CasKAS: direct profiling of genome-wide dCas9 and Cas9 specificity using ssDNA mapping

被引：4

作者：

Marinov, Georgi K. ^{[1
]}

Kim, Samuel H. ^{[2
]}

Bagdatli, S. Tansu ^{[1
]}

Higashino, Soon Il ^{[1
]}

Trevino, Alexandro E. ^{[3
,4
]}

Tycko, Josh ^{[1
]}

Wu, Tong ^{[5
]}

Bintu, Lacramioara ^{[4
]}

Bassik, Michael C. ^{[1
,6
]}

He, Chuan ^{[5
,7
,8
,9
]}

Kundaje, Anshul ^{[1
,10
]}

Greenleaf, William J. ^{[1
,11
,12
,13
]}

机构：

[1] Stanford Univ, Sch Med, Dept Genet, Stanford, CA 94305 USA

[2] Stanford Univ, Sch Med, Canc Biol Program, Stanford, CA 94305 USA

[3] Stanford Univ, Ctr Personal Dynam Regulomes, Stanford, CA 94305 USA

[4] Stanford Univ, Dept Bioengn, Stanford, CA 94305 USA

[5] Univ Chicago, Dept Chem, Chicago, IL 60637 USA

[6] Stanford Univ, Chem Engn & Med Human Hlth ChEM H, Stanford, CA 94305 USA

[7] Univ Chicago, Dept Biochem & Mol Biol, Chicago, IL 60637 USA

[8] Univ Chicago, Inst Biophys Dynam, Chicago, IL 60637 USA

[9] Univ Chicago, Howard Hughes Med Inst, Chicago, IL 60637 USA

[10] Stanford Univ, Dept Comp Sci, Stanford, CA 94305 USA

[11] Stanford Univ, Dept Appl Phys, Stanford, CA 94305 USA

[12] Stanford Univ, Ctr Personal Dynam Regulomes, Stanford, CA 94305 USA

[13] Chan Zuckerberg Biohub, San Francisco, CA 94158 USA

来源：

GENOME BIOLOGY | 2023年 / 24卷 / 01期

基金：

美国国家卫生研究院;

关键词：

MULTIPLE SEQUENCE ALIGNMENT; OFF-TARGET SITES; CRISPR; SEQ; RNA; ACTIVATION; LANDSCAPE;

D O I：

10.1186/s13059-023-02930-z

中图分类号：

Q81 [生物工程学（生物技术）]; Q93 [微生物学];

学科分类号：

071005 ; 0836 ; 090102 ; 100705 ;

摘要：

Detecting and mitigating off-target activity is critical to the practical application of CRISPR-mediated genome and epigenome editing. While numerous methods have been developed to map Cas9 binding specificity genome-wide, they are generally time-consuming and/or expensive, and not applicable to catalytically dead CRISPR enzymes. We have developed CasKAS, a rapid, inexpensive, and facile assay for identifying off-target CRISPR enzyme binding and cleavage by chemically mapping the unwound single-stranded DNA structures formed upon binding of a sgRNA-loaded Cas9 protein. We demonstrate this method in both in vitro and in vivo contexts.

引用

页数：15

共 39 条

[1] The ENCODE Blacklist: Identification of Problematic Regions of the Genome [J].

Amemiya, Haley M. ;

Kundaje, Anshul ;

Boyle, Alan P. .

SCIENTIFIC REPORTS, 2019, 9 (1)

[2] Cas-OFFinder: a fast and versatile algorithm that searches for potential off-target sites of Cas9 RNA-guided endonucleases [J].

Bae, Sangsu ;

Park, Jeongbin ;

Kim, Jin-Soo .

BIOINFORMATICS, 2014, 30 (10) :1473-1475

[3]

Cameron P, 2017, NAT METHODS, V14, P600, DOI [10.1038/NMETH.4284, 10.1038/nmeth.4284]

[4] Nucleotide-resolution DNA double-strand break mapping by next-generation sequencing [J].

Crosetto, Nicola ;

Mitra, Abhishek ;

Silva, Maria Joao ;

Bienko, Magda ;

Dojer, Norbert ;

Wang, Qi ;

Karaca, Elif ;

Chiarle, Roberto ;

Skrzypczak, Magdalena ;

Ginalski, Krzysztof ;

Pasero, Philippe ;

Rowicka, Maga ;

Dikic, Ivan .

NATURE METHODS, 2013, 10 (04) :361-+

[5] Orthogonal gene knockout and activation with a catalytically active Cas9 nuclease [J].

Dahlman, James E. ;

Abudayyeh, Omar O. ;

Joung, Julia ;

Gootenberg, Jonathan S. ;

Zhang, Feng ;

Konermann, Silvana .

NATURE BIOTECHNOLOGY, 2015, 33 (11) :1159-U147

[6] Precision digital mapping of endogenous and induced genomic DNA breaks by INDUCE-seq [J].

Dobbs, Felix M. ;

van Eijk, Patrick ;

Fellows, Mick D. ;

Loiacono, Luisa ;

Nitsch, Roberto ;

Reed, Simon H. .

NATURE COMMUNICATIONS, 2022, 13 (01)

[7] Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9 [J].

Doench, John G. ;

Fusi, Nicolo ;

Sullender, Meagan ;

Hegde, Mudra ;

Vaimberg, Emma W. ;

Donovan, Katherine F. ;

Smith, Ian ;

Tothova, Zuzana ;

Wilen, Craig ;

Orchard, Robert ;

Virgin, Herbert W. ;

Listgarten, Jennifer ;

Root, David E. .

NATURE BIOTECHNOLOGY, 2016, 34 (02) :184-+

[8] MUSCLE: multiple sequence alignment with high accuracy and high throughput [J].

Edgar, RC .

NUCLEIC ACIDS RESEARCH, 2004, 32 (05) :1792-1797

[9] Identifying ChIP-seq enrichment using MACS [J].

Feng, Jianxing ;

Liu, Tao ;

Qin, Bo ;

Zhang, Yong ;

Liu, Xiaole Shirley .

NATURE PROTOCOLS, 2012, 7 (09) :1728-1740

[10] Genome-wide detection of DNA double-stranded breaks induced by engineered nucleases [J].

Frock, Richard L. ;

Hu, Jiazhi ;

Meyers, Robin M. ;

Ho, Yu-Jui ;

Kii, Erina ;

Alt, Frederick W. .

NATURE BIOTECHNOLOGY, 2015, 33 (02) :179-186

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