Generation of Knock-In Primary Human T Cells Using Cas9 Ribonucleoproteins
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Generation of knock-in primary human T cells using Cas9 ribonucleoproteins Kathrin Schumanna,b,1, Steven Linc,1, Eric Boyera,b, Dimitre R. Simeonova,b,d, Meena Subramaniame,f, Rachel E. Gatee,f, Genevieve E. Haliburtona,b, Chun J. Yee, Jeffrey A. Bluestonea, Jennifer A. Doudnac,g,h,i,j,2, and Alexander Marsona,b,g,2 aDiabetes Center, University of California, San Francisco, CA 94143; bDivision of Infectious Diseases, Department of Medicine, University of California, San Francisco, CA 94143; cDepartment of Molecular and Cell Biology, University of California, Berkeley, CA 94720; dBiomedical Sciences Graduate Program, University of California, San Francisco, CA 94143; eDepartment of Epidemiology and Biostatistics, Department of Bioengineering and Therapeutic Sciences, Institute for Human Genetics, University of California, San Francisco, CA 94143; fBiological and Medical Informatics Graduate Program, University of California, San Francisco, CA 94158; gInnovative Genomics Initiative, University of California, Berkeley, CA 94720; hHoward Hughes Medical Institute, University of California, Berkeley, CA 94720; iDepartment of Chemistry, University of California, Berkeley, CA 94720; and jPhysical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720 Contributed by Jennifer A. Doudna, June 29, 2015 (sent for review January 23, 2015) T-cell genome engineering holds great promise for cell-based therapies with specific replacement sequence, rather than deleted (14). Effi- for cancer, HIV, primary immune deficiencies, and autoimmune dis- cient technology to promote homologous recombination in T cells eases, but genetic manipulation of human T cells has been challenging. could eventually allow therapeutic correction of mutations that affect Improved tools are needed to efficiently “knock out” genes and “knock specialized T-cell functions. in” targeted genome modifications to modulate T-cell function and Recent reports in mammalian cell lines demonstrate that correct disease-associated mutations. CRISPR/Cas9 technology is facili- Cas9 ribonucleoproteins (RNPs; recombinant Cas9 protein tating genome engineering in many cell types, but in human T cells its complexed with an in vitro-transcribed single-guide RNA) can efficiency has been limited and it has not yet proven useful for targeted accomplish efficient and specific genome editing (15–17). Here nucleotide replacements. Here we report efficient genome engineering we show that electroporation of Cas9 RNPs leads to efficient + + in human CD4 T cells using Cas9:single-guide RNA ribonucleoproteins genome editing of CD4 Tcells.Wewereabletoablatea (Cas9RNPs).Cas9RNPsallowedablation of CXCR4, a coreceptor for HIV target gene with the random insertion and deletion mutations ∼ entry. Cas9 RNP electroporation caused up to 40% of cells to lose that likely result from nonhomologous end joining (NHEJ) high-level cell-surface expression of CXCR4, and edited cells could be repair of a Cas9-induced double-stranded DNA break (DSB). enriched by sorting based on low CXCR4 expression. Importantly, Cas9 Cells with genomic edits in CXCR4 could be enriched by sorting RNPs paired with homology-directed repair template oligonucleotides basedonlowCXCR4expression.Wewerealsoabletoin- generated a high frequency of targeted genome modifications in pri- troduce precisely targeted nucleotide replacements in primary mary T cells. Targeted nucleotide replacement was achieved in CXCR4 T cells at CXCR4 and PD-1 by homology-directed repair and PD-1 (PDCD1), a regulator of T-cell exhaustion that is a validated target for tumor immunotherapy. Deep sequencing of a target site (HDR) using Cas9 RNPs and exogenous single-stranded DNA templates. This technology enabled Cas9-mediated generation confirmed that Cas9 RNPs generated knock-in genome modifications “ ” with up to ∼20% efficiency, which accounted for up to approximately of knock-in primary human T cells. Deep sequencing of a one-third of total editing events. These results establish Cas9 RNP tech- nology for diverse experimental and therapeutic genome engineering Significance applications in primary human T cells. T-cell genome engineering holds great promise for cancer immu- CRISPR/Cas9 | genome engineering | Cas9 ribonucleoprotein | RNP | notherapies and cell-based therapies for HIV, primary immune primary human T cells deficiencies, and autoimmune diseases, but genetic manipulation of human T cells has been inefficient. We achieved efficient ge- he CRISPR/Cas9 system has been used increasingly to edit nome editing by delivering Cas9 protein pre-assembled with Tmammalian germline sequence and cell lines (1, 2). Considerable guide RNAs. These active Cas9 ribonucleoproteins (RNPs) enabled efforts are underway to use this powerful system directly in primary successful Cas9-mediated homology-directed repair in primary human tissues, but efficiency has been limited, especially in human human T cells. Cas9 RNPs provideaprogrammabletooltoreplace + CD4 T cells. Plasmid delivery of cas9 and single-guide RNAs specific nucleotide sequences in the genome of mature immune — (sgRNAs) was efficient in other cell types, but ablated only 1–5% of cells a longstanding goal in the field. These studies establish + target protein expression in CD4 T cells (3). Improved ability to Cas9 RNP technology for diverse experimental and therapeutic ablate key targets and correct pathogenic genome sequence in human genome engineering applications in primary human T cells. T cells would have direct therapeutic applications, eventually allowing Author contributions: K.S., S.L., J.A.D., and A.M. designed research; K.S., S.L., E.B., and D.R.S. T cells to be edited ex vivo and then reintroduced into patients. performed research; K.S., S.L., E.B., D.R.S., M.S., R.E.G., G.E.H., C.J.Y., J.A.B., J.A.D., and A.M. Multiple scientific and clinical trials are underway to manipulate analyzed data; and K.S., S.L., and A.M. wrote the paper. T-cell genomes with available technologies, including gene de- Conflict of interest statement: J.A.D. is a co-founder of Caribou Biosciences Inc., Editas letions with transcription activator-like effector nucleases and zinc Medicine, and Intellia and is on the scientific advisory board of Caribou Biosciences Inc. finger nucleases and exogenous gene introduction by viral trans- The A.M. laboratory receives sponsored research support from Epinomics. A patent has been filed based on the findings described here. duction (4, 5). Genetic manipulations have been attempted to “knock out” HIV coreceptors CXCR4 and CCR5 in T cells to gain Freely available online through the PNAS open access option. resistance to HIV infection (6–8). There also has been marked Data deposition: The sequence reported in this paper has been deposited in the National Center for Biotechnology Information (NCBI) BioSample database, www.ncbi.nlm.nih.gov/ success in engineering T cells to recognize and kill hematological biosample/ (accession no. SAMN03850749). malignancies, but additional genetic modifications appear necessary 1K.S. and S.L. contributed equally to this work. – for solid organ tumor immunotherapy (9 11). Deletion of genes 2To whom correspondence may be addressed. Email: [email protected] or that encode key immune checkpoints such as PD-1 could prove [email protected]. useful for these efforts (12, 13). Further therapeutic opportunities This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. INFLAMMATION IMMUNOLOGY AND would be possible if targeted T-cell genomic loci could be corrected 1073/pnas.1512503112/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1512503112 PNAS | August 18, 2015 | vol. 112 | no. 33 | 10437–10442 Downloaded by guest on September 29, 2021 target site confirmed that Cas9 RNPs promoted knock-in the CXCR4 gene were likely repaired by NHEJ, a predominant genome modifications with up to ∼20% efficiency (∼22% was DNA repair pathway in cells that gives rise to variable insertions achieved with 50 pmol and ∼18% with 100 pmol of HDR template), and deletions (indels) and often results in frameshift mutations and which accounted for up to approximately one-third of the total loss of gene function (22). Flow cytometry revealed a Cas9 RNP editing events. These findings suggest that Cas9 RNP-mediated nu- dose-dependent increase in the percentage of T cells expressing low cleotide replacement could eventually prove useful for therapeutic levels of CXCR4, consistent with mutation of the CXCR4 gene correction of disease-associated mutations. Our study establishes (Fig. 1C). The T7 endonuclease I (T7E1) assay is a convenient Cas9 RNP technology for experimental and therapeutic knock-out method to assess editing at specific sites in the genome. Here, T7E1 and knock-in editing of the genome in primary human T cells. assay confirmed genomic DNA editing at the CXCR4 locus in cells treated with CXCR4 Cas9 RNPs, but not in control cells treated Results with Cas9 protein alone (no sgRNA; CTRL). Cas9 RNP-treated We aimed to overcome long-standing challenges in genetic ma- cells were separated based on CXCR4 expression with fluorescence- nipulation of primary T cells and establish an efficient genome activated cell sorting (FACS). Using the T7E1 assay, we found engineering toolkit. Recent reports in mammalian cell lines an enrichment of editing in the CXCR4lo cells (15–17%) suggest that Cas9 RNPs can accomplish efficient and specific compared with CXCR4hi cells (4–12% with varying doses of Cas9 genome editing (15–18). Given