CRISPR Editing Tools Efficient Immune Cell Genome Engineering

Efficient Immune Cell Genome Engineering with Enhanced CRISPR Editing Tools

Waipan Chan, Rachel A. Gottschalk, Yikun Yao, Joel L. Pomerantz and Ronald N. Germain Downloaded from ImmunoHorizons 2021, 5 (2) 117-132 doi: https://doi.org/10.4049/immunohorizons.2000082 http://www.immunohorizons.org/content/5/2/117 This information is current as of October 2, 2021. http://www.immunohorizons.org/

Supplementary http://www.immunohorizons.org/content/suppl/2021/02/19/5.2.117.DCSupp Material lemental References This article cites 45 articles, 9 of which you can access for free at: http://www.immunohorizons.org/content/5/2/117.full#ref-list-1 Email Alerts Receive free email-alerts when new articles cite this article. Sign up at:

http://www.immunohorizons.org/alerts by guest on October 2, 2021

ImmunoHorizons is an open access journal published by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 All rights reserved. ISSN 2573-7732. RESEARCH ARTICLE

Adaptive Immunity

Efficient Immune Cell Genome Engineering with Enhanced CRISPR Editing Tools

Waipan Chan,* Rachel A. Gottschalk,*,1 Yikun Yao,† Joel L. Pomerantz,‡ and Ronald N. Germain* *Lymphocyte Biology Section, Laboratory of Immune System Biology, National Institute of Allergy and Infectious Diseases, National Institutes of †

Health, Bethesda, MD 20892; Molecular Development of the Immune System Section, Laboratory of Immune System Biology, National Institute Downloaded from of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892; and ‡Department of Biological Chemistry and Institute for Cell Engineering, The Johns Hopkins University School of Medicine, Baltimore, MD 21205

ABSTRACT http://www.immunohorizons.org/ Clustered regularly interspaced short palindromic repeats (CRISPR)–based methods have revolutionized genome engineering and the study of gene-phenotype relationships. However, modifying cells of the innate immune system, especially macrophages, has been challenging because of cell pathology and low targeting efficiency resulting from nucleic acid activation of intracellular sensors. Likewise, lymphocytes of the adaptive immune system are difficult to modify using CRISPR-enhanced homology-directed repair because of inefficient or toxic delivery of donor templates using transient transfection methods. To overcome these challenges and limitations, we modified existing tools and developed three alternative methods for CRISPR-based genome editing using a hit-and- run transient expression strategy, together with a convenient system for promoting gene expression using CRISPRa. Overall, our CRISPR tools and strategies designed to tackle both murine and human immune cell genome engineering provide efficient alternatives to existing methods and have wide application not only in terms of hematopoietic cells but also other mammalian by guest on October 2, 2021 cell types of interest. ImmunoHorizons, 2021, 5: 117–132.

INTRODUCTION effects in its unmodified form (5–7). Many groups have now developed new methods to minimize this problem (8–14). The The bacterial innate defense system clustered regularly inter- Cas9 nickase technique is one useful approach with a strict spaced short palindromic repeats (CRISPR) was discovered more pairing distance requirement that makes off-target action very than three decades ago (1) but only harnessed for gene editing unlikely. Another strategy to reduce CRISPR-Cas9 off-target applications in a series of landmarkstudiespublishedin2013 effects is to limit the duration of Cas9 or guide-RNA (gRNA) (2–4). Since then, there has been an exponential increase of expression in the target cells (13, 15). Although such temporal CRISPR-related publications, with many of the initial studies control can be achieved using transient transfection methods employing the Cas9 nuclease that can yield variable off-target such as RNA electroporation, the on-target editing efficiency

Received for publication January 28, 2021. Accepted for publication January 28, 2021. Address correspondence and reprint requests to: Dr. Waipan Chan and Dr. Ronald N. Germain, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Building 4, Room 109, 4 Memorial Drive, Bethesda, MD 20892-0421. E-mail addresses: [email protected] (W.C.) and [email protected] (R.N.G.) ORCIDs: 0000-0002-9109-6583 (W.C.); 0000-0002-5248-8816 (R.A.G.); 0000-0002-5030-1018 (J.L.P.); 0000-0003-1495-9143 (R.N.G.). 1Current address: Department of Immunology, University of Pittsburgh School of Medicine, Pittsburgh, PA. This work was supported by the Intramural Research Program of the National Institute of Allergy and Infectious Diseases, National Institutes of Health. Abbreviations used in this article: AAV, adeno-associated virus; AUC, area under the curve; BMDM, bone marrow–derived macrophage; CRISPR, clustered regularly interspaced short palindromic repeats; DUSP, dual-specificity phosphatase; EV, empty vector; gRNA, guide-RNA; HDR, homology-directed repair; iCE, pCW-Cas9-2A- EGFP; indel, insertion/deletion; KO, knockout; LGB, LentiGuide-TagBFP-2A-BSD; mNG, mNeonGreen; NHEJ, nonhomologous end joining; NIAID, National Institute of Allergy and Infectious Diseases; NIH, National Institutes of Health; NIL, nonintegrating lentiviral; PuroR, puromycin resistance; RAW, RAW264.7 macrophage; RAWiCE, iCE-transduced and puromycin-selected RAW cell; T7EN, T7 endonuclease I. The online version of this article contains supplemental material. This article is distributed under the terms of the CC BY 4.0 Unported license. Copyright © 2021 The Authors https://doi.org/10.4049/immunohorizons.2000082 117

ImmunoHorizons is published by The American Association of Immunologists, Inc. 118 CRISPR METHODS FOR MACROPHAGE AND T CELL ENGINEERING ImmunoHorizons can be compromised (16) as compared with stable lentiviral engineering using 1 3 106 multiplicity of infection of adeno- transduction methods. In addition, transient transfection ap- associated virus (AAV) type 6 transduction to deliver a high copy proaches do not permit easy regulation of Cas9 or gRNA expression number of HDR donor templates per target cell (26). AAV is a duration, whereas cytotoxicity created by electroporation small ssDNA virus that infrequently integrates into the host methods remains a concern for many sensitive cell types. genome, making it a safe delivery platform for the HDR donor These issues are amplified for hematopoietic cells, with only a template. However, the production and purification process of few reports using the Cas9 nickase strategy for immune cell AAV particles is relatively labor intensive, increasing the cost and (myeloid cell or lymphocyte) genome engineering. reducing the flexibility of preliminary experiments in the Macrophages have been especially difficult to modify using academic laboratory setting. In addition, the very limited payload available CRISPR methods. All-in-one CRISPR-Cas9 techniques of the AAV vector makes it impossible to fit Cas9, gRNA, and are inefficient (17), and although newer methods have yielded donor template in one AAV backbone. The alternative is the use improved results (18–20), some of these approaches require of lentiviruses. Lentiviral particles are easy to produce and antibiotic selection and clonal isolation that delay the phenotyping purify in most laboratories with relatively low cost. Further- timeline and risk the loss of original cell properties. To enable high- more, the payload of lentiviral particles is large enough to carry

throughput CRISPR-Cas9 genome-wide knockout (KO) library Cas9, gRNA, and donor template all in one. Despite these Downloaded from screens, the murine RAW264.7 macrophage (RAW) cell line has advantages, random integration of lentiviral DNA into the host been modified to constitutively express Cas9 for efficient genome genome makes it problematic as a platform to deliver the HDR editing (21). However, constitutive Cas9 and gRNA expression can donor template, leaving an opportunity for further improve- lead to the accumulation of off-target mutations. A doxycycline- ment in vector design. inducible system pCW-Cas9 (22) has been reported to resolve both All the above methods involve either gene inactivation or issues by controlling the genome editing time window to minimize modification. There are numerous situations in which augmented http://www.immunohorizons.org/ long-term off-target effects and allowing immediate phenotyping expression of a given gene would be desirable in an experimental before the onset of potential compensating mechanisms. Nonethe- setting. Although methods for gene activation using the CRISPRa less, the lack of a live cell marker in current versions of the vectors strategy have been reported (27–29), efficient induction of used for such experiments makes it difficult to identify and isolate selective gene expression is not yet a commonly used method as the Cas9-expressing population for further analysis. we lack simple tools for this purpose. Primary bone marrow–derived macrophages (BMDM) are In this study, we have addressed each of the problems outlined particularly problematic to engineer because of their innate above and developed new vectors and strategies that enable more immune sensors. Lentiviral particles produced from packaging effective genetic modifications of macrophages and lymphocytes. cells such as the HEK293T cell line are usually filtered or spun to To control the duration of Cas9 expression and track cell with this remove cell debris. However, this process does not remove many enzymepresent,wedescribeamodified method to enable by guest on October 2, 2021 small molecules and exosomes secreted from the exhausted and doxycycline-induced Cas9 expression to be tracked by EGFP dying packaging cells. Lentiviral supernatant containing these fluorescence in live single cells and use this system to effectively uncharacterized materials is often directly applied to cell culture, engineer the RAW cell line. To deal with the issue of activation of and the potential side effects of the treatment due to sensor intracellular sensors, we show that purified lentiviral particles signaling are often ignored. enhance primary macrophage cell viability posttransduction. To In contrast to macrophages, all-in-one lentiviral-mediated achieve efficient and more-precise genome modifications in CRISPR-KO is highly efficient in human T lymphocytes (21, 22). humanJurkatTcellsasamodelformaturelymphocyte However, this approach is not suitable for CRISPR–homology- engineering, we adopted the Cas9 nickase strategy and describe directed repair (HDR) applications because of random genome an antibiotic selection strategy that helps enrich specifically for integration of lentiviral vectors carrying the HDR donor template rare HDR clones following lipid-based plasmid cotransfection. that can lead to undesired genomic instability. Transient trans- In addition, we further improved this method to minimize the fection delivery methods such as lipid-based reagents and genomic footprint created by the antibiotic resistance marker electroporation systems either yield low transfection efficiency andmakepossiblefutureroundsofgenomeeditinginthesame or a high percentage of cell death. Furthermore, compared with cells using the same strategy. We overcome the limitation the high targeting efficiency (80–90%) of CRISPR-induced associated with AAV and with integrating lentiviruses by the nonhomologous end joining (NHEJ), the targeting efficiency of use of integrase-deficient lentiviral particles (30–32) and CRISPR-induced HDR is generally inefficient (10–20% for large report two examples of this approach using all-in-one Cas9- fragment insertion) (23). Although the NHEJ inhibitor SCR7 gRNA-HDR-donor design in human Jurkat T cells. Finally, to (24) and the HDR stimulatory compound RS-1 (25) have been enable gene activation rather than inactivation or modification, reported to enhance CRISPR-Cas9–induced HDR efficiency, the we modified an existing two-vector system (29) to create an all- key to shifting the balance between the NHEJ and the HDR DNA in-one lentiviral-based CRISPRa system and show its utility as repair mechanisms in favor of the latter outcome seems to rely applied to the human Jurkat T cell model. These various new on a high molar ratio of donor DNA to Cas9-gRNA ribonucleo- vectors and strategies constitute an effective suite of methods protein. This was demonstrated in human primary T cell for the manipulation of both innate (macrophage) and adaptive

https://doi.org/10.4049/immunohorizons.2000082 ImmunoHorizons CRISPR METHODS FOR MACROPHAGE AND T CELL ENGINEERING 119

(lymphocyte) immune cells that offer benefits when applied to application, we selected a gRNA pair with the best score that a wide variety of cell types. targets near the start codon. For the BCL10 HDR application, we selected top five scoring gRNA pairs that target near the start codon. In addition, we selected a gRNA pair (D3) that MATERIALS AND METHODS confers a small offset distance of 3 bp. Both the CD3E gRNA and the RelA gRNA for the nonintegrating lentiviral (NIL) delivery Gibson assembly cloning of donor and lentiviral applications were designed to overlap with the start codon in expression plasmids each case to avoid further editing following the HDR event. The The pCW-Cas9-2A-EGFP (iCE) plasmid was constructed by RelA gRNA A1 was adopted from the human GeCKOv2 library replacing the FseI-BamHI fragment in the pCW-Cas9 plasmid A (33). The PX461 and PX462 plasmids were gifts from (plasmid no. 50661; Addgene) (22) with the T2A-EGFP fragment. F. Zhang (plasmid nos. 48140 and 48141; Addgene) (35). The The LCv2B plasmid was constructed by replacing the BamHI- pKLV-BFP plasmid was a gift from K. Yusa (plasmid no. 50946; MluI fragment in the LentiCRISPRv2 plasmid (plasmid no. 52961; Addgene) (36). All lentiviral and other gRNA expression Addgene) (33) with the P2A-BSD fragment. The LentiGuide- plasmids were prepared by sticky-end ligation before any fi TagBFP-2A-BSD (LGB) and the LentiGuide-TagRFP-2A-BSD further modi cation described. The PX462-hBCL10-gRNA-A1 Downloaded from (LGR) plasmids were constructed by replacing the XbaI-Cas9- plasmid was constructed by Gibson Assembly in the PciI-linearized BamHI fragment in the LCv2B plasmid with the TagBFP and the PX461 backbone, followed by subcloning of the double gRNA TagRFP-T fragment, respectively.The STAT3 HDR donor plasmid expression fragment into the PX462 backbone with PvuI-XbaI was constructed by replacing the NotI-XhoI fragment in the sticky-end ligation. CRISPRa gRNAs were adopted from the pcDNA3.1 plasmid with the indicated fragments. The BCL10 HDR Human CRISPR Activation Library (SAM–2 plasmid system) donor plasmid was assembled based on a minimal PciI-BglII from F. Zhang (29). http://www.immunohorizons.org/ pcDNA3 fragment that contains only the ColE1 origin and the ampicillin resistance expression cassette. To construct the LCv2B- Production, purification, and concentration of HDR-mCherry-2A-hCD3E plasmid, LCv2B plasmid containing lentiviral particles the CD3E gRNA was linearized at the KpnI site upstream of the U6 Lentiviral particles were produced in HEK293T cells following promoter before the assembly of the donor template fragments. transient transfection of the packaging plasmids pVSVg (plasmid The LCv2S plasmid (Supplemental Fig. 3A) was constructed by no. 8454; Addgene) and psPAX2 (plasmid no. 12260; Addgene) and replacing the BamHI-MluI puromycin resistance (PuroR) frag- the lentiviral expression plasmid in a 1:10:10 ratio using Lipofect- ment in the LentiCRISPRv2 plasmid with a 24-bp bridging amine 3000. Lentiviral supernatant was harvested 44–52 h fragment via sticky-end ligation. To construct the LCv2S-HDR- posttransfection and spun at 500 3 g for 5 min to clear cell debris. tBFP-hRelA plasmid, LCv2S plasmid containing the RelA gRNA NILparticleswereproducedthesameway,exceptwiththeuseof by guest on October 2, 2021 was linearized at the same KpnI site to allow for the assembly of the psPAX2-D64V plasmid (plasmid no. 63586; Addgene) (30) the donor template fragments. The LCv2R plasmid was assembled instead of the psPAX2 plasmid. All lentiviral particles other than by adding the P2A-mScarlet-I fragment to the BamHI-linearized those used in the RAW cell transductions and Fig. 2B (top panel) LCv2S backbone. The LCv2E plasmid was assembled using In- were further purified and concentrated with the Lenti-X Concen- Fusion cloning by adding the P2A-tEGFR fragment to the BamHI- trator reagent (Takara) according to the manufacturer’sinstructions. linearized LCv2S backbone. The LES2A-CRE plasmid was assembled by replacing the XbaI-EcoRI fragment in the lentiCas9-Blast Cell culture, transfection, and antibiotic plasmid (plasmid no. 52962; Addgene) (33) with the mScarlet-I- selection conditions P2A-CRE fragment. The Lenti-CMV-mCherry-P2A-CRE (also HEK293T, RAW, and Jurkat cell lines were purchased from the known as pLM-CMV-R-Cre) plasmid was a gift from M. Sadelain American Type Culture Collection. Cell culture was performed in

(plasmid no. 27546; Addgene) (34). The LentiSAMPHv2 plasmid ahumidified environment with 5% CO2 at 37°C. Both HEK293T was assembled by adding the P2A-MS2-p65-HSF1 fragment and RAW cell lines were cultured in DMEM supplemented with amplified from the lentiMPH v2 plasmid (plasmid no. 89308; 10% FBS, glutamine, and 18 mM HEPES buffer. Jurkat cell lines Addgene) (29) to the BsrGI-linearized lentiSAMv2 backbone were cultured in RPMI 1640 supplemented with 10% FBS, (plasmid no. 75112; Addgene) (29). All constructs described in this penicillin/streptomycin, glutamine, and 50 mM2-ME(complete article will be deposited with Addgene and become available to RPMI medium). For plasmid transfections of HEK293T and qualified investigators. Jurkat cells, Lipofectamine 3000 (Thermo Fisher Scientific) was used according to manufacturer’s protocol. Puromycin (0.5–3mg/ml) CRISPR gRNA target design and cloning or blasticidin (1–3 mg/ml) was added to cell culture medium All gRNAs were designed based on the analysis results arising from 1–2 d after transduction of both RAW and Jurkat cell lines. use of a previously available online platform (crispr.mit.edu). For the dual-specificity phosphatase (DUSP) gene KO application, we Genomic DNA extraction, T7EN assay, and sequencing selected the gRNA with the best score that targets downstream Genomic DNA was extracted from RAW, HEK293T, or Jurkat of the start codon of each DUSP gene. For the STAT3 HDR cells using the QIAmp DNA Mini Kit (Qiagen) following the

https://doi.org/10.4049/immunohorizons.2000082 120 CRISPR METHODS FOR MACROPHAGE AND T CELL ENGINEERING ImmunoHorizons manufacturer’s protocol. Genomic regions of interest were then surface protein staining was analyzed using anti–PD-1 (clone amplified by PCR for follow-up analysis. For the T7EN assay, equal MIH4; Thermo Fisher Scientific) or anti–PD-L1 (clone MIH1; amounts of genomic PCR products were used within each Thermo Fisher Scientific) Ab, respectively. Live cell confocal comparison group for the hybridization reaction in a thermocycler time-lapse imaging data were collected using a Leica SP8 programmed to 95°C for 10 min, 22°C per second ramp to 85°C, microscope with a 63x NA 1.4 oil objective (Biological Imaging 20.1°C per second ramp to 25°C, and then held at 4°C until a Section, Research Technologies Branch of NIAID). The imaging subsequent T7 endonuclease I (T7EN) (New England BioLabs) chamber (155411; Thermo Fisher Scientific) was coated with poly- treatment at 37°C for 30–90 min. Twenty-five millimolars final D-lysine (P7280; Sigma-Aldrich) for 1 h at 37°C and washed twice concentration of EDTA was added to each reaction before agarose with PBS. Cells were imaged in a heated 37°C environment with gel electrophoresis analysis. Insertion/deletion (indel) percentage 5% CO2. Imaging data were processed by Imaris (Bitplane). was determined as described previously (8). For the genomic DNA sequencing, each DUSP gene locus surrounding the CRISPR Chemical reagents, lysis buffer, and Western blots gRNA target site (;2500 bp to +500 bp) was amplified and cloned Doxycycline hydrochloride (D3447; Sigma-Aldrich) was recon- into a XhoI-NotI linearized pcDNA3.1 backbone by Gibson stituted in DMSO at 50 mg/ml and stored at 220°C. Jurkat T cells

Assembly. Sequencing was performed with the BGH-R (Table I) were stimulated with 50 ng/ml PMA (Santa Cruz Biotechnology) Downloaded from primer on 20 clones per gene. and 1 mM ionomycin (Sigma-Aldrich) (37). RAW cells were lysed in a buffer containing 50 mM Tris pH 7.5, 150 mM NaCl, 1 mM Bone marrow isolation and intracellular Ki67 staining EDTA, 10% glycerol, and 1% Igepal. Western blots were analyzed Mice were maintained in specificpathogen–free conditions, and using anti-DUSP3 Ab (clone EPR5492; Abcam), anti-DUSP1 Ab all procedures were approved by the National Institute of Allergy (07-535; Millipore), and anti-FLAG Ab (sc-166355; Santa Cruz and Infectious Diseases (NIAID) Animal Care and Use Committee Biotechnology). http://www.immunohorizons.org/ (National Institutes of Health [NIH], Bethesda, MD, ASP LISB- 4E). Bone marrow progenitors isolated from C57BL/6 mice (The Human PBMC isolation and transduction Jackson Laboratory) and Rosa26-Cas9 B6J mice (no. 026179; The Human whole blood from healthy anonymous volunteer donors Jackson Laboratory) were differentiated into BMDM during a 7-d was purchased from an NIH blood bank. This was exempted from culture in complete DMEM (10% FBS, 100 U/ml penicillin, 100 U/ml the need for informed consent and Institutional Review Board streptomycin, 2 mM L-Glutamine, 20 mM HEPES) supplemented review, as determined by the NIH Office of Human Subjects with 60 ng/ml recombinant mouse M-CSF (R&D Systems). For Research Protection. Human PBMCs were isolated from whole analysis of proliferation, cells were harvested at the indicated blood by Ficoll density gradient separation, cultured in complete day of the BMDM culture, processed using BD Cytofix/Cytoperm RPMI medium, and stimulated with 1 mg/ml anti-CD3 (300334; reagents as directed, blocked, and stained with anti–Ki-67 (BD BioLegend) and 1 mg/ml anti-CD28 (302944; BioLegend) soluble by guest on October 2, 2021 Pharmingen). Abs for 18 h. Purified and concentrated lentiviral particles were then added to the human PBMC culture, with 8 mg/ml Polybrene Macrophage TLR4 activation and intracellular phospho- (TR-1003; Millipore Sigma) and 100 U/ml recombinant human p38 staining IL-2 (TECIN [teceleukin]). These cells were then spun at 400 3 g BMDM or RAW cells were stimulated with TLR4 ligand Kdo2- for 90 min in a prewarmed centrifuge at 34°C. Lipid A (Avanti Polar Lipids). Ligand addition was staggered so that for all time points, cells were fixed at the same time by addition of paraformaldehyde to cell cultures at a final concentration of RESULTS 1.6% (10 min). After one wash with PBS 1% FBS, cells were gently harvested from plates by scraping, permeabilized overnight using Dual-color inducible CRISPR-Cas9 editing system in ice-cold MeOH at 220°C, blocked using 5% goat serum and Fc RAW cells receptor–specific Ab, and stained for 1 h at room temperature with To achieve a controllable balance between on-target and off-target anti-p38 (phospho-Thr180/Try182, 36/p38; BD) Ab. For both mutations induced by CRISPR, we adopted the doxycycline- BMDM and RAW macrophages, two independent time-course inducible pCW-Cas9 system to enable precise manipulation of the experiments were pooled using normalized area under the curve genome editing time period. In pCW-Cas9–transduced RAW (AUC). AUC was calculated using GraphPad Prism, and the AUC macrophages, a range of Cas9 protein expression was induced by for each condition was normalized to the empty vector (EV) doxycycline dose titration (Supplemental Fig. 1A). We modified control from the same experiment. this existing system to enable single-cell flow cytometric analysis and developed the iCE lentiviral expression plasmid (Fig. 1A). Cell sorting, FACS analysis, and live cell confocal imaging Doxycycline titration in the iCE-transduced and puromycin- Cell sorting was conducted by the Flow Cytometry Section, selected RAW cells (RAWiCE) revealed saturating Cas9-2A-EGFP Research Technologies Branch of NIAID. FACS analysis data were protein expression at 1.0 mg/ml doxycycline (Fig. 1B). Single clone collected using a BD LSRII or BD Fortessa Cell Analyzer and isolation from the RAWiCE line yielded cells that fully responded further processed with FlowJo. Human PDCD1 or PDL1 cell to doxycycline induction (Fig. 1C), indicating the digital nature of

https://doi.org/10.4049/immunohorizons.2000082 ImmunoHorizons CRISPR METHODS FOR MACROPHAGE AND T CELL ENGINEERING 121 Downloaded from http://www.immunohorizons.org/ by guest on October 2, 2021

FIGURE 1. Dual-color inducible CRISPR strategy efficiently modifies DUSP genes in RAW cells. (A) Lentiviral vector encoding inducible Cas9-T2A-EGFP under the control of the tTRE promoter. (B) EGFP expression by RAWiCE cells treated with doxycycline. (C) EGFP expression by RAWiCE clones treated with doxycycline. (D) Lentiviral vector for gRNA expression with TagBFP fluorescent marker and blasticidin resistance gene BSD. (E) Analysis of DUSP3, EGFP, and tBFP by RAWiCE cells transduced with the indicated LGB lentiviral particles and selected with blasticidin after doxycycline treatment. Results are representative of three independent experiments. (F) DUSP protein expression by RAWiCE cells transduced with the indicated LGB targeting DUSP2 or DUSP3. Data are representative of three independent experiments. (G) Indel analysis of genomic DNA from RAWiCE lines. (H) Sequence analysis of the genomic DNA from the RAWiCE lines. https://doi.org/10.4049/immunohorizons.2000082 122 CRISPR METHODS FOR MACROPHAGE AND T CELL ENGINEERING ImmunoHorizons

TABLE I. Summary of oligonucleotide sequences and gRNA targeting sequences Oligonucleotide Sequence Related to Fig. mDUSP1-E1F 59-CGTCTTTGCTTTTGGCTTTGGCGGCGCCAAGCTCGGTAGG-39 1G mDUSP1-E1R 59-GGTGGACTGTTTGCTGCACAGCTCAGGGCAGGAAGCCGAA-39 mDUSP2-E1F 59-CCTGGGAGAAAATGGGAAAAGTGAGGAAGCTGAAGACCTG-39 1G mDUSP2-E1R 59-GACAAGAAAGGGTATGATTAGCCAAGAGGTCGGGAACGGG-39 mDUSP3-E1F 59-GTGTTTGGGGTGAATGAATGGATGCCCACTCTGCTGGTGT-39 1G mDUSP3-E1R 59-GGGTATTTGGATTTCCTGACACGGACAGGAAAAGCTCCAC-39 mDUSP4-E1F 59-CTTGCGTTGGGGGCGGGGCCTGGCGAGGTAGTAATGACTC-39 1G mDUSP4-E1R 59-CCGCCAAGTCCCTTACACTACCTACCCTCCCACCTCGCTC-39 mDUSP5-E1F 59-GTTCCTGACACTCCACCGGTAGTCCGGGGTGATGCGTGAG-39 1G mDUSP5-E1R 59-GACGTGTTGCAAGGACCTCCCAAGCGCACGTCAAACCCCG-39 BGH-R 59-TAGAAGGCACAGTCGAGG-39 1H and S1C hSTAT3-Exon1-F 59-GGAGCTGTGATTATCCCAAGGTGGGGATTGTGAATGTGTT-39 3B and 3F hSTAT3-Exon1-R 59-CCACATCACTGGATTATCAGAATATATAGAGGATCTGCCC-39 hBcl10-E1F 59-TGGCGTCGATAGGCTGCC-39 4B hBcl10-E1R 59-TGGTACAGCCTTCGCTCTAGGCTG-39 Downloaded from hCD3E-F 59-GAGGATTAAATAAGATATATCAGAAAAGCTCTTAGAACGGTGC-39 5E 59-TTGGATATAGTAAGCATTTGATTAATGCATGC-39 hCD3E-R 59-CCATTTTGCCTCATGTGGTGCAGCCATTTAGAGTAC-39 Target Gene gRNA Targeting Sequence Related to Fig.

mDUSP1 59-ATCGTGCGGCGCCGCGCCAA-39 1, 2, S1, and S2 mDUSP2 59-TTGCGATGCCGATCGCCATG-39 http://www.immunohorizons.org/ mDUSP3 59-CCTCGGGACGACCTCGTTGC-39 mDUSP4 59-CGGCGACATGGTGACGATGG-39 mDUSP5 59-ATGAAGGTCACGTCGCTCGA-39 hSTAT3 (gRNA-A) 59-TGGGCCATCCTGCTAAAATC-39 3 hSTAT3 (gRNA-1) 59-GCAGCTTGACACACGGTACC-39 hBCL10 (gRNA-A) 59-GATGGCGCTTCTTCCGGGTC-39 4 hBCL10 (gRNA-B) 59-ATGGCGCTTCTTCCGGGTCC-39 hBCL10 (gRNA-C) 59-GCGGGAGATGGCGCTTCTTC-39 hBCL10 (gRNA-D) 59-TCGGTGAGGGACGGTGCGGT-39 hBCL10 (gRNA-1) 59-CGCACCGTCCCTCACCGAGG-39 hBCL10 (gRNA-2) 59-CACCGCACCGTCCCTCACCG-39 hBCL10 (gRNA-3) 59-GGACCTCACTGAAGTGAAGA-39 by guest on October 2, 2021 hCD3E 59-AGATGCAGTCGGGCACTCAC-39 5 hRelA 59-GACCCCGGCCATGGACGGTG-39 5 hRelA (gRNA-A1) 59-AGCGCCCCTCGCACTTGTAG-39 hPDCD1 (gRNA-1) 59-GACAGAGGCAGTGCTGGGGG-39 6 hPDCD1 (gRNA-2) 59-GGGAGACAGAGGAGATGGGG-39 hPDCD1 (gRNA-3) 59-GGGTGAGGAGGGGGTAGGAC-39 hPDL1 (gRNA-1) 59-CTATACACAGCTTTATTCCT-39 6 hPDL1 (gRNA-2) 59-CTGACCTTCGGTGAAATCGG-39 hPDL1 (gRNA-3) 59-TCAGTTTAGGTATCTAGTGT-39

this inducible expression system. To track single cells that constitu- doxycycline induction at 1.0 mg/ml for 6 d, we analyzed DUSP3 tively express a targeting gRNA with a different set of antibiotic expression by intracellular staining and EGFP+ tBFP+ gating (Fig. selection and live cell fluorescent markers, we developed the 1E), choosing this specific protein for analysis among the set LGB plasmid (Fig. 1D) based on the LentiCRISPRv2 backbone, targeted because of the availability of a well-characterized Ab for which had been optimized to produce high-titer lentiviral single-cell analysis by flow cytometry. We observed substantial particles. For applications that require simultaneous tracking of (.95%) reduction in total DUSP3 protein expression (Fig. 1F) two targeting gRNAs, we also developed the LGR plasmid within the CRISPR-targeted EGFP+ tBFP+ population, indicating (Supplemental Fig. 1B) to offer a different color option and that the majority of targeted cells (Fig. 1E) were indeed complete enhance the overall flexibility of this inducible CRISPR-Cas9 KO of DUSP3 expression. Because specificAbswerenotavailable editing system. for direct single-cell protein level analysis of the other targeted To test this method, we targeted members of the DUSP family DUSP loci, we performed T7EN assays to confirm the presence of in RAW cells. The RAWiCE cells transduced with LGB targeting indel mutations at the targeted DUSP genomic loci (Fig. 1G, Table each chosen DUSP genomic locus were first selected by blasticidin I). Because the T7EN assay does not yield the actual DUSP KO to maximize the gRNA-expressing (tBFP+) population. Following percentage, we performed genomic DNA sequencing to better

https://doi.org/10.4049/immunohorizons.2000082 ImmunoHorizons CRISPR METHODS FOR MACROPHAGE AND T CELL ENGINEERING 123 estimate the CRISPR-editing outcomes of all five DUSP genes. The expression loss in these cells. In addition to expression of Cas9n sequencing results revealed that most if not all targeted DUSP and a pair of gRNAs lying in close proximity, a DNA donor alleles had been mutated at the expected loci surrounding the template is required to induce precise knock-in modification via protospacer adjacent motif (PAM) sites (Fig. 1H, Supplemental the HDR repair mechanism. A STAT3 HDR donor plasmid (Fig. Fig. 1C). Overall, these results strongly suggest that our dual- 3C) was designed with 2000-bp homology arms flanking the color inducible CRISPR strategy confers high genome editing insert fragment containing the PuroR gene, followed by the T2A efficiency in an extremely sensitive, albeit transformed, innate sequence and the mNeonGreen (mNG) fluorescent protein- immune cell type. coding gene. Following cotransfection of PX461-STAT3-gRNA-A, Upon stimulating the various DUSP-KO RAW cells with TLR4 pKLV-BFP-STAT3-gRNA-1, and STAT3 HDR donor plasmids, ligand, we did not observe sustained p38 phosphorylation in DUSP1- ;6 3 105 total (,0.5% cotransfection efficiency) Jurkat T cells KO RAW cells (Supplemental Fig. 2A), an expected phenotype based were selected with puromycin and separated by dilution cloning on a previous study using primary BMDM derived from DUSP1-KO before further validation (Fig. 3D). Based on the low HDR mice (38). Thus, we examined the effects of such a KO in BMDM efficiency for large fragment insertion (,10%), it is technically using the same gRNA used to target DUSP1 in RAW cells. Although challenging to isolate this tiny (,0.05% or ,300 cells) population ffi lentiviruses can transduce nondividing cells, transduction e ciency of successful HDR cell clones without using any selection method. Downloaded from is optimized in cells undergoing active replication. Based on the After puromycin selection, flow cytometric analysis of 40 Ki67 proliferative marker expression profile of bone marrow puromycin-resistant Jurkat cell clones revealed that most of cells cultured with M-CSF (Supplemental Fig. 2B), we de- the selected population had showed mNG expression (Fig. 3E). veloped a lentiviral gRNA transduction protocol (Fig. 2A) to Further genomic PCR analysis of seven clones that express create target gene KO BMDM for further analysis. Unlike RAW medium to high level of mNG showed that six out these seven cells, BMDM are more sensitive to crude lentiviral supernatant clones were indeed the expected knock-in for all STAT3 alleles http://www.immunohorizons.org/ from HEK293T packaging cells seen as a dramatic posttrans- (Fig.3F).Basedontheseresults,weestimatedtheclonal duction decline in the live BMDM percentage (Fig. 2B, top distribution of the parental mNG-STAT3 Jurkat cell line (Fig. panel). To optimize BMDM survival, we further purified and 3G).Thehighpercentage(82.5%)ofpartialorcompletemNG- concentrated lentiviral particles to remove potentially toxic STAT3 knock-in clones suggests that this type of antibiotic materials from the crude supernatant. This approach improved selection strategy efficiently enriches the desired HDR cell clones. the live BMDM percentage as the LGB titration reached a peak We extended studies of this method by targeting the human transduction efficiency (Fig. 2B, bottom panel). Using this improved BCL10 locus to express a fluorescent protein-tagged fusion transduction method for LGB expression in Cas9+ EGFP+ BMDM, protein, using six nickase gRNA pairs (Fig. 4A) and measuring we performed intracellular protein staining and validated the editing the targeting efficiency by T7EN assay (Fig. 4B) as in Fig. 3B. efficiency of the method using a DUSP3 gRNA, again because of Despite the higher indel frequency induced by the D3 gRNA by guest on October 2, 2021 reliable Ab availability (Fig. 2C). Consistent with previous report combination (Fig. 4A, 4B), we selected the A1 gRNA combination using BMDM from DUSP1-KO mice (38), when examining BMDM to minimize potential off-target effects, based on a then-available with the DUSP1 loci targeted in this manner, we observed sustained algorithm (crispr.mit.edu). To enhance cotransfection efficiency, TLR4-induced p38 phosphorylation, as compared with EV and we created a double gRNA expression plasmid (Fig. 4C) based on the DUSP4 controls (Fig. 2D). Increased p38 phosphorylation the PX462 backbone. Furthermore, we attempted to minimize the phenotype at the 80-min timepoint generally correlated with the genome editing footprint by incorporating loxP sites (39) flanking LGB-DUSP1 gRNA expression level, as indicated by tBFP the PuroR-T2A selection cassette in the BCL10 HDR donor plasmid fluorescence in Cas9+ EGFP+ BMDM(Fig.2E).Ourresultssuggest (Fig. 4D). Upon CRE-mediated excision of the loxP-PuroR-T2A- that TLR4-induced p38 is differentially regulated between primary loxP fragment, further genome editing with the same strategy can be BMDM and the transformed RAW macrophages and illustrate the applied because puromycin sensitivity is restored (Fig. 4E). After utility of our method for study of these otherwise hard-to-modify plasmid cotransfection and puromycin selection, most Jurkat cells cells. had acquired mNG-BCL10 expression (Fig. 4F). After NIL-CRE (see Fig. 5A for details) treatment, we observed enhanced mNG-BCL10 An efficient selection strategy for CRISPR-induced HDR expression (Fig. 4G) and a substantially higher frequency of mNG- clones in Jurkat T cells BCL10 aggregate formation upon Ag receptor stimulation, in accord We next turned to development of efficient strategies for editing with previous observations (40). Puromycin retreatment killed the genome of lymphocytes. To minimize off-target effects, we ;99.8% of the NIL-CRE–treated mNG-BCL10 Jurkat cells but did designed a pair of gRNAs for targeting with the Cas9-D10A nickase not affect the survival of the original puromycin-resistant mNG- (Cas9n) strategy, choosing the human STAT3 locus as an exemplar BCL10 line. Finally, based on this NIL-CRE–treated or Puro- (Fig. 3A). This pair of gRNAs together with Cas9n expression in T2A–excised mNG-BCL10 Jurkat parental cell line, we repeated HEK293T cells was confirmed to create indel mutations at the STAT3 genome editing as in Fig. 3 but using a modified STAT3 HDR targeted STAT3 loci, as measured by the T7EN assay (Fig. 3B), donor plasmid (Fig. 4H) in which the mScarlet element replaces the following cotransfection of the PX461 and the pKLV-BFP plasmids. mNG element (Fig. 3C). Following plasmid cotransfection and However, our goal was more ambitious than just achieving gene puromycin selection, more than 75% of mNG-BCL10+ Jurkat cells

https://doi.org/10.4049/immunohorizons.2000082 124 CRISPR METHODS FOR MACROPHAGE AND T CELL ENGINEERING ImmunoHorizons Downloaded from http://www.immunohorizons.org/ by guest on October 2, 2021

FIGURE 2. LGB-mediated CRISPR in BMDM recapitulates reported role for DUSP1 in control of TLR4-p38 signaling. (A) Workflow diagram for generating CRISPR-KO BMDM for TLR4 signaling pathway analysis. (B) Lentiviral titration in BMDM for cell toxicity. (C) DUSP3 protein expression following LGB-DUSP3 transduction of Cas9+ BMDM. (D) Phospho-p38 induced by TLR4 engagement of DUSP1-KO BMDM as compared with the EV and the DUSP4-KO controls. Representative data (left) and AUC pooled from two independent time-course experiments (right); AUC was normalized to the EV control from the same experiment. (E) Examination of TLR4-induced phospho-p38 signaling together with LGB-DUSP1 gRNA expression as indicated by tBFP fluorescence.

gained targeted mScarlet-STAT3 expression (Fig. 4I). With minimal All-in-one nonintegrating lentiviral delivery of the oﬀ-target editing concerns, these results demonstrated a highly CRISPR-HDR blueprint eﬃcient yet sustainable selection method to enrich targeted knock- As an alternative approach to achieve targeted engineering of cells, in cell population from very few recombinant clones created by we sought to deliver Cas9, gRNA, and the HDR donor template to CRISPR-mediated HDR. the entire T cell culture in a transient manner. We therefore turned

https://doi.org/10.4049/immunohorizons.2000082 ImmunoHorizons CRISPR METHODS FOR MACROPHAGE AND T CELL ENGINEERING 125 Downloaded from http://www.immunohorizons.org/ by guest on October 2, 2021

FIGURE 3. In-frame PuroR HDR donor design allows highly specific selection of mNG-STAT3 knock-in Jurkat T cells. (A) Cas9 nickase gRNA pair design targeting human STAT3 locus to induce HDR events near the start codon. (B) T7EN assay for locus-specific editing in HEK293T cells. (C) STAT3 HDR donor plasmid. Start codon and the original second codon are labeled as shown in (A). (D) Strategy for puromycin selection of clonal Jurkat cells with the STAT3 locus-targeted Jurkat T cells. (E) mNG expression by clones of puromycin-resistant mNG- STAT3 Jurkat cells. (F) PCR analysis of the hSTAT3 locus in targeted mNG-STAT3 Jurkat cells. (G) Targeting efficiency in the mNG-STAT3 line. to the use of integrase-deficient lentiviral particles. We packaged 5B) with a gRNA targeting the human CD3E locus (Fig. 5C). the Lenti-CMV-mCherry-P2A-CRE viral vector either with the Seven days after the NIL-CRISPR-Cas9-HDR delivery, we indeed wild-type integrase in the psPAX2 plasmid to produce integrating observed a distinct population of mCherry+ Jurkat cells by flow lentiviral particles or with the integrase-deficientmutantinthe cytometry (Fig. 5D). Using primers that distinguish the CD3E locus psPAX2-D64V plasmid to produce NIL particles. Although the from randomly integrated HDR template sequences, genomic integrating lentiviral transduction created stable mCherry expres- PCR analysis confirmed the presence of targeted knock-in alleles sion in Jurkat T cells, the NIL delivery failed to integrate into the within the mCherry-sorted Jurkat population (Fig. 5E). genome and led to transient mCherry expression (Fig. 5A). To Using NIL delivery of the LentiCRISPRv2S-HDR-tBFP-hRelA test whether NIL delivery of the entire CRISPR-HDR instruc- viral vector (Fig. 5F), we attempted to generate TagBFP-RelA tion can generate targeted knock-in cells, we assembled the fusion knock-in Jurkat cells, employing a gRNA targeting the LentiCRISPRv2B-HDR-mCherry-2A-hCD3E viral vector (Fig. human RelA locus (Fig. 5G). This HDR attempt yielded a distinct

https://doi.org/10.4049/immunohorizons.2000082 126 CRISPR METHODS FOR MACROPHAGE AND T CELL ENGINEERING ImmunoHorizons Downloaded from http://www.immunohorizons.org/ by guest on October 2, 2021

FIGURE 4. CRE-loxP excision strategy minimizes genomic footprint of the PuroR selection cassette and allows for multiple rounds of genome editing. (A) Cas9 nickase gRNA pair design targeting the human BCL10 locus. (B) T7EN assay for locus-specific editing in HEK293T cells. (C) Double gRNAs and Cas9-D10A nickase expression plasmid derived from the PX462 backbone. (D) BCL10 HDR donor plasmid design. (E) Strategy for creating puromycin-selected Jurkat T cells with postselection excision of the Puro-T2A cassette. (F) mNG expression by puromycin-selected mNG-BCL10 knock-in Jurkat line (G) treated with or without NIL-CRE. (H) Modified STAT3 HDR donor plasmid. (I) mNG and mScarlet coexpression by puromycin-selected mScarlet-STAT3/mNG-BCL10 double knock-in Jurkat line.

https://doi.org/10.4049/immunohorizons.2000082 ImmunoHorizons CRISPR METHODS FOR MACROPHAGE AND T CELL ENGINEERING 127 Downloaded from http://www.immunohorizons.org/ by guest on October 2, 2021

FIGURE 5. All-in-one NIL-CRISPR-Cas9-HDR delivery enables targeted genome modifications in human Jurkat T cells. (A) Time course of mCherry-P2A-CRE expression after NIL versus integrating lentiviral transduction of Jurkat T cells. (B) All-in-one lentiviral design for mCherry-P2A-CD3E knock-in via CRISPR-Cas9-HDR. (C) Human CD3E locus showing the gRNA target site used in (B). (D) mCherry expression by Jurkat cells transduced with NIL-LCv2B-HDR-mCherry-2A-hCD3E. Results are representative of two independent experiments. (E) Genomic PCR analysis of the human CD3E locus using primers outside both homology arms. (F) All-in-one lentiviral design for tBFP-RelA knock-in via CRISPR-Cas9-HDR. Start codon and the original second codon are labeled as highlighted in (G). (G) Human RelA locus showing (Continued)

https://doi.org/10.4049/immunohorizons.2000082 128 CRISPR METHODS FOR MACROPHAGE AND T CELL ENGINEERING ImmunoHorizons and stable tBFP+ Jurkat population that was further purified interest, such as macrophages and lymphocytes, because of several by FACS (Fig. 5H). We used live cell confocal time-lapse imaging unique features of these hematopoietic elements. To our to visualize the RelA nuclear translocation response to PMA- knowledge, in this study, we have developed new or modified ionomycin stimulation (Supplemental Video 1). As expected, in the versions of existing reagents or methods to improve such cell steady-state, tBFP-RelA was mostly excluded from the H2B- perturbation, providing new tools that enable more-efficient sfCherry–defined nuclear region. Upon NF-kBpathwayactiva- KO of genes in macrophages, HDR targeting in lymphocytes, or tion, a rapid but transient increase in the nuclear to cytoplasmic gene activation in these cells. ratio of tBFP-RelA was observed in ;85% of the Jurkat population We have encountered substantial challenges in our initial (Fig. 5I). To distinguish targeted genomic knock-in from random attempts to use commonly available CRISPR-KO methods to lentiviral integration, we transduced the original tBFP-RelA engineer the widely-studied RAW macrophage cell line. Using the knock-in Jurkat line with LentiCRISPRv2B (Fig. 5J) carrying LentiCRISPR system, we consistently observed high amounts of RelA gRNA-A1 (Fig. 5K) that can inactivate up to 75% of the cell death following lentiviral transduction and puromycin endogenous RelA expression based on our previous data. After selection, likely because of a combinatorial effect of low viral titer 5 d of blasticidin selection, ;70% of the gated population had lost and cytotoxic materials from the crude lentiviral supernatant. In + ffi tBFPexpression(Fig.5L),indicatingthatmostofthetBFP cells contrast, the high transduction e ciency of our LGB gRNA Downloaded from were indeed created by the HDR mechanism at the targeted expression system ensures that a majority of the transduced RAW human RelA locus and did not result from random genomic cells survive under blasticidin selection, providing targeted cells integration of the tBFP-coding sequence. Taken together, these that are a good representation of the original cell line. Inducible results provide proof-of-concept data to support the all-in-one Cas9 expression allows more-precise control of the genome NIL-Cas9-gRNA–donor-template delivery approach as a powerful editing time window and therefore minimizes long-term off-target alternative to the existing CRISPR-HDR methods in transformed effects that cannot be avoided by constitutive Cas9 expression in http://www.immunohorizons.org/ human T cells. many lentiviral-based CRISPR expression systems. For gRNAs that efficiently KO essential cell survival genes or oncogenes, Selective gene expression induction with all-in-one percentage changes in EGFP+ tBFP+ (Cas9+ gRNA+) cells over lentiviral CRISPRa system doxycycline treatment time help quantify the impacts of these The vectors and methods described above are useful for either gRNAs.Thehighgenomeeditingefficiency and the rapid single-cell eliminating expression of a particular gene or modifying an phenotyping option of our dual-color inducible CRISPR system in already-expressed gene in a given cell type, but there are numerous RAW cells makes this platform suitable for high-throughput situations in which it would be useful to induce expression of an genome-wide gRNA library screening. Inducible CRISPR system otherwise silent locus in a specific cell type. To achieve this end, we also allows for stable uninduced cell lines to be frozen for long-term created an all-in-one lentiviral CRISPRa system that we call storage and future repeat analysis. by guest on October 2, 2021 LentiSAMPHv2 (Fig. 6A), which is based on an existing two- Lentiviral or retroviral supernatant produced from HEK293T plasmid system (29). LentiSAMPHv2 also contains blasticidin packaging cells often influences target cell growth rate and resistance marker for efficient selection of transduced cells. With morphology, especially when a high-volume ratio of viral super- this system, we tested six gRNAs from an existing CRISPRa library natant to cell culture medium is applied to the target cell line. Our (29) targeting the promoter region of the human PDCD1 or PDL1 data illustrate that further purification of lentiviral particles helps genomic locus. These gRNAs induced various levels of target minimize BMDM cell death. Thus, our methods achieve high gene expression (Fig. 6B), as compared with the corresponding transduction efficiency without sacrificing cell health in culture. no gRNA control condition. For the gRNA that induces the highest With these approaches, we were able to identify Cas9 and gRNA level of PDCD1 expression, we also tested the specificity of this double-positive macrophages from both the RAW murine cell line induction by measuring the cell surface expression level of PDL1, and primary BMDM, allowing us to focus on those CRISPR- which was not increased under these conditions (Fig. 6C). targeted populations for downstream analysis. Interestingly, these studies revealed distinct p38-MAPK regulation in immortalized versus primary macrophages. CRISPR deletion of the phosphatase DISCUSSION DUSP1 in BMDM resulted in sustained p38 phosphorylation, consistent with a previous report using BMDM derived from Immunologists have had more difficulty than investigators in other DUSP1-deficient mice (38). In contrast, DUSP1 deletion in RAW areas of biology using CRISPR in attempts to modify cells of cells, using the same gRNA, did not lead to substantial changes in

the target site of gRNA used in (F). (H) tBFP expression of Jurkat cells untreated or treatedwithLCv2S-HDR-tBFP-hRelA,beforeoraftertBFP+ cell sorting. Results are representative of two independent experiments. (I) Live cell confocal time-lapse imaging of tBFP-RelA knock-in Jurkat cells stimulated with PMA-ionomycin. Pre-existing H2B-sfCherry expression created by lentiviral transduction was used as nuclear marker. (J) Modified LentiCRISPRv2 plasmid LentiCRISPRv2B replacing PuroR with blasticidin resistance gene BSD. (K) Human RelA locus showing the target site of gRNA A1 near exon 3. (L)tBFP expression by the Jurkat tBFP-RelA knock-in line either untreated or transduced with LCv2B carrying RelA gRNA-A1 to KO endogenous RelA expression.

https://doi.org/10.4049/immunohorizons.2000082 ImmunoHorizons CRISPR METHODS FOR MACROPHAGE AND T CELL ENGINEERING 129 Downloaded from http://www.immunohorizons.org/ by guest on October 2, 2021

FIGURE 6. Lentiviral-mediated CRISPRa with the LentiSAMPHv2 System. (A) The all-in-one LentiSAMPHv2 (LSv2) plasmid for CRISPR-based gene activation applications. (B) Flow cytometric analysis of human PDCD1 (top panel) or PDL1 (bottom panel) cell surface protein expression after LSv2 transduction with EV control or the indicated CRISPRa targeting gRNA. (C) Flow cytometric analysis of human PDCD1 (left panel) or PDL1 (right panel) cell surface protein expression in untransduced control cells versus cells transduced with LSv2 targeting the human PDCD1 promoter region. p38 activation dynamics. This adds to our previous report of The Cas9 nickase strategy requires two gRNA targets to be in dysregulated TLR4-induced MAPK activity in RAW cells com- close proximity to eﬃciently induce DNA double-strand break pared with BMDM (41). repair mechanisms (8, 9). Therefore, it is extremely unlikely to ﬁnd

https://doi.org/10.4049/immunohorizons.2000082 130 CRISPR METHODS FOR MACROPHAGE AND T CELL ENGINEERING ImmunoHorizons another off-target pairing site in the genome. Our data support chemicals such as RS-1 (25), and cell cycle inhibitors for G2/M previous reports that the pair of Cas9 nickases is optimized with phase arrest (44) could be tested to enhance knock-in efficiency. minimized offset of the gRNA pairs (8, 9). By omitting an upstream Furthermore, eSpCas9 (14) or more-specific Cas9 variant could promoter in the donor template design, our PuroR selection be used instead of Cas9 nuclease to minimize off-target effects. strategy reduces the probability of selecting randomly integrated Finally, the use of both NIL and AAV systems could be combined clones. As a potential drawback, this strategy relies on targeting to deliver few copies of NIL-Cas9-gRNA particles but high copy genomic loci that are well expressed to generate sufficient expres- number of AAV-HDR donor template (26) per target cell to sion of the drug resistance gene. Among the target genes that we maximize HDR efficiency and donor template accuracy. have tested, we observed that this strategy worked best with Although inactivation or mutation of a target gene is the the N-terminal PuroR-2A but not the C-terminal 2A-PuroR predominant focus of CRISPR editing to date, the selective fusion expression. When combined with a CRE-lox or FLP-FRT induction of expression of a particular molecule using this method recombination design, our PuroR selection strategy can be further has many applications. To this end we also introduce an all-in-one optimized to sustain many rounds of genome editing, enabling vector system for activation of selected gene loci in immune cells. sequential engineering of multiplex reporters, significantly re- Using a single lentiviral vector containing gRNA that targets either

ducing the genomic footprint to a short single recombination site. the human PDCD1 or PDL1 genomic locus, we demonstrate high- Downloaded from Following the NIL-mediated mCherry-P2A-CRE or mScarlet- level expression of a previously silent locus in Jurkat cells and the P2A-CRE (Supplemental Fig. 3D) delivery, target cells that value of this induced expression in assays of immune function. transiently express CRE recombinase can be isolated from the This strategy builds on pre-existing methods for large-scale gene untreated population via FACS. For relatively small proteins such induction (45) by providing experimentalists with a simple method as BCL10, the protein expression level is dramatically reduced by for selective, one-vector-at-a-time alteration of cell phenotype in a the genomic insertion of a PuroR selection cassette together with a positive rather than negative manner. http://www.immunohorizons.org/ fluorescent protein. Our data suggest that reduced BCL10 protein Insummary,toourknowledge,thisreportprovidesasetof expression can lead to the loss of BCL10 oligomerization upon Ag tools and methods for immunologists seeking to engineer hemato- receptor signaling, which justifies the need to remove PuroR poietic cell types such as macrophages and lymphocytes that are selection cassettes from the targeted genomic loci to achieve more more difficult to modify using CRISPR methods than the epithelial functional BCL10 reporter Jurkat T cells. or mesenchymal cells studied in other fields. Our improvements to Lentiviruses such as HIV have evolved to efficiently infect available vectors and methods enhance the ease and efficiency of human T cells, making them perfect delivery vehicles for human achieving targeted gene inactivation, selective gene editing, and T cell genome engineering. In Jurkat cells, lentiviral delivery is precisegeneactivationandshouldprovevaluableinawiderangeof much more efficient than any existing transfection method investigations. including electroporation, which often leads to substantial cell by guest on October 2, 2021 death upon electric shock and reduces cell growth and overall DISCLOSURES protein production for the surviving population. In addition, lentiviral particles are easy to prepare by most laboratories, carry The authors have no financial conflicts of interest. double to triple the payload as compared with the AAV system, and therefore enable all-in-one design and delivery of Cas9, gRNA expression, and HDR donor template together. Although there is ACKNOWLEDGMENTS an existing report (42) that demonstrates the feasibility of NIL delivery of the HDR donor template, in this study, we demonstrate We thank Clinton Bradfield and Iain Fraser for critical reading of the the utility of this method using an all-in-one Cas9-gRNA–donor manuscript. We thank Cindi Pfannkoch, Bin Lin, Caleb Ng, and Sebastian HDR blueprint in an especially difficult-to-engineer type of cell, Montalvo for helpful discussions and advice. T lymphocytes. Our experiments using human primary T cells showed that the NIL-LCv2R (Supplemental Fig. 3B) particles REFERENCES efficiently delivered a payload of over 10,000 bp to more than 80% of the total population (Supplemental Fig. 3E). 1. Ishino, Y., H. Shinagawa, K. Makino, M. Amemura, and A. Nakata. As one of the first studies, to our knowledge, to report the use 1987. Nucleotide sequence of the iap gene, responsible for alkaline fi of NIL as delivery vehicle for CRISPR-HDR applications, we sug- phosphatase isozyme conversion in Escherichia coli, and identi cation of the gene product. J. Bacteriol. 169: 5429–5433. gest many optimization possibilities for this method. First of all, 2. Jinek, M., A. East, A. Cheng, S. Lin, E. Ma, and J. Doudna. 2013. RNA- sensitive marker expression, such as mScarlet in the LCv2R back- programmed genome editing in human cells. Elife 2: e00471. bone (Supplemental Fig. 3B) or tEGFR in the LCv2E backbone 3. Cong, L., F. A. Ran, D. Cox, S. Lin, R. Barretto, N. Habib, P. D. Hsu, (Supplemental Fig. 3C), could be included to eliminate residual X.Wu,W.Jiang,L.A.Marraffini, and F. Zhang. 2013. Multiplex random integration events from NIL particles. Viral titers could genome engineering using CRISPR/Cas systems. Science 339: 819–823. be enhanced by incorporating two copies of the Sp1 binding site 4. Mali, P., L. Yang, K. M. Esvelt, J. Aach, M. Guell, J. E. DiCarlo, upstream of the U6 promoter (32). In addition, various NHEJ- J. E. Norville, and G. M. Church. 2013. RNA-guided human genome inhibiting chemicals such as SCR7 (43), HDR-stimulating engineering via Cas9. Science 339: 823–826.

https://doi.org/10.4049/immunohorizons.2000082 ImmunoHorizons CRISPR METHODS FOR MACROPHAGE AND T CELL ENGINEERING 131

5. Kuscu, C., S. Arslan, R. Singh, J. Thorpe, and M. Adli. 2014. Genome- 22. Wang, T., J. J. Wei, D. M. Sabatini, and E. S. Lander. 2014. Genetic wide analysis reveals characteristics of off-target sites bound by the screens in human cells using the CRISPR-Cas9 system. Science 343: Cas9 endonuclease. Nat. Biotechnol. 32: 677–683. 80–84. 6. Tsai, S. Q., Z. Zheng, N. T. Nguyen, M. Liebers, V. V. Topkar, 23. Yang, H., H. Wang, and R. Jaenisch. 2014. Generating genetically V. Thapar, N. Wyvekens, C. Khayter, A. J. Iafrate, L. P. Le, et al. 2015. modified mice using CRISPR/Cas-mediated genome engineering. Nat. GUIDE-seq enables genome-wide profiling of off-target cleavage by Protoc. 9: 1956–1968. CRISPR-Cas nucleases. Nat. Biotechnol. 33: 187–197. 24.Maruyama,T.,S.K.Dougan,M.C.Truttmann,A.M.Bilate,J.R.Ingram, 7. Wang, X., Y. Wang, X. Wu, J. Wang, Y. Wang, Z. Qiu, T. Chang, andH.L.Ploegh.2015.Increasingtheefficiency of precise genome H. Huang, R.-J. Lin, and J.-K. Yee. 2015. Unbiased detection of off- editing with CRISPR-Cas9 by inhibition of nonhomologous end joining. target cleavage by CRISPR-Cas9 and TALENs using integrase- [Published erratum appears in 2016 Nat. Biotechnol. 34: 210.] Nat. Bio- defective lentiviral vectors. Nat. Biotechnol. 33: 175–178. technol. 33: 538–542. 8. Ran, F. A., P. D. Hsu, C. Y. Lin, J. S. Gootenberg, S. Konermann, 25. Song, J., D. Yang, J. Xu, T. Zhu, Y. E. Chen, and J. Zhang. 2016. RS-1 A. E. Trevino, D. A. Scott, A. Inoue, S. Matoba, Y. Zhang, and F. Zhang. enhances CRISPR/Cas9- and TALEN-mediated knock-in efficiency. 2013. Double nicking by RNA-guided CRISPR Cas9 for enhanced ge- Nat. Commun. 7: 10548. nome editing specificity. [Published erratum appears in 2013 Cell 155: 26. Eyquem, J., J. Mansilla-Soto, T. Giavridis, S. J. C. van der Stegen, 479–480.] Cell 154: 1380–1389. M. Hamieh, K. M. Cunanan, A. Odak, M. Gonen,¨ and M. Sadelain. 9. Shen, B., W. Zhang, J. Zhang, J. Zhou, J. Wang, L. Chen, L. Wang, 2017. Targeting a CAR to the TRAC locus with CRISPR/Cas9 enhances A. Hodgkins, V. Iyer, X. Huang, and W. C. Skarnes. 2014. Efficient tumour rejection. Nature 543: 113–117. Downloaded from genome modification by CRISPR-Cas9 nickase with minimal off- 27.Maeder,M.L.,S.J.Linder,V.M.Cascio,Y.Fu,Q.H.Ho,andJ.K.Joung. target effects. Nat. Methods 11: 399–402. 2013. CRISPR RNA-guided activation of endogenous human genes. Nat. 10. Fu, Y., J. D. Sander, D. Reyon, V. M. Cascio, and J. K. Joung. 2014. Methods 10: 977–979. Improving CRISPR-Cas nuclease specificity using truncated guide 28.Konermann,S.,M.D.Brigham,A.E.Trevino,J.Joung,O.O.Abudayyeh, RNAs. Nat. Biotechnol. 32: 279–284. C. Barcena, P. D. Hsu, N. Habib, J. S. Gootenberg, H. Nishimasu, et al.

11. Guilinger, J. P., D. B. Thompson, and D. R. Liu. 2014. Fusion of cat- 2015. Genome-scale transcriptional activation by an engineered CRISPR- http://www.immunohorizons.org/ alytically inactive Cas9 to FokI nuclease improves the specificity of Cas9 complex. Nature 517: 583–588. genome modification. Nat. Biotechnol. 32: 577–582. 29.Joung,J.,S.Konermann,J.S.Gootenberg,O.O.Abudayyeh,R.J.Platt, 12. Komor, A. C., Y. B. Kim, M. S. Packer, J. A. Zuris, and D. R. Liu. 2016. M. D. Brigham, N. E. Sanjana, and F. Zhang. 2017. Genome-scale Programmable editing of a target base in genomic DNA without CRISPR-Cas9 knockout and transcriptional activation screening. double-stranded DNA cleavage. Nature 533: 420–424. [Published erratum appears in 2019 Nat. Protoc. 14: 2259.] Nat. Protoc. 13. Pawluk, A., N. Amrani, Y. Zhang, B. Garcia, Y. Hidalgo-Reyes, J. Lee, 12: 828–863. A. Edraki, M. Shah, E. J. Sontheimer, K. L. Maxwell, and A. R. 30.Certo,M.T.,B.Y.Ryu,J.E.Annis,M.Garibov,J.Jarjour,D.J.Rawlings, Davidson. 2016. Naturally occurring off-switches for CRISPR-Cas9. and A. M. Scharenberg. 2011. Tracking genome engineering outcome at Cell 167: 1829–1838.e9. individual DNA breakpoints. Nat. Methods 8: 671–676. 14. Chen, J. S., Y. S. Dagdas, B. P. Kleinstiver, M. M. Welch, A. A. Sousa, 31. Hu, J., S. Schokrpur, M. Archang, K. Hermann, A. C. Sharrow, L.B.Harrington,S.H.Sternberg,J.K.Joung,A.Yildiz,and P. Khanna, J. Novak, S. Signoretti, R. S. Bhatt, B. S. Knudsen, et al.

J. A. Doudna. 2017. Enhanced proofreading governs CRISPR-Cas9 2018. A non-integrating lentiviral approach overcomes Cas9-induced by guest on October 2, 2021 targeting accuracy. Nature 550: 407–410. immune rejection to establish an immunocompetent metastatic renal 15. Harrington, L. B., K. W. Doxzen, E. Ma, J. J. Liu, G. J. Knott, cancer model. Mol. Ther. Methods Clin. Dev. 9: 203–210. A. Edraki, B. Garcia, N. Amrani, J. S. Chen, J. C. Cofsky, et al. 2017. A 32. Ortinski, P. I., B. O’Donovan, X. Dong, and B. Kantor. 2017. Integrase- broad-spectrum inhibitor of CRISPR-Cas9. Cell 170: 1224–1233.e15. deficient lentiviral vector as an all-in-one platform for highly efficient 16. Ren, J., X. Liu, C. Fang, S. Jiang, C. H. June, and Y. Zhao. 2017. CRISPR/Cas9-mediated gene editing. Mol. Ther. Methods Clin. Dev. 5: Multiplex genome editing to generate universal CAR T cells resistant 153–164. to PD1 inhibition. Clin. Cancer Res. 23: 2255–2266. 33. Sanjana, N. E., O. Shalem, and F. Zhang. 2014. Improved vectors and 17. Ferri, F., A. Parcelier, V. Petit, A.-S. Gallouet, D. Lewandowski, genome-wide libraries for CRISPR screening. Nat. Methods 11: M. Dalloz, A. van den Heuvel, P. Kolovos, E. Soler, M. L. Squadrito, 783–784. et al. 2015. TRIM33 switches off Ifnb1 gene transcription during the 34.Papapetrou,E.P.,G.Lee,N.Malani,M.Setty,I.Riviere,L.M.S.Tirunagari, late phase of macrophage activation. Nat. Commun. 6: 8900. K.Kadota,S.L.Roth,P.Giardina,A.Viale,etal.2011.Genomicsafeharbors 18. Tang, S., T. Chen, Z. Yu, X. Zhu, M. Yang, B. Xie, N. Li, X. Cao, and permit high b-globin transgene expression in thalassemia induced plurip- J. Wang. 2014. RasGRP3 limits toll-like receptor-triggered inflam- otent stem cells. Nat. Biotechnol. 29: 73–78. matory response in macrophages by activating Rap1 small GTPase. 35. Ran, F. A., P. D. Hsu, J. Wright, V. Agarwala, D. A. Scott, and Nat. Commun. 5: 4657. F. Zhang. 2013. Genome engineering using the CRISPR-Cas9 system. 19. Jing, W., X. Zhang, W. Sun, X. Hou, Z. Yao, and Y. Zhu. 2015. Nat. Protoc. 8: 2281–2308. CRISPR/CAS9-mediated genome editing of miRNA-155 inhibits 36. Koike-Yusa, H., Y. Li, E.-P. Tan, M. C. Velasco-Herrera, and K. Yusa. proinflammatory cytokine production by RAW264.7 cells. Biomed Res. 2014. Genome-wide recessive genetic screening in mammalian cells Int. 2015: 326042. with a lentiviral CRISPR-guide RNA library. Nat. Biotechnol. 32: 20. Li, X., M. Yang, Z. Yu, S. Tang, L. Wang, X. Cao, and T. Chen. 2017. 267–273. The tyrosine kinase Src promotes phosphorylation of the kinase TBK1 37. Chan, W., T. B. Schaffer, and J. L. Pomerantz. 2013. A quantitative to facilitate type I interferon production after viral infection. Sci. signaling screen identifies CARD11 mutations in the CARD and Signal. 10: eaae0435. LATCH domains that induce Bcl10 ubiquitination and human lym- 21.Napier,B.A.,S.W.Brubaker,T.E.Sweeney,P.Monette,G.H.Rothmeier, phoma cell survival. Mol. Cell. Biol. 33: 429–443. N. A. Gertsvolf, A. Puschnik, J. E. Carette, P. Khatri, and D. M. 38.Zhao,Q.,X.Wang,L.D.Nelin,Y.Yao,R.Matta,M.E.Manson,R.S.Baliga, Monack. 2016. Complement pathway amplifies caspase-11-dependent X. Meng, C. V. Smith, J. A. Bauer, et al. 2006. MAP kinase phos- cell death and endotoxin-induced sepsis severity. J. Exp. Med. 213: phatase 1 controls innate immune responses and suppresses endotoxic 2365–2382. shock. J. Exp. Med. 203: 131–140.

https://doi.org/10.4049/immunohorizons.2000082 132 CRISPR METHODS FOR MACROPHAGE AND T CELL ENGINEERING ImmunoHorizons

39. Celis-Gutierrez, J., P. Blattmann, Y. Zhai, N. Jarmuzynski, K. Ruminski, through homology-directed repair in human embryonic stem cells. C. Grégoire, Y. Ounoughene, F. Fiore, R. Aebersold, R. Roncagalli, et al. Nucleic Acids Res. 45: e29. 2019. Quantitative interactomics in primary T cells provides a rationale 43. Chu, V. T., T. Weber, B. Wefers, W. Wurst, S. Sander, K. Rajewsky, for concomitant PD-1 and BTLA coinhibitor blockade in cancer immu- and R. Kuhn.¨ 2015. Increasing the efficiency of homology-directed notherapy. Cell Rep. 27: 3315–3330.e7. repair for CRISPR-Cas9-induced precise gene editing in mammalian 40. Paul, S., A. K. Kashyap, W. Jia, Y. W. He, and B. C. Schaefer. 2012. cells. [Published erratum appears in 2018 Nat. Biotechnol. 36: 196.] – Selective autophagy of the adaptor protein Bcl10 modulates T cell Nat. Biotechnol. 33: 543 548. receptor activation of NF-kB. Immunity 36: 947–958. 44. Yang, D., M. A. Scavuzzo, J. Chmielowiec, R. Sharp, A. Bajic, and M. Borowiak. 2016. Enrichment of G2/M cell cycle phase in human 41. Gottschalk, R. A., A. J. Martins, B. R. Angermann, B. Dutta, C. E. Ng, pluripotent stem cells enhances HDR-mediated gene repair with S. Uderhardt, J. S. Tsang, I. D. C. Fraser, M. Meier-Schellersheim, and customizable endonucleases. Sci. Rep. 6: 21264. k R. N. Germain. 2016. Distinct NF- B and MAPK activation thresholds 45.Simeonov,D.R.,B.G.Gowen,M.Boontanrart,T.L.Roth,J.D.Gagnon, uncouple steady-state microbe sensing from anti-pathogen in- M.R.Mumbach,A.T.Satpathy,Y.Lee,N.L.Bray,A.Y.Chan,etal.2017. fl – ammatory responses. Cell Syst. 2: 378 390. Discovery of stimulation-responsive immune enhancers with CRISPR 42. Wang, Y., Y. Wang, T. Chang, H. Huang, and J.-K. Yee. 2017. activation. [Published erratum appears in 2018 Nature 559: E13.] Nature Integration-defective lentiviral vector mediates efficient gene editing 549: 111–115. Downloaded from http://www.immunohorizons.org/ by guest on October 2, 2021

https://doi.org/10.4049/immunohorizons.2000082

Supplemental Figure 1. Inducible genome editing system in RAW264.7 macrophages. (A) RAW cells transduced with pCW-Cas9 were treated with titrating doses of doxycycline. Cell lysates were analyzed by western blot with anti-FLAG antibody. (B) Lentiviral vector for gRNA expression with TagRFP-T fluorescent marker and blasticidin resistance gene BSD. (C) Sequence analysis of the genomic DNA from the RAWiCE lines.

Supplemental Figure 2. DUSP1-KO RAW cell phenotype and Ki67 profile of B6 bone marrow. (A) After blasticidin selection and doxycycline induction, RAWiCE cells transduced with the indicated LGB variant were stimulated with TLR4 ligand and mean phospho-p38 was quantified over time. Representative data (left) and AUC pooled from two independent experiments (right), normalized to the appropriate EV control. (B) Ki67 staining profile of C57BL/6 mouse bone marrow cells after 30 ng/mL M-CSF treatment.

Supplemental Figure 3. LES2A-CRE, LentiCRISPRv2 variants LCv2S, LCv2R and LCv2E, and lentiviral transduction efficiency in human PBMC. (A) The LentiCRISPRv2S plasmid offers reduced lentiviral vector size and flexible cloning of the marker of interest. (B) The LentiCRISPRv2R plasmid contains mScarlet-I for sensitive detection of Cas9 expression. (C) The LentiCRISPRv2E plasmid contains the tEGFR cell surface marker to report Cas9 expression. (D) The Lenti-EF1a-mScarlet-2A-CRE plasmid allows sensitive reporting of CRE recombinase expression via the mScarlet-I fluorescent marker. (E) mScarlet expression by human PBMC transduced with LCv2R or NIL-LCv2R.

Video 1. Live cell confocal time-lapse imaging data of tBFP-RelA translocations following

PMA-ionomycin stimulation in human Jurkat T cells. PMA-ionomycin activates the NF-kB pathway and induces nuclear translocations of tBFP-RelA.