QuickExtract DNA extraction solution (Nordic
 Biolabs). The <em>TRAC </em>and<em> CD123</em>
 genomic region covering the sgRNA target site(s) were PCR-amplified using
 the following primer pairs: (<em>TRAC</em>) Fw, 5’-
 ATCACGAGCAGCTGGTTTCT -3’; Rv, 5’-CCCGTGTCATTCTCTGGACT -3’,
 (<em>CD123</em>) Fw, 5’- ACTGTAACCTCCTCCGCCTC -3’; Rv,
 5’-GATATCTTCCCGTGTGCGCT -3’. PCR products were either run on a 1% agarose
 gel and purified using the GeneJet Gel Extraction Kit (Thermo Fisher
 Scientific) or using an enzyme-based PCR clean-up method. 10 µL of
 post-PCR reaction mixture was mixed with 7 µL nuclease-free water, 0.5 µL
 Exonuclease I (Thermo Fisher Scientific, #EN0581) for a final
 concentration of 0.556U/µL and 0.5µL of FastAP Thermosensitive Alkaline
 Phosphatase (Thermo Fisher Scientific, #EF0651) for a final concentration
 of 0,0278U/µL. The reaction mixture was placed in a C1000 Thermal Cycler
 (Bio-Rad) running the following program: 37<sup>o</sup>C, 15
 min., 80<sup>o</sup>C, 15 min. The purified PCR products were
 Sanger-sequenced (Eurofins Genomics) and the sequencing files were
 analyzed by ICE CRISPR analysis tool (Synthego) to validate indel
 formations using a mock-electroporated sample as a wildtype
 control.
 <strong> </strong><strong>Flow
 cytometry</strong> Between 0.8 x
 10<sup>5</sup> and 3 x 10<sup>5</sup> cells were
 collected and spun down at 300xg for 5 min. Cells were washed in PBS, and
 then stained with a viability dye for 30 min to exclude dead cells from
 the analysis. The cells were then washed in PBS, and resuspended in
 staining buffer (PBS, 2% FCS, 2mM EDTA). Cells were stained with
 fluorochrome-conjugated antibodies (Table S4) in concentrations
 recommended by the supplier, and then incubated for 30 min. The cells were
 analyzed by flow cytometry on a NovoCyte Quanteon 4025 flow cytometer
 equipped with four lasers (405 nm, 488 nm, 561 nm, and 637 nm) and 25
 fluorescence detectors (Agilent, Santa Clara, Ca) or a CytoFLEX S flow
 cytometer equipped with four lasers (405 nm, 488 nm, 561 nm, and 638 nm)
 and 13 fluorescence detectors (Beckman Coulter, Brea, Ca). All flow
 cytometry experiments were individually compensated by individually
 stained compensation beads and analyzed in FlowJo (version 10.8.1). A
 representative gating scheme is shown in Figure S11. Surface
 marker-positive cells were gated based on combinations of unstained,
 viability stain only, or target knockout. For counting human T cells to
 assess proliferation, the cells were counted on the flow cytometer using
 CountBright absolute counting beads (Invitrogen).
 <strong> </strong><strong>RNA-seq
 analysis</strong> Jurkat cells were
 electroporated in biological triplicates for each condition as described
 previously. The RNA amounts used for each condition in the electroporation
 mix were: (1) 0.095 µg/µL dSpCas9 + 0.095 µg/µL dSaCas9; (2) 0.095 µg/µL
 dSpCas9-VPR + 0.095 µg/µL dSaCas9-KOX1; and (3) 0.095 µg/µL dSpCas9-VPR +
 0.095 µg/µL dSaCas9-KOX1 + 0.0125 µg/µL of each of the four
 sgRNA<em> </em>against <em>CD123</em> and 0.0167
 of each of the three sgRNA targeting <em>CD5</em>. Three days
 following incubation, the cells were lysed, and total RNA was purified
 using the ReliaPrep RNA cell miniprep system following the supplied
 protocol. The quality of the RNA was verified on a Bioanalyzer and
 thereafter sent to Novogene for the RNA sequencing workflow. Here,
 messenger RNA was purified from total RNA using poly-T oligo-attached
 magnetic beads. After fragmentation, the first strand cDNA was synthesized
 using random hexamer primers followed by second strand cDNA synthesis. The
 library was ready after end repair, A-tailing, adapter ligation, size
 selection, amplification, and purification. The library was checked with
 Qubit and real-time PCR for quantification and bioanalyzer for size
 distribution detection. Quantified libraries were pooled and sequenced on
 an Illumina NovaSeq X Plus. A minimum of 63M raw reads were obtained from
 each sample. Raw sequences were screened using fastqc (v0.12.1) to ensure
 high sequence quality. Reads were aligned to Genome Reference Consortium
 Human Build 38 patch release 14 (GRCh38.p14) using HISAT2 (v2.2.1).
 Quantification of transcripts was performed using Subread (v2.0.6). After
 quantification, the data was filtered to remove low-count features. We
 retained only those genes where at least one sample had a read count
 greater than 5. Differentially expressed genes were determined using
 DESeq2(v1.40.2) where statistically significance is classified by a
 p-value ≤ 0.05 and a log2FC &gt; 2 or &lt; -2 (Table S5). All the
 computing for this project was performed on the GenomeDK cluster. For
 off-target analysis, each of the four CD123 SpCas9 sgRNAs used for CRISPRa
 and each of the three CD5 SaCas9 sgRNAs used for CRISPRi were analyzed for
 potential off-targets in the genome using COSMID and CRISPOR (Table S5).
 The protospacer sequences were inputted and the default parameters were
 used. For COSMID, this allows up to 2 mismatches between the sgRNA and the
 protospacer or 1bp bulge in either the protospacer or in the sgRNA. For
 SaCas9 PAM, the more relaxed 5'-NNGRRN-3' PAM was used. For
 CRISPOR, up to 4 mismatches were allowed by default. Here, the canonical
 SaCas9 PAM 5'-NNGRRT-3' was used. The identified potential
 off-target sites were then checked to see if they were situated within a
 window of 10,000 bp of the TSS of any of the DEGs. TSSs were extracted
 using BioMart in Ensembl except for non-coding RNAs where TSSs were
 manually extracted using NCBI. <strong>Cell
 stimulation and ELISA</strong> Four days after
 electroporation with the trimodal CRISPR system, 500,000 primary human T
 cells from each donor were seeded into 96-well plates. Cells were
 stimulated with 25 ng/mL of phorbol 12-myristate 13-acetate (PMA) and 1
 µg/mL ionomycin for a duration of 5 hours to induce cytokine production.
 Post-stimulation, cell culture supernatants were collected for cytokine
 quantification. TNF-α and IFN-γ concentrations were measured using the
 ELISA Max Deluxe Human TNF-α and IFN-γ kits, respectively (BioLegend). The
 experiment was conducted in technical duplicates or triplicates for each
 of the three T cell donors. Cytokine concentrations were interpolated from
 a standard curve, and supernatants were serially diluted to fit the
 assay's dynamic range.
 <strong>Statistics</strong> All
 statistical analyses were performed in GraphPad Prism (version 10.2.0).
 Statistical parameters are reported in the figure legends. Expression
 analyses were performed in biological triplicates unless otherwise
 specified. The significance of differences in multiple groups was
 determined by one-way or two-way analysis of variance (ANOVA) with Tukey’s
 multiple comparison’s test. <em>P</em> values<em>
 </em>&lt; 0.05 was considered statistically significant. ns –
 nonsignificant (P &gt; 0.05), * - P ≤ 0.05, ** - P
 ≤ 0.01, *** - P ≤ 0.001, **** - P ≤ 0.0001.
description: # Data files from the "Orthogonal transcriptional modulation and gene
 editing using multiple CRISPR/Cas systems" article
 [https://doi.org/10.5061/dryad.r4xgxd2q0](https://doi.org/10.5061/dryad.r4xgxd2q0) ## Description of the data and file structure **RNA-seq data:** *Naming of RNA-seq samples:* CD123_CD5: Jurkat cells treated with simultaneous CRISPRa (dSpCas9-VPR mRNA + CD123 sgRNAs) and CRISPRi (dSaCas9-KRAB mRNA CD5 sgRNAs) VPR_KRAB: Jurkat cells treated with dSpCas9-VPR and dSaCas9-KRAB mRNA Sp_Sa: Jurkat cells treated with dSpCas9 and dSaCas9 mRNA The samples are performed in triplicates (hence the numbers 1, 2 and 3 after sample name) **RNA-seq analysis** Jurkat cells were electroporated in biological triplicates for each condition as described previously. The RNA amounts used for each condition in the electroporation mix were: (1) 0.095 µg/µL dSpCas9 + 0.095 µg/µL dSaCas9; (2) 0.095 µg/µL dSpCas9-VPR + 0.095 µg/µL dSaCas9-KOX1; and (3) 0.095 µg/µL dSpCas9-VPR + 0.095 µg/µL dSaCas9-KOX1 + 0.0125 µg/µL of each of the four sgRNA* *against *CD123* and 0.0167 of each of the three sgRNA targeting *CD5*. Three days following incubation, the cells were lysed, and total RNA was purified using the ReliaPrep RNA cell miniprep system following the supplied protocol. The quality of the RNA was verified on a Bioanalyzer and thereafter sent to Novogene for the RNA sequencing workflow. Here, messenger RNA was purified from total RNA using poly-T oligo-attached magnetic beads. After fragmentation, the first strand cDNA was synthesized using random hexamer primers followed by second strand cDNA synthesis. The library was ready after end repair, A-tailing, adapter ligation, size selection, amplification, and purification. The library was checked with Qubit and real-time PCR for quantification and bioanalyzer for size distribution detection. Quantified libraries were pooled and sequenced on an Illumina NovaSeq X Plus. A minimum of 63M raw reads were obtained from each sample. Raw sequences were screened using fastqc (v0.12.1) to ensure high sequence quality. Reads were aligned to Genome Reference Consortium Human Build 38 patch release 14 (GRCh38.p14) using HISAT2 (v2.2.1). Quantification of transcripts was performed using Subread (v2.0.6). After quantification, the data was filtered to remove low-count features. We retained only those genes where at least one sample had a read count greater than 5. Differentially expressed genes were determined using DESeq2(v1.40.2) where statistically significance is classified by a p-value ≤ 0.05 and a log2FC &gt; 2 or &lt; -2 (Table S5). All the computing for this project was performed on the GenomeDK cluster. For off-target analysis, each of the four CD123 SpCas9 sgRNAs used for CRISPRa and each of the three CD5 SaCas9 sgRNAs used for CRISPRi were analyzed for potential off-targets in the genome using COSMID and CRISPOR (Table S5). The protospacer sequences were inputted and the default parameters were used. For COSMID, this allows up to 2 mismatches between the sgRNA and the protospacer or 1bp bulge in either the protospacer or in the sgRNA. For SaCas9 PAM, the more relaxed 5'-NNGRRN-3' PAM was used. For CRISPOR, up to 4 mismatches were allowed by default. Here, the canonical SaCas9 PAM 5'-NNGRRT-3' was used. The identified potential off-target sites were then checked to see if they were situated within a window of 10,000 bp of the TSS of any of the DEGs. TSSs were extracted using BioMart in Ensembl except for non-coding RNAs where TSSs were manually extracted using NCBI. **data sets from article file:** The Excel spreadsheet includes all data points corresponding to the graphs presented in the article, including those from the supplemental figures. Each worksheet is designated for a specific figure, with sheet names reflecting their respective figure numbers. Above each data table, annotations specify whether the data represent surface expression values or expression levels (measured as mean fluorescence intensity, MFI). Additionally, the Cas variant associated with each dataset is clearly indicated. The tables are formatted for straightforward copying, enabling seamless integration into graphing software for further analysis and visualization. **ELISA** Cytokine concentrations were interpolated from a standard curve, and supernatants were serially diluted to fit the assay's dynamic range. **Statistics** All statistical analyses were performed in GraphPad Prism (version 10.2.0). Statistical parameters are reported in the figure legends. Expression analyses were performed in biological triplicates unless otherwise specified. The significance of differences in multiple groups was determined by one-way or two-way analysis of variance (ANOVA) with Tukey’s multiple comparison’s test. *P* values* *&lt; 0.05 was considered statistically significant. ns – nonsignificant (P &gt; 0.05), * - P ≤ 0.05, ** - P ≤ 0.01, *** - P ≤ 0.001, **** - P ≤ 0.0001. **Graphs** All graphs are made in prism using grouped tables. Normality tests was performed on all data prior to statistical analysis.  **Flow cytometry plots** All data analysis of flow cytometry data was performed in FlowJo V10.10.0.