Digenome-seq: genome-wide profiling of CRISPR-Cas9 off-target effects in human cells

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.

[1]  Dana Carroll,et al.  Enhancing Gene Targeting with Designed Zinc Finger Nucleases , 2003, Science.

[2]  David Baltimore,et al.  Chimeric Nucleases Stimulate Gene Targeting in Human Cells , 2003, Science.

[3]  Jeffrey C. Miller,et al.  Highly efficient endogenous human gene correction using designed zinc-finger nucleases , 2005, Nature.

[4]  Thomas D. Schmittgen,et al.  Analyzing real-time PCR data by the comparative CT method , 2008, Nature Protocols.

[5]  Fyodor Urnov,et al.  Chromosomal translocations induced at specified loci in human stem cells , 2009, Proceedings of the National Academy of Sciences.

[6]  Seung Woo Cho,et al.  Targeted genome editing in human cells with zinc finger nucleases constructed via modular assembly. , 2009, Genome research.

[7]  Eunji Kim,et al.  Targeted chromosomal deletions in human cells using zinc finger nucleases. , 2010, Genome research.

[8]  J. Dye,et al.  Ebola virus entry requires the cholesterol transporter Niemann-Pick C1 , 2011, Nature.

[9]  Helga Thorvaldsdóttir,et al.  Integrative Genomics Viewer , 2011, Nature Biotechnology.

[10]  Elo Leung,et al.  A TALE nuclease architecture for efficient genome editing , 2011, Nature Biotechnology.

[11]  Jin-Soo Kim,et al.  Surrogate reporters for enrichment of cells with nuclease-induced mutations , 2011, Nature Methods.

[12]  Jeffrey C. Miller,et al.  An unbiased genome-wide analysis of zinc-finger nuclease specificity , 2011, Nature Biotechnology.

[13]  Eunji Kim,et al.  Targeted chromosomal duplications and inversions in the human genome using zinc finger nucleases. , 2012, Genome research.

[14]  Eli J. Fine,et al.  DNA targeting specificity of RNA-guided Cas9 nucleases , 2013, Nature Biotechnology.

[15]  Seung Woo Cho,et al.  Targeted genome engineering in human cells with the Cas9 RNA-guided endonuclease , 2013, Nature Biotechnology.

[16]  Dana Carroll,et al.  Heritable Gene Knockout in Caenorhabditis elegans by Direct Injection of Cas9–sgRNA Ribonucleoproteins , 2013, Genetics.

[17]  Daehyun Baek,et al.  TALEN-based knockout library for human microRNAs , 2013, Nature Structural &Molecular Biology.

[18]  David R. Liu,et al.  High-throughput profiling of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity , 2013, Nature Biotechnology.

[19]  Feng Zhang,et al.  CRISPR-assisted editing of bacterial genomes , 2013, Nature Biotechnology.

[20]  Semyon Kruglyak,et al.  Isaac: ultra-fast whole-genome secondary analysis on Illumina sequencing platforms , 2013, Bioinform..

[21]  Le Cong,et al.  Multiplex Genome Engineering Using CRISPR/Cas Systems , 2013, Science.

[22]  Jeffry D. Sander,et al.  Efficient In Vivo Genome Editing Using RNA-Guided Nucleases , 2013, Nature Biotechnology.

[23]  Duhee Bang,et al.  A library of TAL effector nucleases spanning the human genome , 2013, Nature Biotechnology.

[24]  Gang Bao,et al.  CRISPR/Cas9 systems targeting β-globin and CCR5 genes have substantial off-target activity , 2013, Nucleic acids research.

[25]  G. Church,et al.  CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering , 2013, Nature Biotechnology.

[26]  Jiyeon Kweon,et al.  TALENs and ZFNs are associated with different mutation signatures , 2013, Nature Methods.

[27]  Jennifer Doudna,et al.  RNA-programmed genome editing in human cells , 2013, eLife.

[28]  James E. DiCarlo,et al.  RNA-Guided Human Genome Engineering via Cas9 , 2013, Science.

[29]  David A. Scott,et al.  Double Nicking by RNA-Guided CRISPR Cas9 for Enhanced Genome Editing Specificity , 2013, Cell.

[30]  J. Keith Joung,et al.  High frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells , 2013, Nature Biotechnology.

[31]  Martin J. Aryee,et al.  Dimeric CRISPR RNA-guided FokI nucleases for highly specific genome editing , 2014, Nature Biotechnology.

[32]  Chad A. Cowan,et al.  Low incidence of off-target mutations in individual CRISPR-Cas9 and TALEN targeted human stem cell clones detected by whole-genome sequencing. , 2014, Cell stem cell.

[33]  Mazhar Adli,et al.  Genome-wide analysis reveals characteristics of off-target sites bound by the Cas9 endonuclease , 2014, Nature Biotechnology.

[34]  Jin-Soo Kim,et al.  Genotyping with CRISPR-Cas-derived RNA-guided endonucleases , 2014, Nature Communications.

[35]  Daesik Kim,et al.  Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins , 2014, Genome research.

[36]  H. Kim,et al.  A guide to genome engineering with programmable nucleases , 2014, Nature Reviews Genetics.

[37]  David R. Liu,et al.  Fusion of catalytically inactive Cas9 to FokI nuclease improves the specificity of genome modification , 2014, Nature Biotechnology.

[38]  J. Keith Joung,et al.  Improving CRISPR-Cas nuclease specificity using truncated guide RNAs , 2014, Nature Biotechnology.

[39]  Kun Zhang,et al.  Whole-genome sequencing analysis reveals high specificity of CRISPR/Cas9 and TALEN-based genome editing in human iPSCs. , 2014, Cell stem cell.

[40]  Suresh Ramakrishna,et al.  Gene disruption by cell-penetrating peptide-mediated delivery of Cas9 protein and guide RNA , 2014, Genome research.

[41]  Gang Bao,et al.  CRISPR/Cas9 systems have off-target activity with insertions or deletions between target DNA and guide RNA sequences , 2014, Nucleic acids research.

[42]  Jin-Soo Kim,et al.  Analysis of off-target effects of CRISPR/Cas-derived RNA-guided endonucleases and nickases , 2014, Genome research.

[43]  David A. Scott,et al.  Genome-wide binding of the CRISPR endonuclease Cas9 in mammalian cells , 2014, Nature Biotechnology.

[44]  Jin-Soo Kim,et al.  Cas-OFFinder: a fast and versatile algorithm that searches for potential off-target sites of Cas9 RNA-guided endonucleases , 2014, Bioinform..

[45]  J. Joung,et al.  What's changed with genome editing? , 2014, Cell stem cell.

[46]  S. Ha,et al.  Highly efficient gene knockout in mice and zebrafish with RNA-guided endonucleases , 2014, Genome research.

[47]  Martin J. Aryee,et al.  GUIDE-Seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases , 2014, Nature Biotechnology.

[48]  Richard L. Frock,et al.  Genome-wide detection of DNA double-stranded breaks induced by engineered nucleases , 2014, Nature Biotechnology.