title: Targeted and genome-wide sequencing reveal single nucleotide variations impacting specificity of Cas9 in human stem cells.
abstract: CRISPR/Cas9 has demonstrated a high-efficiency in site-specific gene targeting. However, potential off-target effects of the Cas9 nuclease represent a major safety concern for any therapeutic application. Here, we knock out the Tafazzin gene by CRISPR/Cas9 in human-induced pluripotent stem cells with 54% efficiency. We combine whole-genome sequencing and deep-targeted sequencing to characterise the off-target effects of Cas9 editing. Whole-genome sequencing of Cas9-modified hiPSC clones detects neither gross genomic alterations nor elevated mutation rates. Deep sequencing of in silico predicted off-target sites in a population of Cas9-treated cells further confirms high specificity of Cas9. However, we identify a single high-efficiency off-target site that is generated by a common germline single-nucleotide variant (SNV) in our experiment. Based on in silico analysis, we estimate a likelihood of SNVs creating off-target sites in a human genome to be ~1.5-8.5%, depending on the genome and site-selection method, but also note that mutations might be generated at these sites only at low rates and may not have functional consequences. Our study demonstrates the feasibility of highly specific clonal ex vivo gene editing using CRISPR/Cas9 and highlights the value of whole-genome sequencing before personalised CRISPR design.
authors.affiliation: 1] Department of Genetics, Harvard Medical School, Boston, Massachusetts 02115, USA [2] Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, Massachusetts 02115, USA.
authors.forename: Luhan
authors.initials: L
authors.lastname: Yang
authors.affiliation: Department of Genetics, Harvard Medical School, Boston, Massachusetts 02115, USA.
authors.forename: Dennis
authors.initials: D
authors.lastname: Grishin
authors.affiliation: Department of Cardiology, Boston Children's Hospital, Boston, Massachusetts 02115, USA.
authors.forename: Gang
authors.initials: G
authors.lastname: Wang
authors.affiliation: Department of Genetics, Harvard Medical School, Boston, Massachusetts 02115, USA.
authors.forename: John
authors.initials: J
authors.lastname: Aach
authors.affiliation: Broad Institute of MIT and Harvard, Cambridge, Massachusetts 02142, USA.
authors.forename: Cheng-Zhong
authors.initials: CZ
authors.lastname: Zhang
authors.affiliation: Department of Genetics, Harvard Medical School, Boston, Massachusetts 02115, USA.
authors.forename: Raj
authors.initials: R
authors.lastname: Chari
authors.affiliation: Department of Genetics, Harvard Medical School, Boston, Massachusetts 02115, USA.
authors.forename: Jason
authors.initials: J
authors.lastname: Homsy
authors.affiliation: Illumina, San Diego, California 92122, USA.
authors.forename: Xuyu
authors.initials: X
authors.lastname: Cai
authors.affiliation: Illumina, San Diego, California 92122, USA.
authors.forename: Yue
authors.initials: Y
authors.lastname: Zhao
authors.affiliation: Illumina, San Diego, California 92122, USA.
authors.forename: Jian-Bing
authors.initials: JB
authors.lastname: Fan
authors.affiliation: Department of Genetics, Harvard Medical School, Boston, Massachusetts 02115, USA.
authors.forename: Christine
authors.initials: C
authors.lastname: Seidman
authors.affiliation: Department of Genetics, Harvard Medical School, Boston, Massachusetts 02115, USA.
authors.forename: Jonathan
authors.initials: J
authors.lastname: Seidman
authors.affiliation: Department of Cardiology, Boston Children's Hospital, Boston, Massachusetts 02115, USA.
authors.forename: William
authors.initials: W
authors.lastname: Pu
authors.affiliation: 1] Department of Genetics, Harvard Medical School, Boston, Massachusetts 02115, USA [2] Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, Massachusetts 02115, USA.
authors.forename: George
authors.initials: G
authors.lastname: Church
mesh_terms: D000217:Acyltransferases; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D001483:Base Sequence; D004720:Endonucleases / Q000737:chemistry / Q000378:metabolism*; D018390:Gene Targeting; D015894:Genome, Human; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000378:metabolism*; D008969:Molecular Sequence Data; D059645:Mutation Rate; D020641:Polymorphism, Single Nucleotide; D017422:Sequence Analysis, DNA; D013297:Streptococcus pyogenes / Q000201:enzymology*; D013379:Substrate Specificity; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D014157:Transcription Factors; D000217:Acyltransferases; C108675:TAFAZZIN protein, human; D004720:Endonucleases