title: Generation of knock-in primary human T cells using Cas9 ribonucleoproteins.
abstract: T-cell genome engineering holds great promise for cell-based therapies for cancer, HIV, primary immune deficiencies, and autoimmune diseases, but genetic manipulation of human T cells has been challenging. Improved tools are needed to efficiently "knock out" genes and "knock in" targeted genome modifications to modulate T-cell function and correct disease-associated mutations. CRISPR/Cas9 technology is facilitating genome engineering in many cell types, but in human T cells its efficiency has been limited and it has not yet proven useful for targeted nucleotide replacements. Here we report efficient genome engineering in human CD4(+) T cells using Cas9:single-guide RNA ribonucleoproteins (Cas9 RNPs). Cas9 RNPs allowed ablation of CXCR4, a coreceptor for HIV entry. Cas9 RNP electroporation caused up to ∼40% of cells to lose high-level cell-surface expression of CXCR4, and edited cells could be enriched by sorting based on low CXCR4 expression. Importantly, Cas9 RNPs paired with homology-directed repair template oligonucleotides generated a high frequency of targeted genome modifications in primary T cells. Targeted nucleotide replacement was achieved in CXCR4 and PD-1 (PDCD1), a regulator of T-cell exhaustion that is a validated target for tumor immunotherapy. Deep sequencing of a target site confirmed that Cas9 RNPs generated knock-in genome modifications with up to ∼20% efficiency, which accounted for up to approximately one-third of total editing events. These results establish Cas9 RNP technology for diverse experimental and therapeutic genome engineering applications in primary human T cells.
authors.affiliation: Diabetes Center, University of California, San Francisco, CA 94143; Division of Infectious Diseases, Department of Medicine, University of California, San Francisco, CA 94143;
authors.forename: Kathrin
authors.initials: K
authors.lastname: Schumann
authors.affiliation: Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720;
authors.forename: Steven
authors.initials: S
authors.lastname: Lin
authors.affiliation: Diabetes Center, University of California, San Francisco, CA 94143; Division of Infectious Diseases, Department of Medicine, University of California, San Francisco, CA 94143;
authors.forename: Eric
authors.initials: E
authors.lastname: Boyer
authors.affiliation: Diabetes Center, University of California, San Francisco, CA 94143; Division of Infectious Diseases, Department of Medicine, University of California, San Francisco, CA 94143; Biomedical Sciences Graduate Program, University of California, San Francisco, CA 94143;
authors.forename: Dimitre R
authors.initials: DR
authors.lastname: Simeonov
authors.affiliation: Department of Epidemiology and Biostatistics, Department of Bioengineering and Therapeutic Sciences, Institute for Human Genetics, University of California, San Francisco, CA 94143; Biological and Medical Informatics Graduate Program, University of California, San Francisco, CA 94158;
authors.forename: Meena
authors.initials: M
authors.lastname: Subramaniam
authors.affiliation: Department of Epidemiology and Biostatistics, Department of Bioengineering and Therapeutic Sciences, Institute for Human Genetics, University of California, San Francisco, CA 94143; Biological and Medical Informatics Graduate Program, University of California, San Francisco, CA 94158;
authors.forename: Rachel E
authors.initials: RE
authors.lastname: Gate
authors.affiliation: Diabetes Center, University of California, San Francisco, CA 94143; Division of Infectious Diseases, Department of Medicine, University of California, San Francisco, CA 94143;
authors.forename: Genevieve E
authors.initials: GE
authors.lastname: Haliburton
authors.affiliation: Department of Epidemiology and Biostatistics, Department of Bioengineering and Therapeutic Sciences, Institute for Human Genetics, University of California, San Francisco, CA 94143;
authors.forename: Chun J
authors.initials: CJ
authors.lastname: Ye
authors.affiliation: Diabetes Center, University of California, San Francisco, CA 94143;
authors.forename: Jeffrey A
authors.initials: JA
authors.lastname: Bluestone
authors.affiliation: Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720; Innovative Genomics Initiative, University of California, Berkeley, CA 94720; Howard Hughes Medical Institute, University of California, Berkeley, CA 94720; Department of Chemistry, University of California, Berkeley, CA 94720; Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720 alexander.marson@ucsf.edu doudna@berkeley.edu.
authors.forename: Jennifer A
authors.initials: JA
authors.lastname: Doudna
authors.affiliation: Diabetes Center, University of California, San Francisco, CA 94143; Division of Infectious Diseases, Department of Medicine, University of California, San Francisco, CA 94143; Innovative Genomics Initiative, University of California, Berkeley, CA 94720; alexander.marson@ucsf.edu doudna@berkeley.edu.
authors.forename: Alexander
authors.initials: A
authors.lastname: Marson
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics*; D015496:CD4-Positive T-Lymphocytes / Q000166:cytology; D002460:Cell Line; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D018274:Electroporation; D004720:Endonucleases / Q000737:chemistry / Q000235:genetics*; D055879:Gene Knock-In Techniques; D005818:Genetic Engineering / Q000379:methods; D016678:Genome; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000166:cytology; D019718:Receptors, CXCR4 / Q000378:metabolism; D012261:Ribonucleoproteins / Q000737:chemistry / Q000235:genetics*; D013601:T-Lymphocytes / Q000166:cytology*
chemical_list: D001426:Bacterial Proteins; C527276:CXCR4 protein, human; D019718:Receptors, CXCR4; D012261:Ribonucleoproteins; D004720:Endonucleases
keywords: CRISPR/Cas9; Cas9 ribonucleoprotein; RNP; genome engineering; primary human T cells