Efficient CRISPR/Cas9 Gene Editing in Uncultured Naive Mouse T Cells for in Vivo Studies

Efficient CRISPR/Cas9 Gene Editing in Uncultured Naive Mouse T Cells for In Vivo Studies

This information is current as Simone Nüssing, Imran G. House, Conor J. Kearney, of October 1, 2021. Amanda X. Y. Chen, Stephin J. Vervoort, Paul A. Beavis, Jane Oliaro, Ricky W. Johnstone, Joseph A. Trapani and Ian A. Parish J Immunol published online 9 March 2020 http://www.jimmunol.org/content/early/2020/03/06/jimmun ol.1901396 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2020 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published March 9, 2020, doi:10.4049/jimmunol.1901396 The Journal of Immunology

Efﬁcient CRISPR/Cas9 Gene Editing in Uncultured Naive Mouse T Cells for In Vivo Studies

Simone Nu¨ssing,* Imran G. House,*,† Conor J. Kearney,*,† Amanda X. Y. Chen,* Stephin J. Vervoort,*,† Paul A. Beavis,*,† Jane Oliaro,*,† Ricky W. Johnstone,*,† Joseph A. Trapani,*,† and Ian A. Parish*,†

CRISPR/Cas9 technologies have revolutionized our understanding of gene function in complex biological settings, including T cell immunology. Current CRISPR-mediated gene editing strategies in T cells require in vitro stimulation or culture that can both preclude the study of unmanipulated naive T cells and alter subsequent differentiation. In this study, we demonstrate highly efﬁcient gene editing within uncultured primary naive murine CD8+ T cells by electroporation of recombinant Cas9/sgRNA ribonucleoprotein immediately prior to in vivo adoptive transfer. Using this approach, we generated single and double gene

knockout cells within multiple mouse infection models. Strikingly, gene deletion occurred even when the transferred cells were left Downloaded from in a naive state, suggesting that gene deletion occurs independent of T cell activation. Finally, we demonstrate that targeted mutations can be introduced into naive CD8+ T cells using CRISPR-based homology-directed repair. This protocol thus expands CRISPR-based gene editing approaches beyond models of robust T cell activation to encompass both naive T cell homeostasis and models of weak activation, such as tolerance and tumor models. The Journal of Immunology, 2020, 204: 000–000.

elineating the molecular mechanisms that underpin it can thus be difficult to definitively prove that loss of a KO cell http://www.jimmunol.org/ cellular pathways or processes is a major goal of most population is due to the function of the gene being examined D biological studies and is typically achieved by probing rather than rejection of the transferred cell population. the function of gene-deficient cells. However, generating such cells Clustered, regularly interspaced short palindromic repeats is often a bottleneck in research progress. Traditional approaches (CRISPR)/CRISPR-associated protein 9 (Cas9)-based gene use whole genome (complete) knockout (KO) mice or conditional deletion techniques have revolutionized our ability to rapidly gen- KO mouse models but are often slow and laborious. In particular, erate gene-deficient (2) or -edited (3) cells. In CRISPR/Cas9-based generating compound KO mice can take many months to years, systems, guide RNAs, or more recently single guide RNAs depending on the number of KO alleles being combined. This is (sgRNAs), target the Cas9 nuclease to a genomic region of by guest on October 1, 2021 even more problematic when the KO strain is on the incorrect interest, leading to dsDNA breaks that can be resolved by two genetic background, as back-crossing mice to a different back- possible DNA repair mechanisms: nonhomologous end joining ground takes many years. Furthermore, where there is no existing (NHEJ) and homology-directed repair (HDR) (4). NHEJ fre- KO mouse line, the creation of a new KO line is both costly and quently results in insertion or deletion of several nucleotides, time consuming. Finally, cells from KO mice are often not suitable leading to disruption of gene function, which is beneficial in for adoptive transfer studies because of cell rejection due to minor KO studies. In contrast, HDR can occur when a DNA template histocompatibility mismatches (1). In studies in which gene loss with homology to the site of double-stranded breaks is codelivered is proposed to compromise survival of adoptively transferred cells, with sgRNA/Cas9. The provision of an appropriate template can enable precise DNA editing within genomic loci, including insertion of large DNA fragments, or editing of single nucleotides *Peter MacCallum Cancer Centre, Melbourne, Victoria 3000, Australia; and (3, 5). Viral transduction or plasmid electroporation of Cas9- †Sir Peter MacCallum Department of Oncology, The University of Melbourne, expressing cells with vectors encoding guide RNAs is often Parkville, Victoria 3052, Australia used to induce gene deletion or modification (6–9). Although ORCID: 0000-0003-3528-478X (I.A.P.). this strategy has worked well for T cells (6, 9), it has drawbacks Received for publication November 22, 2019. Accepted for publication February 10, that limit its utility. First, transduction with viral vectors typi- 2020. cally requires in vitro T cell activation and culture, which both This work was supported by Human Frontier Science Program Young Investigators alter in vivo differentiation (particularly in settings involving Grant RGY0065/2018 and by program, fellowship, and project grant support from the National Health and Medical Research Council of Australia. weak activation, such as tumor and tolerance models) and pre- Address correspondence and reprint requests to Dr. Ian A. Parish, Peter MacCallum clude the study of naive T cell homeostasis. Second, persistent Cancer Centre, 305 Grattan Street, Melbourne, VIC 3000, Australia. E-mail address: Cas9 expression both increases off-target effects (10), and is not [email protected] suitable for mouse in vivo adoptive transfer approaches, as the The online version of this article contains supplemental material. immunogenic Cas9 protein causes cell rejection (11). Abbreviations used in this article: Cas9, CRISPR-associated protein 9; CRISPR, Electroporation of cells with recombinant Cas9/sgRNA ri- clustered, regularly interspaced short palindromic repeats; F, forward; HDR, homology-directed repair; KO, knockout; LCMV-Cl13, lymphocytic choriomeningi- bonucleoprotein (RNP) can overcome these problems (12, 13), tis virus Clone 13 strain; LM-OVA, OVA-transgenic Listeria monocytogenes; NHEJ, and chemically modified guides can further increase editing nonhomologous end joining; p.i., postinfection; R, reverse; sgRNA, single guide efficacy in this setting (13). This approach works well in vitro in RNA; SNP, single nucleotide polymorphism. naive mouse and human T cells (14), suggesting that editing Copyright Ó 2020 by The American Association of Immunologists, Inc. 0022-1767/20/$37.50 could be achieved without in vitro T cell activation using this

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1901396 2 GENE EDITING IN ADOPTIVELY TRANSFERRED NAIVE T CELLS strategy. Nevertheless, resting murine T cells have to be either forward (F) and reverse (R) primers were used: 227-bp product, F: 59- activated via aCD3/aCD28 shortly after electroporation or CCAACTTCACCACCAAGGAT-39,R:59-CAGGAGACCAGCAGTTA- precultured in IL-7 for efficient gene deletion in vitro (14). GGG-39; 500-bp product, F: 59-TCTGCAAGTGGTGGAACATC-39,R: 59-TTCTGGGAACCAGTCTCACC-39; 984-bp product, F: 59-GAAGC- Coupled with previous studies, this has lead to the assumption CAGCCTGGTCTACAG-39,R:59-TCAGCTGTTCAAGGCAGCTA-39 that in vitro T cell culture is an absolute requirement for high (primers were custom synthesized by Sigma-Aldrich). efficiency CRISPR/Cas9 gene editing. It is not known whether CRISPR/Cas9 gene editing, adoptive transfer, and this approach works in vivo within mouse T cell adoptive mouse infection transfer systems, where activation signals are not as rapidly delivered. Moreover, although the IL-7 culture step required for See part B and C of the detailed protocol in the Supplemental Material for this approach did not overtly activate T cells (14), it may be more information. Note that the 10-min cell rest period at 37˚C and 5% CO2 after electroporation is substantially shorter than the 2 h rest in Seki incompatible with in vivo models in which exogenous IL-7 can et al. (14). For KO experiments, sgRNAs targeting the murine Cd3e alter differentiation (e.g., exhaustion) (15). Finally, gene editing (59-AGGGCACGUCAACUCUACAC-39), Thy1 (encoding CD90) (59- via HDR has not been previously described in naive T cells, CCUUGGUGUUAUUCUCAUGG-39), or Pdcd1 (59-GACACACGGCG- with existing literature suggesting that this may not be feasible. CAAUGACAG-39) genes and the mouse genome nontargeting Ctrl sgRNA It is well established that HDR preferentially occurs in the S and (59-GCACUACCAGAGCUAACUCA-39) were obtained from Synthego (CRISPRevolution sgRNA EZ Kit; Synthego). In HDR experiments, G2 phases of the cell cycle (16, 17), and it is thus unclear Ctrl sgRNA Cas9 RNPs with no template or Cd90.2 sgRNA (59-CT- whether quiescent, noncycling T cells are capable of the ap- TTTGTGAGCTTCAAGTCT-39; sourced from Synthego) Cas9 RNPs propriate DNA repair processes. with 3 3 1012 copies of 227-, 500-, or 984-bp-long homologous template DNA were electroporated into P14 T cells. T cells were adoptively In this study, we challenge the idea that T cell culture is 3 transferred into B6 recipient mice, with 5 3 10 P14 T cells transferred Downloaded from required for efficient CRISPR/Cas9 gene editing. We demon- per mouse for LCMV-Cl13 infection, 5 3 104 OT-I cells per mouse for strate highly efficient CRISPR/Cas9-mediated gene editing LM-OVA infection, and 1–2 3 106 cells per mouse for naive cell within in vivo models by sgRNA/Cas9 RNP electroporation into “parking” experiments. For infections, mice were i.v. injected with ei- 6 4 primary, uncultured, and unstimulated naive TCR-transgenic ther 2.5 3 10 PFU LCMV-Cl13 or 5 3 10 CFU LM-OVA. + CD8 T cells prior to adoptive transfer. We validate this Flow cytometric analysis model using multiple genes and TCR transgenic T cell speci- ficities in two infection models: chronic lymphocytic chorio- For cell surface staining, cells were stained on ice in PBS containing 2.5% http://www.jimmunol.org/ FCS and 0.1% sodium azide using the following anti-mouse Abs and meningitis virus Clone 13 (LCMV-Cl13) and OVA-transgenic reagents: Fixable Viability Stain 620, CD8-BUV395 (Clone 53-6.7), Listeria monocytogenes (LM-OVA). In gene deletion studies, CD45.1-FITC or –Pacific Blue (Clone A20), CD3-allophycocyanin transferred cells underwent clonal expansion postinfection (Clone 145-2C11), CD90.2-PE or –Alexa Fluor 647 (Clone 53-2.1 and (p.i.) and lost target protein expression at high efficiency, with Clone 30-H12) (all from BD Biosciences, except CD45.1-PB and CD90.2-Alexa Fluor 647 from BioLegend) and PD-1-BV785 (Clone the electroporation procedure otherwise having little impact on 29F.1A12), LAG3-PE (Clone C9B7W) (both from BioLegend), KLRG1- T cell expansion, differentiation, and function. Strikingly, gene allophycocyanin (Clone 2F1), and CD90.1-PE (Clone HIS51) (both from deletion was equally efficient when naive T cells were injected eBioscience). For intracellular staining, the eBioscience Foxp3/Transcription into recipients and “parked” without stimulation. We extended factor staining buffer set (ThermoFisher Scientific) and the following anti- by guest on October 1, 2021 this technique to HDR-based gene editing, where we success- mouse Abs were used: TOX-PE (Clone TXRX10; eBioscience), GzmB- allophycocyanin (Clone GB11; ThermoFisher Scientific), and rabbit TCF-1 fully introduced a single nucleotide polymorphism (SNP) into mAb (Cell Signaling Technology, detected with a secondary anti-rabbit IgG the Thy1 (encoding CD90) gene with no prior T cell activation. AlexaFluor594 Ab; ThermoFisher Scientific). For peptide restimulation These findings validate naive T cell electroporation for in vivo and cytokine staining, splenocytes were incubated with 10 ng/ml LCMV- studies, enable rapid probing of gene function without the ca- Cl13 GP33–41 peptide (Biomolecular Resource Facility, John Curtin School of Medical Research, Australian National University) and Bre- veats of in vitro cell culture, and provide the first evidence, to feldin (eBioscience) for 5 h at 37˚C as described previously (20); surface our knowledge, that HDR-mediated gene editing can be suc- stained with Fixable Viability Stain, anti-CD8, and anti-CD45.1 as cessfully achieved in uncultured naive T cells. above; fixed with the BioLegend fixation buffer; and stained intracellu- larly with TNFa-PE (Clone MP6-XT22; BioLegend), anti–IFN-g–PE- Cy7 (Clone XMG1.2), and IL-2–allophycocyanin (Clone JES6-5H4) Materials and Methods (both from eBioscience). Samples were acquired on a BD LSRFortessa Mice X-20 (BD Biosciences) and analyzed using FlowJo software (TreeStar) and GraphPad Prism (GraphPad Software). All p values were calculated Six- to ten-week-old male or female mice were used for all experi- using an unpaired One-way ANOVAwith a Tukey posttest, except Figs. 2 ments. CD45.2+ C57BL/6 (B6) mice were obtained from the Walter and5Cinwhichatwo-tailedunpairedStudentt test was used. and Eliza Hall Institute Kew Animal Facility (Kew, VIC, Australia), whereas CD45.1+ P14 (18) and CD45.1+ OT-I (19) mice were bred in house. Results All animal work was in accordance with protocols approved by the Peter Effective gene KO in CRISPR-edited adoptively transferred MacCallum Cancer Centre Animal Experimentation Ethics Committee + (Protocol E597) and current guidelines from the Australian Code of Practice naive CD8 P14 T cells after LCMV-Cl13 infection for the Care and Use of Animals for Scientific Purposes. sgRNA/Cas9 RNP electroporation into cultured naive T cells can Naive CD8+ T cell enrichment result in efficient gene deletion (14). We modified this strategy to

+ + avoid in vitro culture and test its utility within in vivo mouse CD8 T cells were enriched using the EasySep Mouse CD8 T Cell Iso- + + lation Kit (STEMCELL Technologies). See part A of the detailed protocol models. Naive CD8 LCMV-specific CD45.1 P14 cells were in the Supplemental Material for more information. Note that our T cell immediately electroporated after enrichment using commer- preparation differed from that in Seki et al. (14) through use of a different cially synthesized, chemically modified sgRNAs optimized for T cell isolation kit, and we omitted the described dead cell removal step. electroporation (13) and a commercially available recombinant PCR amplification and purification of template DNA Streptococcus pyogenes Cas9 nuclease. Of note, the chemically modified guides are known to enhance KO efficacy, so should See part C of the detailed protocol in the Supplemental Material for more information. Thy1.1 plasmids were obtained from commercially available boost efficiency relative to the previous study, and the use of DH5a bacterial stocks (p229_LTJ_2kbCD90.1Template; Addgene) (9) sgRNAs simplifies the protocol (Seki and Rutz predominantly grown on ampicillin-containing agar plates. For the PCR, the following used the two component crRNA and tracrRNA system) (14). The Journal of Immunology 3

Electroporated P14 cells were immediately transferred into recipient CD45.2+ B6 mice that were simultaneously infected with chronic LCMV-Cl13. At day 8 p.i., we measured splenic P14 cell expansion and gene deletion by flow cytometry (Fig.1A).Wechosethreedifferentclassesofgenetotarget:a gene required for clonal expansion (Cd3e), one that restrains expansion in this model (Pdcd1), and another whose deletion shouldhavenoeffectonexpansion(Cd90; Thy1 gene). Consistent with the known phenotypes associated with gene deficiency, relative to nontargeting (Ctrl) sgRNA treated P14 cells, Cd3e sgRNA-treated cells had a severe expansion deficiency and Cd90 sgRNA-treated cells expanded normally, whereas Pdcd1 sgRNA-treated cells exhibited augmented expansion (Fig. 1B, 1C). This was associated with high deletion efficiency within Cd90- and Pdcd1-targeted cells relative to control cells (86 and 99% KO cells, respectively) (Fig. 1D). Notably, only 14.2% of Cd3e targeted P14 cells lost CD3 protein expression; however, this is likely because cells that failed to delete Cd3e preferentially expanded. Consistent with this idea, the number of P14 T cells that failed to undergo gene deletion (“nondeleted”) did not sig- Downloaded from nificantly differ between Cd3e targeted P14 cells and Cd90 or Pdcd1 targeted P14 cells (Fig. 1E). Nondeleted cell numbers differed significantly between Cd90 and Pdcd1 targeted cells, although this likely reflected the lower gene editing efficiency of the Cd90 sgRNA. Thus, uncultured sgRNA/Cas9 RNP electroporated naive P14 cells efficiently delete genes when activated by http://www.jimmunol.org/ LCMV-Cl13 in vivo. sgRNA/Cas9 RNP electroporation does not alter CD8+ T cell expansion, differentiation, and function during LCMV-Cl13 infection As electroporation of Cas9 RNPs both transiently disrupts the cell membrane and introduces a foreign protein and nucleic acid into the cell, we next wished to examine whether the RNP by guest on October 1, 2021 electroporation process had any impact upon T cell expansion, differentiation, and function. Naive CD8+ CD45.1+ P14 cells were either electroporated with nontargeting Ctrl sgRNA/Cas9 RNPs as above (Fig. 2; Electroporated) or left untreated (not electroporated) after CD8+ T cell enrichment (Fig. 2; Untreated), with the cells then introduced into CD45.2+ B6 recipient mice that were simultaneously infected with LCMV-Cl13. Electroporated and untreated control CD8+ T cells expanded equally by day 8 p.i. with LCMV-Cl13 (Fig. 2A, 2B). Staining for markers associated with differentiation and exhaustion, including the inhibitory receptors PD-1 (21) and LAG3 (22), the differentiation marker KLRG1 (23), the key transcriptional regulators of exhaustion TCF-1 (24–26) and TOX (27–31), and the cytolytic molecule GzmB (23), revealed no difference between electroporated CD8+ T cells and their nonelectroporated counterparts (Fig. 2C, 2D). To examine the functionality of electroporated CD8+ T cells, we mea- FIGURE 1. Efficient CRISPR/Cas9-mediated gene deletion in adoptively transferred naive P14 T cells after LCMV-Cl13 infection. (A) sured cytokine production by the P14 cells after restimulation with Purified naive CD8+ CD45.1+ P14 cells were electroporated with targeting their specific peptide (LCMV-Cl13 derived GP33–41). Cytokine or nontargeting sgRNA/Cas9 RNPs and immediately transferred into production and T cell exhaustion (23) were comparable between congenic CD45.2+ B6 recipient mice (0 h) simultaneously infected with electroporated and untreated cells when measuring IFN-g, LCMV-Cl13. Gene deletion efficiency and T cell expansion within splenic TNF-a, and IL-2 in both single producers (Fig. 2E, 2F) and P14 cells were analyzed on day 8 p.i. (B) Representative profiles and (C) IFN-g+ TNF-a+ and IL-2+ double and triple producers (Fig. 2G). pooled percentages (left) and numbers (right) of transferred CD45.1+ P14 Thus, the electroporation step has no significant impact on T cell CD8+ T cells electroporated with nontargeting Ctrl, Cd3e-, Cd90-, or functionality. Pdcd1-targeting sgRNA/Cas9 RNPs. (D) Representative histograms (top) and pooled percentages (bottom) of CD3, CD90, and PD-1 surface ex- High efficiency gene deletion within a different target T cell pression in sgRNA/Cas9 electroporated CD45.1+ P14 CD8+ T cells. (E) population and infection model Total number of CD45.1+ P14 CD8+ T cells that retained expression of the respective targeted surface molecule on day 8 p.i. (nondeleted). Data and To test if high gene deletion efficiency was observed in a different + representative FACS plots are from two independent experiments with a model, we performed similar experiments using OVA-specific CD8 total of n = 6 mice per group. Bars depict mean, and error bars represent + CD45.1 OT-I TCR transgenic T cells adoptively transferred into B6 SD. *p , 0.05, **p , 0.01, ***p , 0.001, ****p , 0.0001. 4 GENE EDITING IN ADOPTIVELY TRANSFERRED NAIVE T CELLS

mice simultaneously infected with LM-OVA. As PD-1 expression is only evident early during infection in this model, splenic OT-I cells were analyzed at day 5 p.i. (Fig. 3A). Again, Cd3e targeted cells had an expansion deficiency, whereas Cd90 targeted cells expanded normally (Fig. 3B, 3C). Pdcd1 targeted cells did not exhibit significantly altered expansion, consistent with previous reports describing either a neutral or positive role for the PD-1/PD-L1 axis in OT-I expansion within this model (32). Again, high deletion efficiency was observed within Cd90 and Pdcd1 targeted cells (88 and 85%, respectively) (Fig. 3D). Similar as in Fig. 1E, comparable numbers of nondeleted cells were again observed in all conditions (Fig. 3E), but a much higher proportion (74%) of gene-deficient Cd3e-sgRNA electroporated OT-I T cells were recovered (Fig. 3D). As CD3- deficient cells should not expand, we speculated that this was due to the earlier (day 5) time point in these experiments; there is likely a lag between initial gene deletion and protein loss, meaning KO cells can initiate expansion prior to protein loss. Nevertheless, expansion was still impaired within this group, and overall, these data confirm that this approach is similarly efficient in an independent in vivo setting. Downloaded from Comparable gene deletion in naive P14 T cells transferred into uninfected mice Previous in vitro work has suggested that immediate strong T cell activation is required for efficient gene deletion within electro-

porated, uncultured naive T cells (14). As Cas9 protein is likely http://www.jimmunol.org/ rapidly lost from cells, we sought to define how long infection could be delayed posttransfer without compromising deletion efficiency. To this end, we conducted a time course in which B6 mice given Ctrl or Pdcd1 sgRNA electroporated CD45.1+ P14 cells were infected with LCMV-Cl13 either immediately (0 h) or at 24 or 48 h after P14 transfer, with PD-1 expression analyzed at day 8 p.i. (Fig. 4A). Surprisingly, there was no significant difference in gene deletion efficiency regardless of how long infection was delayed (Fig. 4B). This could be explained by sgRNA/Cas9 by guest on October 1, 2021 RNP persistence in P14 cells, or alternatively, gene editing may occur in naive P14 cells independent of T cell activation. To discriminate between these possibilities, we examined whether deletion of a gene constitutively expressed in naive T cells (Cd90) would still occur within adoptively transferred naive P14 cells kept in a naive state. Ctrl or Cd90 targeted CD45.1+ P14 cells were transferred either into B6 mice immediately infected with LCMV-Cl13 or into B6 mice that were left uninfected (Fig. 4C). Strikingly, when we measured CD90 expression after 8 d, similar proportions of CD90-deficient P14 cells were recovered from infected or uninfected recipients (79.5 versus 79.9% respectively; Fig. 4D). Thus, this approach can be used for efficient gene deletion within naive CD8+ T cells without any prior in vitro culture or conditioning. These findings suggested that delaying infection in this system may be beneficial, as it would enable protein depletion from gene- deficient cells prior to activation. We previously observed expanded

recipient mice that were simultaneously infected with LCMV-Cl13. T cell phenotype and function was analyzed on day 8 p.i. (A) Representative proﬁles and (B) pooled percentages (left) and numbers (right). (C) Representative histograms and (D) pooled percentages of PD-1, LAG3, KLRG1, GzmB, TCF-1, and TOX expression. (E) Representative histograms and (F) pooled percentages of IFN-g,TNF-a,andIL-2(F) single FIGURE 2. Unaltered phenotype and function of electroporated P14 and (G) double or triple producers in electroporated or untreated CD45.1+ + + + T cells in the LCMV-Cl13 infection model. Puriﬁed naive CD8 CD45.1 P14 CD8 T cells restimulated with GP33–41 peptide. FACS plots are from P14 cells were electroporated with nontargeting sgRNA/Cas9 RNPs or left three independent experiments with a total of n = 8–9 mice per group. Bars untreated (not electroporated) and transferred into congenic CD45.2+ B6 depict mean, and error bars represent SD. The Journal of Immunology 5 Downloaded from http://www.jimmunol.org/ by guest on October 1, 2021

FIGURE 3. Efficient CRISPR-mediated gene loss in adoptively transferred naive OT-I T cells after LM-OVA infection. (A) Purified naive CD8+ FIGURE 4. CRISPR/Cas9-mediated gene deletion occurs within trans- CD45.1+ OT-I cells were electroporated with targeting or nontargeting ferred naive CD8+ T cells independent of infection. (A and B) The ex- sgRNA/Cas9 RNPs and transferred into congenic CD45.2+ B6 recipient periment in Fig. 1A was repeated using Ctrl-orPdcd1-targeting sgRNA/ mice simultaneously infected with LM-OVA. Gene deletion efficiency and Cas9 RNPs, except that LCMV-Cl13 infection was either immediate or T cell expansion was analyzed on day 5 p.i. (B) Representative profiles and delayed until 24 or 48 h after transfer (A). (B) illustrates PD-1 levels in the (C) pooled percentages (left) and numbers (right) of transferred CD45.1+ transferred P14 cells at day 8 p.i. (C and D) Naive P14 cells were elec- OT-I CD8+ T cells electroporated with the sgRNAs used in Fig. 1B. (D) troporated with either Ctrl-orCd90-sgRNA/Cas9 RNPs and transferred Representative histograms (top) and pooled percentages (bottom) of CD3, into recipient B6 mice that were either immediately LCMV-Cl13 infected CD90, and PD-1 surface expression in sgRNA/Cas9 electroporated or left uninfected (C). (D) shows representative (left) and pooled (right) CD45.1+ OT-I CD8+ T cells. (E) Total number of nondeleted CD45.1+ OT-I CD90 deletion efficiency on day 8 p.i. (E and F) The experiment in Fig. 2A CD8+ T cells as defined in Fig. 1E. Data and representative FACS plots are was repeated using nontargeting Ctrl or Cd3e-targeting sgRNA/Cas9 from two independent experiments with a total of n = 6 mice per group. RNPs, except that LM-OVA infection was either immediate or delayed Bars depict mean, and error bars represent SD. *p , 0.05, ***p , 0.001, 48 h after transfer (E). (F) shows CD3 expression at day 5 p.i. Data and ****p , 0.0001. representative FACS plots are from two independent experiments with a total of n = 5–6 mice per group. Bars depict mean, and error bars represent SD. **p , 0.01, ***p , 0.001, ****p , 0.0001. 6 GENE EDITING IN ADOPTIVELY TRANSFERRED NAIVE T CELLS

CD3 KO OT-I cells in LM-OVA infection (Fig. 3D), and we HDR. To enable identification of cells that had successfully under- speculated that this KO population would be reduced if infec- gone HDR by flow cytometry, we used a previously designed HDR tion was delayed to enable protein depletion prior to activation. template (9) to introduce a single nucleotide change into CD90.2 that Indeed, when we repeated this experiment but delayed infection converts it into the CD90.1 congenic marker. To test the impact of until 48 h after OT-I transfer (Fig. 4E), a much lower proportion homology arm length on HDR efficiency, we used PCR amplified of CD3 KO cells were recovered (Fig. 4F). Thus, gene deletion HDR templates of three different sizes: 227, 500, and 984 bp. CD8+ within naive T cells via this method can be employed to ensure CD45.1+ CD90.2+ P14 donor cells were electroporated with sgRNA/ protein depletion prior to activation. Cas9 RNPs targeting the SNP site in the Cd90 gene (referred to as Cd90.2) alongside ∼3 3 1012 copies of the 227-, 500-, or 984-bp- CRISPR-mediated deletion of multiple genes within transferred sized DNA templates. T cells were transferred immediately into naive CD8+ T cells CD45.2+ CD90.2+ B6 recipient mice, alongside mice given Ctrl Given the high efficiency of gene deletion using this approach, we sgRNA/Cas9-treated cells, and infected simultaneously with next attempted to generate double KO cells by combined elec- LCMV-Cl13. On day 8 p.i., transferred CD8+ T cells were an- troporation of two guides (targeting Cd90 and Pdcd1) into alyzed for successful SNP modification by staining CD45.1+ + CD45.1 P14 T cells prior to transfer and LCMV-Cl13 infection. CD8+ T cells for both CD90.2 and CD90.1 (Fig. 6). As has been A very high proportion (80%) of cells lacked both proteins observed in previous HDR studies (9), NHEJ-mediated gene deletion (Fig. 5A, 5B), and, importantly, the KO efficiency of each in- dominated in all samples, with 93.9–96.9% CD90 KO efficiency in dividual sgRNA was comparable to when the guides were in- Cd90.2 sgRNA/Cas9 electroporated T cells (regardless of template) troduced alone in previous experiments (dashed lines) (Fig. 5C). over nontargeting Ctrl sgRNA (Fig. 6). There was no HDR-mediated This indicated that there was no competitive inhibition between SNP editing in cells electroporated with Ctrl sgRNA/Cas9 with no Downloaded from sgRNAs simultaneously introduced into the same cell and val- template or Cd90.2 sgRNA/Cas9 RNPs plus a 227-bp-long template idated that this experimental approach can simultaneously target (Fig. 6). However, CD8+ T cells electroporated with either a 500- or multiple genes. 984-bp-long DNA template, and Cd90.2 sgRNA, demonstrated HDR + CRISPR/Cas9-mediated HDR within transferred, uncultured as measured by the appearance of a CD90.1 cell population (3.2% naive CD8+ T cells of P14 cells) (Fig. 6B). Although a low efficiency process, this editing efficiency is similar to that observed with the same template http://www.jimmunol.org/ We next investigated if this approach could be used to introduce precise + in in vitro cultured and activated T cells and, unlike this previous genomic edits into naive CD8 T cells via CRISPR/Cas9-mediated study, did not require any sorting/selection of cells prior to transfer (9). Our results thus provide the first proof-of-principle data, to our knowledge, that CRISPR/Cas9-based HDR can be achieved in uncultured, naive CD8+ T cells.

Discussion

A major limitation of current CRISPR/Cas9-mediated gene editing by guest on October 1, 2021 protocols in T cells has been the requirement for in vitro culture,

FIGURE 5. Efficient CRISPR/Cas9-mediated generation of double KO CD8+ T cells. The experiment outlined in Fig. 1A was repeated, except that P14 cells were electroporated with either nontargeting Ctrl or combined Pdcd1-andCd90-targeting sgRNA/Cas9 RNPs. Representative (A)and FIGURE 6. CRISPR/Cas9-mediated HDR of CD90 in naive, uncultured pooled (B and C) proportions of cells lacking PD-1 and/or CD90 are CD8+ T cells. The experiment outlined in Fig. 1A was repeated, except that shown. (B) shows proportion of cells from each quadrant in the plots P14 cells were electroporated with either nontargeting Ctrl or Thy1.2 SNP illustrated in (A). (C) illustrates the proportion of cells expressing CD90 (Cd90.2)-targeting sgRNA/Cas9 RNPs in the absence (Ctrl) or presence of or PD-1. Dotted lines in the CD90+ and PD-1+ plots indicate the KO a 227-, 500-, or 984-bp-long homologous DNA template encoding the efficiency seen in cells given each guide singly in Fig. 1D. Data and CD90.1 SNP. Representative (A) and pooled (B) proportions of cells representative FACS plots are from two independent experiments with expressing CD90.2 or CD90.1 are shown. Data and representative FACS n = 6 mice per group. Bars depict mean, and error bars represent SD. plots are from two independent experiments with n = 6 mice per group. Bars ****p , 0.0001. depict mean, and error bars represent SD. **p , 0.01, ****p , 0.0001. The Journal of Immunology 7 which restricts the experimental questions that can be addressed. In of multiple guides against the same target can substantially increase this study we adapted a previously described CRISPR-based gene the efficiency of gene deletion (14), so the ability to codeliver editing strategy in murine T cells (14) to achieve high efficiency multiple Cas9 RNPs into the same cell means that it may be pos- gene deletion in adoptively transferred naive CD8+ T cells in vivo. sible to achieve close to 100% gene deletion efficiency using this The high KO efficiency meant that selection or sorting of targeted approach. Our findings also highlight that when working with a cells was not needed, and, importantly, no prior naive T cell protein that has a long half-life, gene deletion within naive T cells culture was required for gene deletion. Notably, electroporation prior to activation should be strongly considered. Conversely, our of Cas9 RNPs into naive T cells had little obvious influence on approach may be useful to determine the in vivo half-life of the T cell functionality. It remains possible that electroporation may protein expressed by a targeted gene. have a greater impact on functionality in other experimental Finally, we provide proof-of-concept data demonstrating that models, but our results suggest that this effect is likely to be CRISPR-mediated HDR can be achieved in adoptively transferred minimal. Finally, we provide the first evidence, to our knowledge, naive T cells without prior cell selection. In addition to enabling that HDR-mediated gene editing can be achieved in uncultured, more complex genetic manipulations in naive T cells, HDR also naive CD8+ T cells, which suggests that precise genomic edits provides an approach for “tagging” KO cells by, for example, (e.g., nucleotide changes and gene knock-ins) are possible in naive replacing the target gene with a fluorescent marker such as GFP T cells using this approach. Collectively, these techniques enable (3). This would enable identification of KO cells in situations in rapid CRISPR-based gene deletion and editing for use in in vivo which there are no flow cytometry Abs available for the protein naive T cell homeostasis studies, as well as in vivo adoptive target. Collectively, the methods reported in this paper are thus a transfer models (e.g., peripheral self-tolerance models) that are powerful new (to our knowledge) approach that enables the use of compromised by the in vitro culture and activation steps required in CRISPR-based techniques in models that were previously not Downloaded from other CRISPR protocols. This is also, to our knowledge, the first amenable to these strategies. report describing the use of uncultured, naive T cell electroporation for gene deletion and HDR-mediated editing within in vivo adoptive Acknowledgments transfer mouse models. We thank the Peter MacCallum Cancer Centre Animal Core facility for Previous CRISPR/Cas9 gene deletion approaches in T cells re-

breeding and maintenance of mice, the Peter MacCallum Cancer Centre http://www.jimmunol.org/ quired either T cell activation or culture with supraphysiological Flow Cytometry Facility, and the Peter MacCallum Cancer Centre Genotyping levels of IL-7 to facilitate retroviral transduction (7), plasmid Laboratory for genotyping of mice. electroporation (9), or efficient gene deletion (14) or editing (9) after sgRNA/Cas9 RNP electroporation. These approaches are unsuitable for adoptive transfer models in which prior activation Disclosures or cytokine culture could alter in vivo T cell differentiation (e.g., The authors have no financial conflicts of interest. tolerance and tumor models). We find that when CRISPR/Cas9 electroporated T cells are used within in vivo mouse adoptive transfer models, not only is activation by in vivo infection suffi- References by guest on October 1, 2021 ciently strong and rapid to facilitate gene deletion but naive cells 1. Simpson, E., and D. Roopenian. 1997. 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