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 September 25, 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

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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. http://www.jimmunol.org/ elineating the molecular mechanisms that underpin it can thus be difficult to definitively prove that loss of a KO cell 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 September 25, 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 tem-

In this study, we challenge the idea that T cell culture is plate DNA were electroporated into P14 T cells. T cells were adoptively Downloaded from transferred into B6 recipient mice, with 5 3 103 P14 T cells transferred 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- http://www.jimmunol.org/ ficities in two infection models: chronic lymphocytic chorio- For cell surface staining, cells were stained on ice in PBS containing 2.5% 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 September 25, 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 Downloaded from that failed to undergo gene deletion (“nondeleted”) did not significantly 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 electro- http://www.jimmunol.org/ porated naive P14 cells efficiently delete genes when activated by 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 September 25, 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 Downloaded from setting. 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- http://www.jimmunol.org/ porated, uncultured naive T cells (14). As Cas9 protein is likely 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 differ-

ence in gene deletion efficiency regardless of how long infection by guest on September 25, 2021 was delayed (Fig. 4B). This could be explained by sgRNA/Cas9 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 September 25, 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 Downloaded from This indicated that there was no competitive inhibition between SNP editing in cells electroporated with Ctrl sgRNA/Cas9 with no 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 http://www.jimmunol.org/ editing efficiency is similar to that observed with the same template 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 September 25, 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 Downloaded from compromised by the in vitro culture and activation steps required in CRISPR-based techniques in models that were previously not 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- http://www.jimmunol.org/ breeding and maintenance of mice, the Peter MacCallum Cancer Centre 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). 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2 Protocol for CRISPR/Cas9 RNP gene deletion and HDR in naïve CD8+ T cells

3 General Materials

4 • Lonza 4D-NucleofectorTM Core Unit (Lonza, Cat# AAF-1002B)

5 • P3 primary cell 4D-NucleofectorTM X kit S electroporation kit (Lonza, Cat# V4XP-

6 3032)

7 • Alt-R® S.p. Cas9 Nuclease (500 µg) (Integrated DNA Technologies (IDT®), Cat#

8 1081059)

9 • EasySepTM mouse CD8+ T cell isolation kit (StemcellTM Technologies, Cat# 19853A)

10 • 20 bp sgRNAs (3 nmol diluted in 10 µl Nuclease free H2O; Synthego,

11 CRISPRevolution sgRNA EZ Kit)

12 • Dulbecco’s PBS free of CaCl2 and MgCl2 (sourced from Peter MacCallum Laboratory

13 Support Services, but commercial sources of PBS can be used instead)

14 • cRPMI (10% Fetal Calf Serum (FCS), 2 mM (1x) GlutaMaxTM (Gibco®, Cat#35050-

15 061), 0.1 mg/ml Streptomycin and 100 Units/ml Penicillin (Sigma, Cat# P3032-

16 100MU) in RPMI Medium 1640 (Gibco®, Cat# 11875-093))

17 • MACS buffer (2 mM EDTA, 0.5% FCS in PBS)

18 • P3 buffer (make fresh on the day, mix 3.6 µl reagent 1 with 16.4 µl diluent per

19 reaction (Lonza 4D-Nucleofector kit))

20 • Spleens and lymph nodes from TCR transgenic mice (e.g. OT-I, or P14 mice)

21 • EASYstrainerTM 70 µm cell strainer (Greiner Bio-One, Cat# 542070)

22 • 15 ml and 50 ml conical tubes (CELLSTAR® Greiner Bio-One, Cat# 188261 and

23 Cat# 227261)

24 • 5 ml round-bottom tubes (Falcon®, Cat# 352054)

25 • 1.5 ml Microtube (Axygen®, Cat# MCT-150-C-S)

26 • EasyEights™ EasySep™ Magnet (StemcellTM Technologies, Cat# 18103)

27 • 3 ml Syringes (Terumo, Cat# SS+03L)

28 • 60/15 mm cell culture dish (Greiner Bio-One, Cat# 628160)

29 • Neubauer counting chamber (Hirschmann®, Cat# 8100203)

30 • Trypan Blue diluted in PBS to 0.1% (Sigma, Cat# T8154-100ML)

31 Additional Material for HDR

32 • LB Media (5 g NaCl, 5 g yeast extract, 10 g Casein Hydrolysate in 1 L H2O) (in-

33 house, Peter MacCallum Laboratory Support Services)

34 • NucleoBond® Xtra Maxi Plus kit (Macherey-Nagel, Cat# 740416.50)

35 • NucleoSpin® Gel and PCR Clean-up kit (Macherey-Nagel, Cat# 740609.250)

36 • Agarose (Bioline, Cat# BIO-41025)

37 • AMPure XP beads (Beckman Coulter, Cat# A63880)

38 • Phusion High-Fidelity 2x PCR Master Mix (ThermoFisher Scientific, Cat# F-531S)

39 • NanoDrop® (ThermoFisher Scientific)

40 • DynaMagTM-2 magnet (ThermoFisher Scientific, Cat# 12321D)

41 (A) Isolation of CD8+ T cells from TCR transgenic donor mice

42 1. Euthanize TCR transgenic donor mouse (e.g. OT-I, P14) by CO2 asphyxiation

43 2. Harvest spleen and pooled lymph nodes (inguinal, axillary, brachial, cervical,

44 mesenteric) for maximum yield of CD8+ T cells and pool them into a 5 ml round-

45 bottom tube containing 4 ml of MACS buffer on ice.

46 3. Mash spleen and lymph nodes through a 70 µm cell strainer into a 60/15 mm cell

47 culture dish using the plunger of a 3 ml syringe to generate a single cell suspension.

48 4. Wash cell strainer by passing 10 ml of MACS buffer through the strainer and

49 collecting the flow through into the same dish containing the cell suspension.

50 5. Transfer cell suspension into a 15 ml conical tube.

51 6. Centrifuge cells to pellet (all cell centrifugation steps are performed for 5 min at 524 g

52 and 4oC if not otherwise specified) and discard the supernatant.

53 7. Resuspend cell pellet in 1 ml MACS buffer using a pipette and filter through a 70 µm

54 cell strainer into a 5 ml round-bottom tube (depending on size of the magnetic stand

55 the cell enrichment steps below can also be performed in a 15 ml conical tube).

56 8. Add 50 µl of the CD8+ T cell enrichment antibody-mix (EasySepTM kit) and mix by

57 gentle vortexing.

58 9. Incubate for 10 min at room temperature to allow antibodies to bind to the cells.

59 10. Add 100 µl of beads (EasySepTM kit), mix by vortexing briefly and incubate for 5 min

60 at room temperature.

61 11. Rinse the walls of the tube with 1 ml of MACS buffer using a pipette to wash down

62 beads stuck to the side of the tube, then put the tube with cells and beads onto the

63 EasySepTM magnet.

64 12. Wait for ~1 minute for the beads to attach to the magnet site (or until media is clear)

65 and transfer enriched CD8+ T cells into a fresh 15 ml tube.

66 13. Add 10 ml of PBS, centrifuge cells, discard supernatant and resuspend the pellet in 2

67 ml PBS using a pipette for cell counting.

68 14. Count cells with a Neubauer counting chamber. To exclude dead cells, mix cells with

69 Trypan Blue solution (10 µl cells per 990 µl Trypan Blue) prior to loading onto the

70 counting chamber. Cell yields vary depending on the TCR transgenic donor used. We

71 typically recovered ~4 x 107 enriched CD8+ cells per OT-I donor and ~2 x 107

72 enriched CD8+ cells per P14 donor.

73 15. Resuspend desired amount of T cells per sgRNA target in 500 µl PBS (no loss in KO

74 efficiency was observed between 2-10 x 106 cells per electroporation) and transfer

75 into a 1.5 ml micro tube. Washing and resuspending cells in PBS is important to

76 remove potential RNase contamination from FCS containing media prior to

77 electroporation. 40-50% of T cells will be lost during the electroporation step, so this

78 should be factored in when considering how many cells to electroporate.

79 (B) Electroporation of sgRNA/Cas9 RNPs

80 1. Prepare 20 µl P3 buffer per electroporation reaction by adding 3.6 µl Supplement 1 to

81 16.4 µl P3 Primary Cell Solution (Lonza 4D Nucleofector kit) in a 1.5 ml micro tube.

82 Mix by vortexing and keep at room temperature until use. Buffer P3 needs to be

83 freshly prepared for each experiment.

84 2. For sgRNA/Cas9 RNP complex formation, combine 0.6 µl of Cas9 protein (10 mg/ml

85 in 50% glycerol) with 1 µl of sgRNA (0.3 nmol/µl in nuclease-free H2O) and top up

86 with nuclease free water (CRISPRevolution kit) to a final volume of 5 µl in a 1.5ml

87 micro tube. For double KO experiments where two sgRNA/Cas9 RNP complexes will

88 be introduced into cells, use 1 µl of each sgRNA, 0.6 µl of Cas9 and adjust the added

89 water to final volume of 5 µl. For HDR experiments, use 1 µl of sgRNA, 0.6 µl of

12 90 Cas9 and 3x10 copies of template DNA in up to 3.4 µl H2O for final volume of 5 µl.

91 Preparation of the HDR template is covered in a separate section below.

92 3. Gently flick the 1.5 ml micro tube to mix sgRNAs with Cas9 and spin down briefly

93 using a microfuge to collect drops from the sides of the tube.

94 4. Incubate for 10 min at room temperature for complex formation.

95 5. During complex formation, centrifuge the cells in the micro tube (4 min, 400 x g,

96 room temperature) and switch on the Nucleofector during centrifugation. It is

97 important to prepare everything prior to resuspending the cells in P3 buffer to

98 minimize the time that cells are exposed to the buffer.

99 6. Remove supernatant from cell pellet with a pipette. Ensure that all remaining liquid is

100 removed from the cell pellet to prevent dilution of the 25 µl reaction volume required

101 for electroporation. Steps 7-11 below should be completed quickly and carefully to

102 minimize cell exposure to Buffer P3.

103 7. Resuspend cell pellet in 20 µl P3 buffer by pipetting up and down gently.

104 8. Transfer cells in P3 buffer into the micro tube containing 5 µl of sgRNA/Cas9 RNP

105 and mix gently by pipetting up and down.

106 9. Transfer the 25 µl cell/RNP mix to the bottom hole of a well of the Lonza nuclefector

107 strip. Be careful to not create any bubbles.

108 10. To electroporate the cells, place the nucleofector strip into the Lonza nucleofector,

109 select the appropriate wells that were loaded with cells, and select the “mouse T cell,

110 unstimulated” (Pulse DN100) pre-configured program.

111 11. After electroporation (a few seconds) add 130 µl of 37°C pre-warmed 10% cRPMI to

112 the well containing electroporated cells.

113 12. Rest cells for 10 min in a cell culture incubator (5% CO2, 37°C).

114 13. Resuspend the cells in the electroporation well using a pipette, and transfer the cell

115 suspension into a fresh 15 ml tube. To recover any remaining cells, wash the

116 electroporation well with an additional 150 µl 10% cRPMI by pipetting, and transfer

117 the media into the same collection tube.

118 14. Count cells as in Step (A) 14 using 10 µl cells per 10 µl Trypan Blue, resuspend in

119 PBS at desired concentration and adoptively transfer into recipient mice via

120 intravenous injection. We typically observe 40-50% loss of cells during the

121 electroporation step.

122 (C) Preparation of template DNA for HDR

123 1. The optimal size and concentration of the DNA template for HDR is likely to vary

124 depending on the size of the inserted sequence, sgRNA used and region targeted. For

125 introduction of a point mutation into the Thy1 (Cd90) gene locus, we found that

126 homology arms of at least 250 bp either side of the Cas9 cut site were optimal,

127 however this may need to be optimized independently for each experiment. It is

128 important to mutate the PAM site in the HDR template to ensure that it is not cut by

129 Cas9. For our targeting experiments, the PAM site was already mutated in the

130 commercially sourced plasmid.

131 2. Once the HDR template is designed, synthesised, and cloned into a plasmid vector,

132 grow bacterial stocks containing the plasmid with template DNA sequence spanning

133 the target genomic region, and isolate plasmids using the NucleoBond® Xtra Maxi

134 Plus kit following the manufacturer’s instructions.

135 3. Amplify template DNA with an initial PCR reaction using the Phusion High-Fidelity

136 2x PCR Master Mix and primers spanning the target region. The 20 µl PCR reaction

137 is prepared as follows: 20 ng of plasmid in 1 µl H2O, 1 µl each of 10 µM forward and

138 reverse primer, and 7 µl H2O with 10 µl 2x Phusion Master Mix.

139 4. Amplify the template using the following cycling conditions: 95°C for 1 min, 34

140 cycles of (95°C-30 sec, 59°C-20 sec, 72°C-20sec), 72°C for 5 min. Note that the

141 primer annealing temperature will need to be adjusted depending on the primers used.

142 5. Run the PCR reaction on a 1% agarose gel via electrophoresis alongside the

143 appropriate DNA ladder for size and quality control.

144 6. Excise the DNA band containing the PCR product from the gel over a UV lamp, and

145 purify the template DNA using the NucleoSpin® Gel and PCR Clean-up kit following

146 the manufacturer’s instructions. Elute the DNA template in 200 µl H2O.

147 7. A single round of PCR does not yield sufficient product for HDR, so a second round

148 of amplification is required. The second round of PCR is prepared by making an 800

149 µl (total volume) PCR master mix as per step 3 and using the 200 µl of DNA from

150 step 6 as a template. Aliquot the 800 µl Master Mix into 16 separate 50 µl PCR

151 reactions and follow the PCR cycling conditions above.

152 8. Pool the 16 amplified PCR reactions into a single 1.5 ml micro tube.

153 9. Purify the template DNA using AMPure XP beads by adding beads in a 1:1 (vol:vol)

154 ratio to the PCR product. Mix by pipetting up and down until solution is homogenous

155 (be careful not to create bubbles) and incubate for 5 min at room temperature.

156 10. Place beads onto a DynaMag magnet for 2 min to pellet the beads out of the solution.

157 11. Pipette off and discard supernatant, taking care not to disturb the beads.

158 12. Take the tube with beads off the magnet and wash beads by adding 200 µl of 70%

159 ethanol. Addition of the ethanol should resuspend the beads without any additional

160 mixing or pipetting. Be careful not to touch the beads with the tip of the pipette.

161 13. Place the tube back onto the magnet. The beads will be washed by moving through

162 ethanol to the site of the magnet. Pipette off the ethanol, again taking care not to

163 disturb the beads.

164 14. Repeat wash as above with a further 200 µl of 70% ethanol.

165 15. Remove the tube from the magnet and dry off all remaining ethanol by laying down

166 the micro tube with the lid open for ~1 min (no longer than 4 min). Perform this step

167 in a tissue culture hood to maintain sterility.

168 16. Elute DNA by adding 20-25 µl nuclease free H2O to the beads, and put the tube back

169 onto the magnet. Again, be careful not to touch the beads with the tip of the pipette.

170 17. Transfer the clear eluate containing the template DNA into a fresh 1.5 ml micro tube.

171 18. Measure the DNA concentration of the eluate on a NanoDrop and estimate the copy

172 number.

173 19. Adjust the DNA concentration as needed to achieve 3x1012 copies of template DNA

174 in ≤ 3.4 µl H2O. Note that for bigger sized templates this will equate to a larger

175 quantity of DNA, meaning that it may be necessary to use a lower copy number to

176 limit DNA-induced cytotoxicity (1). However, for up to a 1 kb template, we have not

177 observed significant template-induced toxicity at these DNA concentrations.

178

179 References

180 1. Larkin, B., V. Ilyukha, M. Sorokin, A. Buzdin, E. Vannier, and A. Poltorak. 2017.

181 Cutting Edge: Activation of STING in T Cells Induces Type I IFN Responses and

182 Cell Death. J Immunol 199: 397-402.

183