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Genome-wide target specificities of CRISPR-Cas9 nucleases revealed by multiplex Digenome-seq

Published on Mar 1, 2016in Genome Research 10.10
· DOI :10.1101/gr.199588.115
Daesik Kim12
Estimated H-index: 12
(SNU: Seoul National University),
Sojung Kim10
Estimated H-index: 10
(SNU: Seoul National University)
+ 2 AuthorsJin-Soo Kim52
Estimated H-index: 52
(SNU: Seoul National University)
Abstract
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.
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  • References (36)
  • Citations (75)
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References36
Newest
Published on Jun 15, 2015in Bioinformatics 5.48
Eduard Valera Zorita4
Estimated H-index: 4
(UPF: Pompeu Fabra University),
Pol Cuscó2
Estimated H-index: 2
(UPF: Pompeu Fabra University),
Guillaume J. Filion15
Estimated H-index: 15
(UPF: Pompeu Fabra University)
Motivation: The increasing throughput of sequencing technologies offers new applications and challenges for computational biology. In many of those applications, sequencing errors need to be corrected. This is particularly important when sequencing reads from an unknwon reference such as random DNA barcodes. In this case, error correction can be done by performing a pairwise comparison of all the barcodes, which is computationally complex problem. Results: Here we address this challenge and desc...
36 Citations Source Cite
Published on Apr 1, 2015in Nature 41.58
F. Ann Ran9
Estimated H-index: 9
,
Le Cong15
Estimated H-index: 15
+ 10 AuthorsKira S. Makarova77
Estimated H-index: 77
The physical size of the commonly used Cas9 from Streptococcus pyogenes poses challenges for CRISPR-Cas genome editing systems that use the adeno-associated virus as a delivery vehicle; here, smaller Cas9 orthologues are characterized, and Cas9 from Staphylococcus aureus allowed targeting of the cholesterol regulatory gene Pcsk9 in the mouse liver.
896 Citations Source Cite
Published on Mar 1, 2015in Nature Methods 26.92
Daesik Kim12
Estimated H-index: 12
(SNU: Seoul National University),
Sangsu Bae13
Estimated H-index: 13
(Hanyang University)
+ 6 AuthorsJinsoo Kim49
Estimated H-index: 49
(SNU: Seoul National University)
In vitro digestion of genomic DNA with Cas9 and single guide RNAs (sgRNAs) yields genome-wide off-target sites at frequencies below 0.1%. Off-target sites can be further reduced with modified sgRNAs.
359 Citations Source Cite
Published on Feb 1, 2015in Nature Biotechnology 35.72
Richard L. Frock13
Estimated H-index: 13
,
Jiazhi Hu11
Estimated H-index: 11
+ 3 AuthorsFrederick W. Alt155
Estimated H-index: 155
(Harvard University)
An unbiased genome-wide method reveals on- and off-target DNA cleavage by TALEN and Cas9 nucleases by detecting chromosome translocation events.
297 Citations Source Cite
Published on Feb 1, 2015in Nature Biotechnology 35.72
Xiaoling Wang3
Estimated H-index: 3
,
Yebo Wang5
Estimated H-index: 5
+ 7 AuthorsJiing-Kuan Yee13
Estimated H-index: 13
Off-target cleavage by CAS9 or TALEN genome editing tools is detected by integrase-defective lentiviral vectors.
225 Citations Source Cite
Published on Feb 1, 2015in Nature Biotechnology 35.72
Shengdar Q. Tsai24
Estimated H-index: 24
(Harvard University),
Zongli Zheng24
Estimated H-index: 24
(KI: Karolinska Institutet)
+ 9 AuthorsLong P. Le19
Estimated H-index: 19
(Harvard University)
An unbiased approach for the genome-wide detection of off-target cleavage by CRISPR-Cas9 RNA–guided nucleases reveals wide variability in the off-target activity of different guide RNAs.
662 Citations Source Cite
Published on Feb 1, 2015in Nature Biotechnology 35.72
Richard Gabriel14
Estimated H-index: 14
(DKFZ: German Cancer Research Center),
Christof von Kalle36
Estimated H-index: 36
,
Manfred Schmidt71
Estimated H-index: 71
(DKFZ: German Cancer Research Center)
Genome-wide, unbiased methods provide a comprehensive picture of off-target cleavage by engineered nucleases.
21 Citations Source Cite
Published on Jan 1, 2015in Nature Biotechnology 35.72
John A. Zuris15
Estimated H-index: 15
,
David B. Thompson10
Estimated H-index: 10
+ 7 AuthorsDavid R. Liu69
Estimated H-index: 69
(Harvard University)
Efficient protein delivery using cationic lipid transfection reagents enables high efficiency protein-based genome editing in vivo and in vitro.
463 Citations Source Cite
Published on Jul 1, 2014in Nature Biotechnology 35.72
Cem Kuscu14
Estimated H-index: 14
,
Sevki Arslan4
Estimated H-index: 4
+ 2 AuthorsMazhar Adli25
Estimated H-index: 25
ChIP-seq for Cas9 shows varying amounts of off-target binding with different guide RNAs and low levels of indels at some off-target sites.
386 Citations Source Cite
Published on Jun 17, 2014in Nucleic Acids Research 11.56
Yanni Lin4
Estimated H-index: 4
(Georgia Institute of Technology),
Thomas J. Cradick18
Estimated H-index: 18
(Georgia Institute of Technology)
+ 7 AuthorsGang Bao56
Estimated H-index: 56
(Georgia Institute of Technology)
CRISPR/Cas9 systems are a versatile tool for genome editing due to the highly efficient targeting of DNA sequences complementary to their RNA guide strands. However, it has been shown that RNA-guided Cas9 nuclease cleaves genomic DNA sequences containing mismatches to the guide strand. A better understanding of the CRISPR/Cas9 specificity is needed to minimize off-target cleavage in large mammalian genomes. Here we show that genomic sites could be cleaved by CRISPR/Cas9 systems when DNA sequence...
259 Citations Source Cite
Cited By75
Newest
Published on Jan 25, 2019in Genome Biology 13.21
Jean-Philippe Fortin9
Estimated H-index: 9
(Genentech),
Jenille Tan5
Estimated H-index: 5
(Genentech)
+ 4 AuthorsScott E. Martin4
Estimated H-index: 4
(Genentech)
Background Genome-wide loss-of-function screens using the CRISPR/Cas9 system allow the efficient discovery of cancer cell vulnerabilities. While several studies have focused on correcting for DNA cleavage toxicity biases associated with copy number alterations, the effects of sgRNAs co-targeting multiple genomic loci in CRISPR screens have not been discussed.
4 Citations Source Cite
Published on Jan 8, 2019
D. Ghosh , P. Venkataramani + 1 AuthorsSonali Bhattacharjee5
Estimated H-index: 5
Genome editing allows for the precise manipulation of DNA sequences in a cell making this technology essential for understanding gene function. CRISPR/Cas9 is a targeted genome-editing platform derived from bacterial adaptive immune system and has been repurposed into a genome-editing tool. The RNA-guided DNA endonuclease, Cas9 can be easily programmed to target new sites by altering its guide RNA sequence, making this technology easier, more efficient, scalable and an indispensable tool in biol...
Published on Jan 8, 2019in Nature Communications 12.35
Puping Liang7
Estimated H-index: 7
(SYSU: Sun Yat-sen University),
Xiaowei Xie6
Estimated H-index: 6
(SYSU: Sun Yat-sen University)
+ 9 AuthorsDan Liu26
Estimated H-index: 26
(BCM: Baylor College of Medicine)
The adenine base editor (ABE), capable of catalyzing A•T to G•C conversions, is an important gene editing toolbox. Here, we systematically evaluate genome-wide off-target deamination by ABEs using the EndoV-seq platform we developed. EndoV-seq utilizes Endonuclease V to nick the inosine-containing DNA strand of genomic DNA deaminated by ABE in vitro. The treated DNA is then whole-genome sequenced to identify off-target sites. Of the eight gRNAs we tested with ABE, 2–19 (with an average of 8.0) o...
7 Citations Source Cite
Published on Jan 31, 2019in Genome Biology 13.21
Nur Diana Anuar (ANU: Australian National University), Sebastian Kurscheid14
Estimated H-index: 14
(ANU: Australian National University)
+ 8 AuthorsDavid J. Tremethick32
Estimated H-index: 32
(ANU: Australian National University)
Background: Altering the biochemical makeup of chromatin by the incorporation of histone variants during development represents a key mechanism in regulating gene expression. The histone variant H2A.B, H2A.B.3 in mice, appeared late in evolution and is most highly expressed in the testis. In the mouse, it is encoded by three different genes. H2A.B expression is spatially and temporally regulated during spermatogenesis being most highly expressed in the haploid round spermatid stage. Active genes...
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Published on Apr 15, 2019in Nature Biotechnology 35.72
D. Dewran Kocak4
Estimated H-index: 4
(Duke University),
Eric A. Josephs8
Estimated H-index: 8
(UNCG: University of North Carolina at Greensboro)
+ 3 AuthorsCharles A. Gersbach38
Estimated H-index: 38
(Duke University)
CRISPR (clustered regularly interspaced short palindromic repeat) systems have been broadly adopted for basic science, biotechnology, and gene and cell therapy. In some cases, these bacterial nucleases have demonstrated off-target activity. This creates a potential hazard for therapeutic applications and could confound results in biological research. Therefore, improving the precision of these nucleases is of broad interest. Here we show that engineering a hairpin secondary structure onto the sp...
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Published on Jun 3, 2019in Nature Medicine 32.62
Source Cite
Published on Apr 1, 2019in Nature Biotechnology 35.72
Daesik Kim12
Estimated H-index: 12
(SNU: Seoul National University),
Da-eun Kim3
Estimated H-index: 3
(SNU: Seoul National University)
+ 2 AuthorsJin-Soo Kim52
Estimated H-index: 52
(SNU: Seoul National University)
Adenine base editors1 enable efficient targeted adenine-to-guanine single nucleotide conversions to induce or correct point mutations in human cells, animals, and plants1–4. Here we present a modified version of Digenome-seq, an in vitro method for identifying CRISPR (clustered regularly interspaced short palindromic repeats)-induced double-strand breaks using whole-genome sequencing5–8, to assess genome-wide target specificity of adenine base editors. To produce double-strand breaks at sites co...
8 Citations Source Cite