RESUMO
Programmable nucleases and deaminases, which include zinc-finger nucleases, transcription activator-like effector nucleases, CRISPR RNA-guided nucleases, and RNA-guided base editors, are now widely employed for the targeted modification of genomes in cells and organisms. These gene-editing tools hold tremendous promise for therapeutic applications. Importantly, these nucleases and deaminases may display off-target activity through the recognition of near-cognate DNA sequences to their target sites, resulting in collateral damage to the genome in the form of local mutagenesis or genomic rearrangements. For therapeutic genome-editing applications with these classes of programmable enzymes, it is essential to measure and limit genome-wide off-target activity. Herein, we discuss the key determinants of off-target activity for these systems. We describe various cell-based and cell-free methods for identifying genome-wide off-target sites and diverse strategies that have been developed for reducing the off-target activity of programmable gene-editing enzymes.
Assuntos
Proteína 9 Associada à CRISPR/genética , Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Edição de Genes/métodos , Engenharia de Proteínas/métodos , RNA Guia de Cinetoplastídeos/genética , Desaminases APOBEC/genética , Desaminases APOBEC/metabolismo , Adenosina Desaminase/genética , Adenosina Desaminase/metabolismo , Artefatos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteína 9 Associada à CRISPR/metabolismo , Endonucleases/genética , Endonucleases/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Técnicas de Transferência de Genes , Genoma Humano , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , RNA Guia de Cinetoplastídeos/metabolismo , SoftwareRESUMO
Nucleotide repeat expansion disorders, a group of genetic diseases characterized by the expansion of specific DNA sequences, pose significant challenges to treatment and therapy development. Here, we present a precise and programmable method called prime editor-mediated correction of nucleotide repeat expansion (PE-CORE) for correcting pathogenic nucleotide repeat expansion. PE-CORE leverages a prime editor and paired pegRNAs to achieve targeted correction of repeat sequences. We demonstrate the effectiveness of PE-CORE in HEK293T cells and patient-derived induced pluripotent stem cells (iPSCs). Specifically, we focus on spinal and bulbar muscular atrophy and spinocerebellar ataxia type, two diseases associated with nucleotide repeat expansion. Our results demonstrate the successful correction of pathogenic expansions in iPSCs and subsequent differentiation into motor neurons. Specifically, we detect distinct downshifts in the size of both the mRNA and protein, confirming the functional correction of the iPSC-derived motor neurons. These findings highlight PE-CORE as a precision tool for addressing the intricate challenges of nucleotide repeat expansion disorders, paving the way for targeted therapies and potential clinical applications.
Assuntos
Edição de Genes , Células-Tronco Pluripotentes Induzidas , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Edição de Genes/métodos , Células HEK293 , Neurônios Motores/metabolismo , Diferenciação Celular/genética , Expansão das Repetições de DNA/genética , Expansão das Repetições de Trinucleotídeos/genéticaRESUMO
X-linked adrenoleukodystrophy (ALD), an inherited neurometabolic disorder caused by mutations in ABCD1, which encodes the peroxisomal ABC transporter, mainly affects the brain, spinal cord, adrenal glands, and testes. In ALD patients, very-long-chain fatty acids (VLCFAs) fail to enter the peroxisome and undergo subsequent ß-oxidation, resulting in their accumulation in the body. It has not been tested whether in vivo base editing or prime editing can be harnessed to ameliorate ALD. We developed a humanized mouse model of ALD by inserting a human cDNA containing the pathogenic variant into the mouse Abcd1 locus. The humanized ALD model showed increased levels of VLCFAs. To correct the mutation, we tested both base editing and prime editing and found that base editing using ABE8e(V106W) could correct the mutation in patient-derived fibroblasts at an efficiency of 7.4%. Adeno-associated virus (AAV)-mediated systemic delivery of NG-ABE8e(V106W) enabled robust correction of the pathogenic variant in the mouse brain (correction efficiency: â¼5.5%), spinal cord (â¼5.1%), and adrenal gland (â¼2%), leading to a significant reduction in the plasma levels of C26:0/C22:0. This established humanized mouse model and the successful correction of the pathogenic variant using a base editor serve as a significant step toward treating human ALD disease.
Assuntos
Membro 1 da Subfamília D de Transportadores de Cassetes de Ligação de ATP , Adrenoleucodistrofia , Dependovirus , Modelos Animais de Doenças , Edição de Genes , Terapia Genética , Animais , Adrenoleucodistrofia/terapia , Adrenoleucodistrofia/genética , Camundongos , Humanos , Membro 1 da Subfamília D de Transportadores de Cassetes de Ligação de ATP/genética , Dependovirus/genética , Terapia Genética/métodos , Vetores Genéticos/genética , Vetores Genéticos/administração & dosagem , Adenina , Mutação , Fibroblastos/metabolismo , Ácidos Graxos/metabolismo , Encéfalo/metabolismo , Encéfalo/patologiaRESUMO
A variety of cancers have been found to have chromosomal rearrangements, and the genomic abnormalities often induced expression of fusion oncogenes. To date, a pair of engineered nucleases including ZFNs, TALENs, and CRISPR-Cas9 nucleases have been used to generate chromosomal rearrangement in living cells and organisms for disease modeling. However, these methods induce unwanted indel mutations at the DNA break junctions, resulting in incomplete disease modeling. Here, we developed prime editor nuclease-mediated translocation and inversion (PETI), a method for programmable chromosomal translocation and inversion using prime editor 2 nuclease (PE2 nuclease) and paired pegRNA. Using PETI method, we successfully introduced DNA recombination in episomal fluorescence reporters as well as precise chromosomal translocations in human cells. We applied PETI to create cancer-associated translocations and inversions such as NPM1-ALK and EML4-ALK in human cells. Our findings show that PETI generated chromosomal translocation and inversion in a programmable manner with efficiencies comparable of Cas9. PETI methods, we believe, could be used to create disease models or for gene therapy.
Assuntos
Neoplasias , Translocação Genética , Humanos , Rearranjo Gênico , Genoma , Endonucleases , Genômica , Receptores Proteína Tirosina Quinases , Edição de Genes/métodos , Sistemas CRISPR-CasRESUMO
This corrects the article DOI: 10.1038/nature24033.
RESUMO
Despite their fundamental biological and clinical importance, the molecular mechanisms that regulate the first cell fate decisions in the human embryo are not well understood. Here we use CRISPR-Cas9-mediated genome editing to investigate the function of the pluripotency transcription factor OCT4 during human embryogenesis. We identified an efficient OCT4-targeting guide RNA using an inducible human embryonic stem cell-based system and microinjection of mouse zygotes. Using these refined methods, we efficiently and specifically targeted the gene encoding OCT4 (POU5F1) in diploid human zygotes and found that blastocyst development was compromised. Transcriptomics analysis revealed that, in POU5F1-null cells, gene expression was downregulated not only for extra-embryonic trophectoderm genes, such as CDX2, but also for regulators of the pluripotent epiblast, including NANOG. By contrast, Pou5f1-null mouse embryos maintained the expression of orthologous genes, and blastocyst development was established, but maintenance was compromised. We conclude that CRISPR-Cas9-mediated genome editing is a powerful method for investigating gene function in the context of human development.
Assuntos
Desenvolvimento Embrionário/genética , Edição de Genes , Regulação da Expressão Gênica no Desenvolvimento , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Animais , Blastocisto/metabolismo , Sistemas CRISPR-Cas/genética , Linhagem da Célula , Ectoderma/metabolismo , Embrião de Mamíferos/citologia , Embrião de Mamíferos/embriologia , Embrião de Mamíferos/metabolismo , Feminino , Camadas Germinativas/metabolismo , Células-Tronco Embrionárias Humanas/citologia , Células-Tronco Embrionárias Humanas/metabolismo , Humanos , Masculino , Camundongos , Proteína Homeobox Nanog/genética , Proteína Homeobox Nanog/metabolismo , Fator 3 de Transcrição de Octâmero/deficiência , Especificidade por Substrato , Zigoto/metabolismoRESUMO
Genome editing has potential for the targeted correction of germline mutations. Here we describe the correction of the heterozygous MYBPC3 mutation in human preimplantation embryos with precise CRISPR-Cas9-based targeting accuracy and high homology-directed repair efficiency by activating an endogenous, germline-specific DNA repair response. Induced double-strand breaks (DSBs) at the mutant paternal allele were predominantly repaired using the homologous wild-type maternal gene instead of a synthetic DNA template. By modulating the cell cycle stage at which the DSB was induced, we were able to avoid mosaicism in cleaving embryos and achieve a high yield of homozygous embryos carrying the wild-type MYBPC3 gene without evidence of off-target mutations. The efficiency, accuracy and safety of the approach presented suggest that it has potential to be used for the correction of heritable mutations in human embryos by complementing preimplantation genetic diagnosis. However, much remains to be considered before clinical applications, including the reproducibility of the technique with other heterozygous mutations.
Assuntos
Proteínas de Transporte/genética , Embrião de Mamíferos/metabolismo , Edição de Genes/métodos , Mutação/genética , Adulto , Alelos , Blastocisto/metabolismo , Blastocisto/patologia , Divisão Celular , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Quebras de DNA de Cadeia Dupla , Embrião de Mamíferos/patologia , Marcação de Genes , Teste de Complementação Genética , Heterozigoto , Homozigoto , Humanos , Masculino , Mosaicismo , Reparo de DNA por Recombinação/genética , Fase S , Moldes Genéticos , Zigoto/metabolismo , Zigoto/patologiaRESUMO
Prime editors (PEs) enable targeted precise editing, including the generation of substitutions, insertions and deletions, in eukaryotic genomes. However, their genome-wide specificity has not been explored. Here, we developed Nickase-based Digenome-seq (nDigenome-seq), an in vitro assay that uses whole-genome sequencing to identify single-strand breaks induced by CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 (CRISPR-associated protein 9) nickase. We used nDigenome-seq to screen for potential genome-wide off-target sites of Cas9 H840A nickase, a PE component, targeted to nine human genomic sites. Then, using targeted amplicon sequencing of off-target candidates identified by nDigenome-seq, we showed that only five off-target sites showed detectable PE-induced modifications in cells, at frequencies ranging from 0.1 to 1.9%, suggesting that PEs provide a highly specific method of precise genome editing. We also found that PE specificity in human cells could be further improved by incorporating mutations from engineered Cas9 variants, particularly eSpCas9 and Sniper Cas9, into PE.
Assuntos
Proteína 9 Associada à CRISPR/genética , Sistemas CRISPR-Cas/genética , Quebras de DNA de Cadeia Simples , Edição de Genes/métodos , Genoma Humano/genética , Humanos , Sequenciamento Completo do GenomaRESUMO
Charcot-Marie-Tooth 1A (CMT1A) is the most common inherited neuropathy without a known therapy, which is caused by a 1.4 Mb duplication on human chromosome 17, which includes the gene encoding the peripheral myelin protein of 22 kDa (PMP22). Overexpressed PMP22 protein from its gene duplication is thought to cause demyelination and subsequently axonal degeneration in the peripheral nervous system (PNS). Here, we targeted TATA-box of human PMP22 promoter to normalize overexpressed PMP22 level in C22 mice, a mouse model of CMT1A harboring multiple copies of human PMP22. Direct local intraneural delivery of CRISPR/Cas9 designed to target TATA-box of PMP22 before the onset of disease, downregulates gene expression of PMP22 and preserves both myelin and axons. Notably, the same approach was effective in partial rescue of demyelination even after the onset of disease. Collectively, our data present a proof-of-concept that CRISPR/Cas9-mediated targeting of TATA-box can be utilized to treat CMT1A.
Assuntos
Doença de Charcot-Marie-Tooth/terapia , Terapia de Alvo Molecular/métodos , Proteínas da Mielina/genética , Bainha de Mielina/metabolismo , Células de Schwann/metabolismo , TATA Box , Animais , Axônios , Sistemas CRISPR-Cas , Doença de Charcot-Marie-Tooth/genética , Doença de Charcot-Marie-Tooth/metabolismo , Doença de Charcot-Marie-Tooth/patologia , Duplicação Cromossômica , Cromossomos Humanos Par 17 , Modelos Animais de Doenças , Edição de Genes/métodos , Humanos , Injeções , Camundongos , Proteínas da Mielina/metabolismo , Bainha de Mielina/patologia , Cultura Primária de Células , Regiões Promotoras Genéticas , Células de Schwann/patologia , Nervo Isquiático/metabolismo , Nervo Isquiático/patologiaRESUMO
OBJECTIVE: Dysfunctional resolution of intestinal inflammation and altered mucosal healing are essential features in the pathogenesis of inflammatory bowel disease (IBD). Intestinal macrophages are vital in the process of inflammation resolution, but the mechanisms underlying their mucosal healing capacity remain elusive. DESIGN: We investigated the role of the prostaglandin E2 (PGE2) receptor PTGER4 on the differentiation of intestinal macrophages in patients with IBD and mouse models of intestinal inflammation. We studied mucosal healing and intestinal epithelial barrier regeneration in Csf1r-iCre Ptger4fl/fl mice during dextran sulfate sodium (DSS)-induced colitis. The effect of PTGER4+ macrophage secreted molecules was investigated on epithelial organoid differentiation. RESULTS: Here, we describe a subset of PTGER4-expressing intestinal macrophages with mucosal healing properties both in humans and mice. Csf1r-iCre Ptger4fl/fl mice showed defective mucosal healing and epithelial barrier regeneration in a model of DSS colitis. Mechanistically, an increased mucosal level of PGE2 triggers chemokine (C-X-C motif) ligand 1 (CXCL1) secretion in monocyte-derived PTGER4+ macrophages via mitogen-activated protein kinases (MAPKs). CXCL1 drives epithelial cell differentiation and proliferation from regenerating crypts during colitis. Specific therapeutic targeting of macrophages with liposomes loaded with an MAPK agonist augmented the production of CXCL1 in vivo in conditional macrophage PTGER4-deficient mice, restoring their defective epithelial regeneration and favouring mucosal healing. CONCLUSION: PTGER4+ intestinal macrophages are essential for supporting the intestinal stem cell niche and regeneration of the injured epithelium. Our results pave the way for the development of a new class of therapeutic targets to promote macrophage healing functions and favour remission in patients with IBD.
Assuntos
Doenças Inflamatórias Intestinais/metabolismo , Mucosa Intestinal/citologia , Mucosa Intestinal/metabolismo , Ativação de Macrófagos , Receptores de Prostaglandina E Subtipo EP4/metabolismo , Animais , Diferenciação Celular , Quimiocina CXCL1/metabolismo , Modelos Animais de Doenças , Camundongos , Regeneração , Transdução de SinaisRESUMO
To investigate whether and how CRISPR-Cas9 on-target and off-target activities are affected by chromatin in eukaryotic cells, we first identified a series of identical endogenous DNA sequences present in both open and closed chromatin regions and then measured mutation frequencies at these sites in human cells using Cas9 complexed with matched or mismatched sgRNAs. Unlike matched sgRNAs, mismatched sgRNAs were highly sensitive to chromatin states, suggesting that off-target but not on-target DNA cleavage is hindered by chromatin. We next performed Digenome-seq using cell-free chromatin DNA (now termed DIG-seq) and histone-free genomic DNA in parallel and found that only a subset of sites, cleaved in histone-free DNA, were cut in chromatin DNA, suggesting that chromatin can inhibit Cas9 off-target effects in favor of its genome-wide specificity in cells.
Assuntos
Sistemas CRISPR-Cas , Cromatina/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Sequenciamento de Nucleotídeos em Larga Escala , Linhagem Celular , Edição de Genes , Humanos , Reprodutibilidade dos TestesRESUMO
RNA-guided genome surgery using CRISPR-Cas9 nucleases has shown promise for the treatment of diverse genetic diseases. Yet, the potential of such nucleases for therapeutic applications in nongenetic diseases is largely unexplored. Here, we focus on age-related macular degeneration (AMD), a leading cause of blindness in adults, which is associated with retinal overexpression of, rather than mutations in, the VEGFA gene. Subretinal injection of preassembled, Vegfa gene-specific Cas9 ribonucleoproteins (RNPs) into the adult mouse eye gave rise to mutagenesis at the target site in the retinal pigment epithelium. Furthermore, Cas9 RNPs effectively reduced the area of laser-induced choroidal neovascularization (CNV) in a mouse model of AMD. Genome-wide profiling of Cas9 off-target effects via Digenome-seq showed that off-target mutations were rarely induced in the human genome. Because Cas9 RNPs can function immediately after in vivo delivery and are rapidly degraded by endogenous proteases, their activities are unlikely to be hampered by antibody- and cell-mediated adaptive immune systems. Our results demonstrate that in vivo genome editing with Cas9 RNPs has the potential for the local treatment for nongenetic degenerative diseases, expanding the scope of RNA-guided genome surgery to a new dimension.
Assuntos
Proteínas de Bactérias/metabolismo , Endonucleases/metabolismo , Edição de Genes/métodos , Terapia Genética/métodos , Degeneração Macular/terapia , Fator A de Crescimento do Endotélio Vascular/genética , Células 3T3 , Animais , Proteínas de Bactérias/genética , Proteína 9 Associada à CRISPR , Endonucleases/genética , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteólise , Retina/metabolismo , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
We present multiplex Digenome-seq to profile genome-wide specificities of up to 11 CRISPR-Cas9 nucleases simultaneously, saving time and reducing cost. Cell-free human genomic DNA was digested using multiple sgRNAs combined with the Cas9 protein and then subjected to whole-genome sequencing. In vitro cleavage patterns, characteristic of on- and off-target sites, were computationally identified across the genome using a new DNA cleavage scoring system. We found that many false-positive, bulge-type off-target sites were cleaved by sgRNAs transcribed from an oligonucleotide duplex but not by those transcribed from a plasmid template. Multiplex Digenome-seq captured many bona fide off-target sites, missed by other genome-wide methods, at which indels were induced at frequencies <0.1%. After analyzing 964 sites cleaved in vitro by these sgRNAs and measuring indel frequencies at hundreds of off-target sites in cells, we propose a guideline for the choice of target sites for minimizing CRISPR-Cas9 off-target effects in the human genome.
Assuntos
Sistemas CRISPR-Cas , Marcação de Genes , Genoma Humano , Genômica , Sítios de Ligação , Genômica/métodos , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Mutação INDEL , Taxa de Mutação , Motivos de Nucleotídeos , Matrizes de Pontuação de Posição Específica , Ligação Proteica , Reprodutibilidade dos TestesRESUMO
Duchenne muscular dystrophy (DMD) is a fatal, X-linked muscle-wasting disease caused by mutations in the DMD gene. In 51% of DMD cases, a reading frame is disrupted because of deletion of several exons. Here, we show that CjCas9 derived from Campylobacter jejuni can be used as a gene-editing tool to correct an out-of-frame Dmd exon in Dmd knockout mice. Herein, we used Cas9 derived from S. pyogenes to generate Dmd knockout mice with a frameshift mutation in Dmd gene. Then, we expressed CjCas9, its single-guide RNA, and the EGFP gene in the tibialis anterior muscle of the Dmd knockout mice using an all-in-one adeno-associated virus (AAV) vector. CjCas9 cleaved the target site in the Dmd gene efficiently in vivo and induced small insertions or deletions at the target site. This treatment resulted in conversion of the disrupted Dmd reading frame from out of frame to in frame, leading to the expression of dystrophin in the sarcolemma. Importantly, muscle strength was enhanced in the CjCas9-treated muscles, without off-target mutations, indicating high efficiency and specificity of CjCas9. This work suggests that in vivo DMD frame correction, mediated by CjCas9, has great potential for the treatment of DMD and other neuromuscular diseases.
Assuntos
Campylobacter jejuni/enzimologia , Distrofina/deficiência , Distrofina/genética , Mutação da Fase de Leitura/genética , Animais , Sistemas CRISPR-Cas/genética , Edição de Genes , Terapia Genética , Masculino , Camundongos , Camundongos Knockout , Músculo Esquelético/metabolismo , Distrofia Muscular de Duchenne/genéticaRESUMO
Gene editing can be used to overcome allo-recognition, which otherwise limits allogeneic T cell therapies. Initial proof-of-concept applications have included generation of such "universal" T cells expressing chimeric antigen receptors (CARs) against CD19 target antigens combined with transient expression of DNA-targeting nucleases to disrupt the T cell receptor alpha constant chain (TRAC). Although relatively efficient, transgene expression and editing effects were unlinked, yields variable, and resulting T cell populations heterogeneous, complicating dosing strategies. We describe a self-inactivating lentiviral "terminal" vector platform coupling CAR expression with CRISPR/Cas9 effects through incorporation of an sgRNA element into the ΔU3 3' long terminal repeat (LTR). Following reverse transcription and duplication of the hybrid ΔU3-sgRNA, delivery of Cas9 mRNA resulted in targeted TRAC locus cleavage and allowed the enrichment of highly homogeneous (>96%) CAR+ (>99%) TCR- populations by automated magnetic separation. Molecular analyses, including NGS, WGS, and Digenome-seq, verified on-target specificity with no evidence of predicted off-target events. Robust anti-leukemic effects were demonstrated in humanized immunodeficient mice and were sustained longer than by conventional CAR+TCR+ T cells. Terminal-TRAC (TT) CAR T cells offer the possibility of a pre-manufactured, non-HLA-matched CAR cell therapy and will be evaluated in phase 1 trials against B cell malignancies shortly.
Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Edição de Genes , Receptores de Antígenos de Linfócitos T/genética , Linfócitos T/imunologia , Linfócitos T/metabolismo , Sequências Repetidas Terminais , Animais , Antígenos CD19/imunologia , Modelos Animais de Doenças , Ordem dos Genes , Vetores Genéticos/genética , Humanos , Imunofenotipagem , Imunoterapia Adotiva/métodos , Hibridização in Situ Fluorescente , Lentivirus/genética , Leucemia/genética , Leucemia/imunologia , Leucemia/terapia , RNA Guia de Cinetoplastídeos , Receptores de Antígenos Quiméricos , Resultado do Tratamento , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Although RNA-guided genome editing via the CRISPR-Cas9 system is now widely used in biomedical research, genome-wide target specificities of Cas9 nucleases remain controversial. Here we present Digenome-seq, in vitro Cas9-digested whole-genome sequencing, to profile genome-wide Cas9 off-target effects in human cells. This in vitro digest yields sequence reads with the same 5' ends at cleavage sites that can be computationally identified. We validated off-target sites at which insertions or deletions were induced with frequencies below 0.1%, near the detection limit of targeted deep sequencing. We also showed that Cas9 nucleases can be highly specific, inducing off-target mutations at merely several, rather than thousands of, sites in the entire genome and that Cas9 off-target effects can be avoided by replacing 'promiscuous' single guide RNAs (sgRNAs) with modified sgRNAs. Digenome-seq is a robust, sensitive, unbiased and cost-effective method for profiling genome-wide off-target effects of programmable nucleases including Cas9.
Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Linhagem Celular , Endonucleases/genética , Endonucleases/metabolismo , Genoma Humano , Haploidia , Humanos , Limite de Detecção , Mutação , RNA Guia de Cinetoplastídeos/genética , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
RNA-guided engineered nucleases (RGENs) derived from the prokaryotic adaptive immune system known as CRISPR (clustered, regularly interspaced, short palindromic repeat)/Cas (CRISPR-associated) enable genome editing in human cell lines, animals, and plants, but are limited by off-target effects and unwanted integration of DNA segments derived from plasmids encoding Cas9 and guide RNA at both on-target and off-target sites in the genome. Here, we deliver purified recombinant Cas9 protein and guide RNA into cultured human cells including hard-to-transfect fibroblasts and pluripotent stem cells. RGEN ribonucleoproteins (RNPs) induce site-specific mutations at frequencies of up to 79%, while reducing off-target mutations associated with plasmid transfection at off-target sites that differ by one or two nucleotides from on-target sites. RGEN RNPs cleave chromosomal DNA almost immediately after delivery and are degraded rapidly in cells, reducing off-target effects. Furthermore, RNP delivery is less stressful to human embryonic stem cells, producing at least twofold more colonies than does plasmid transfection.