Identifying genome-wide off-target sites of CRISPR RNA–guided nucleases and deaminases with Digenome-seq

Daesik Kim, Beum-Chang Kang, Jin-Soo Kim

Published: 2021-01-17 DOI: 10.1038/s41596-020-00453-6

Digenome-seq

off-target sites

CRISPR

nucleases

deaminases

AI 解读

Generation of mouse–human chimeric embryos

Genome-wide quantification of transcription factor binding at single-DNA-molecule resolution using methyl-transferase footprinting

Generation of CRISPR–Cas9-mediated genetic knockout human intestinal tissue–derived enteroid lines by lentivirus transduction and single-cell cloning

A puromycin-dependent activity-based sensing probe for histochemical staining of hydrogen peroxide in cells and animal tissues

Decoding personal biotic and abiotic airborne exposome

A clinically relevant model of focal embolic cerebral ischemia by thrombus and thrombolysis in rhesus monkeys

Bioengineering human intestinal mucosal grafts using patient-derived organoids, fibroblasts and scaffolds

Biomimetic tail-to-head terpene cyclizations using the resorcin[4]arene capsule catalyst

Neuroscience robotics for controlled induction and real-time assessment of hallucinations

Scanorama: integrating large and diverse single-cell transcriptomic datasets

Single-cell RNA sequencing (scRNA-seq)（单细胞RNA测序）

Data integration（数据集成）

Heterogeneity（异质性）

查看全部

Sections

Figures

References

Digested genome sequencing (Digenome-seq) is a highly sensitive, easy-to-carry-out, cell-free method for experimentally identifying genome-wide off-target sites of programmable nucleases and deaminases (also known as base editors). Genomic DNA is digested
Introduction
Materials
Procedure
Troubleshooting
Timing
Anticipated results
References
ACKNOWLEDGMENTS

Mali, P. et al. RNA-guided human genome engineering via Cas9. Science 339, 823–826 (2013).
Jinek, M. et al. RNA-programmed genome editing in human cells. eLife 2, e00471 (2013).
Cong, L. et al. Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819–823 (2013).
Cho, S. W., Kim, S., Kim, J. M. & Kim, J. S. Targeted genome engineering in human cells with the Cas9 RNA-guided endonuclease. Nat. Biotechnol. 31, 230–232 (2013).
Kim, H. & Kim, J. S. A guide to genome engineering with programmable nucleases. Nat. Rev. Genet. 15, 321–334 (2014).
Pattanayak, V. et al. High-throughput profiling of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity. Nat. Biotechnol. 31, 839–843 (2013).
Fu, Y. et al. High-frequency off-target mutagenesis induced by CRISPR–Cas nucleases in human cells. Nat. Biotechnol. 31, 822–826 (2013).
Cradick, T. J., Fine, E. J., Antico, C. J. & Bao, G. CRISPR/Cas9 systems targeting beta-globin and CCR5 genes have substantial off-target activity. Nucleic Acids Res. 41, 9584–9592 (2013).
Cho, S. W. et al. Analysis of off-target effects of CRISPR/Cas-derived RNA-guided endonucleases and nickases. Genome Res. 24, 132–141 (2014).
Hsu, P. D. et al. DNA targeting specificity of RNA-guided Cas9 nucleases. Nat. Biotechnol. 31, 827–832 (2013).
Morgens, D. W. et al. Genome-scale measurement of off-target activity using Cas9 toxicity in high-throughput screens. Nat. Commun. 8, 15178 (2017).
Gabriel, R. et al. An unbiased genome-wide analysis of zinc-finger nuclease specificity. Nat. Biotechnol. 29, 816–823 (2011).
Lee, H. J., Kim, E. & Kim, J. S. Targeted chromosomal deletions in human cells using zinc finger nucleases. Genome Res. 20, 81–89 (2010).
Lee, H. J., Kweon, J., Kim, E., Kim, S. & Kim, J. S. Targeted chromosomal duplications and inversions in the human genome using zinc finger nucleases. Genome Res. 22, 539–548 (2012).
Brunet, E. et al. Chromosomal transs induced at specified loci in human stem cells. Proc. Natl Acad. Sci. USA 106, 10620–10625 (2009).
Jinek, M. et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816–821 (2012).
Zetsche, B. et al. Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR–Cas system. Cell 163, 759–771 (2015).
Komor, A. C., Kim, Y. B., Packer, M. S., Zuris, J. A. & Liu, D. R. Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. Nature 533, 420–424 (2016).
Nishida, K. et al. Targeted nucleotide editing using hybrid prokaryotic and vertebrate adaptive immune systems. Science 353, aaf8729 (2016).
Gehrke, J. M. et al. An APOBEC3A-Cas9 base editor with minimized bystander and off-target activities. Nat. Biotechnol. 36, 977–982 (2018).
Wang, X. et al. Efficient base editing in methylated regions with a human APOBEC3A-Cas9 fusion. Nat. Biotechnol. 36, 946–949 (2018).
Gaudelli, N. M. et al. Programmable base editing of A*T to G*C in genomic DNA without DNA cleavage. Nature 551, 464–471 (2017).
Li, X. et al. Base editing with a Cpf1-cytidine deaminase fusion. Nat. Biotechnol. 36, 324–327 (2018).
Kleinstiver, B. P. et al. Engineered CRISPR–Cas12a variants with increased activities and improved targeting ranges for gene, epigenetic and base editing. Nat. Biotechnol. 37, 276–282 (2019).
Kim, D., Kim, D. E., Lee, G., Cho, S. I. & Kim, J. S. Genome-wide target specificity of CRISPR RNA-guided adenine base editors. Nat. Biotechnol. 37, 430–435 (2019).
Jin, S. et al. Cytosine, but not adenine, base editors induce genome-wide off-target mutations in rice. Science 364, 292–295 (2019).
Zuo, E. et al. Cytosine base editor generates substantial off-target single-nucleotide variants in mouse embryos. Science 364, 289–292 (2019).
Kim, D. et al. Genome-wide target specificities of CRISPR RNA-guided programmable deaminases. Nat. Biotechnol. 35, 475–480 (2017).
Kim, D. et al. Digenome-seq: genome-wide profiling of CRISPR–Cas9 off-target effects in human cells. Nat. Methods 12, 237–243 (2015).
Kim, D., Kim, S., Kim, S., Park, J. & Kim, J. S. Genome-wide target specificities of CRISPR–Cas9 nucleases revealed by multiplex Digenome-seq. Genome Res. 26, 406–415 (2016).
Park, J. et al. Digenome-seq web tool for profiling CRISPR specificity. Nat. Methods 14, 548–549 (2017).
Kim, E. et al. In vivo genome editing with a small Cas9 orthologue derived from Campylobacter jejuni. Nat. Commun. 8, 14500 (2017).
Kim, D. et al. Genome-wide analysis reveals specificities of Cpf1 endonucleases in human cells. Nat. Biotechnol. 34, 863–868 (2016).
Liang, P. et al. Genome-wide profiling of adenine base editor specificity by EndoV-seq. Nat. Commun. 10, 67 (2019).
Tsai, S. Q. et al. GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR–Cas nucleases. Nat. Biotechnol. 33, 187–197 (2015).
Tsai, S. Q. et al. CIRCLE-seq: a highly sensitive in vitro screen for genome-wide CRISPR–Cas9 nuclease off-targets. Nat. Methods 14, 607–614 (2017).
Cameron, P. et al. Mapping the genomic landscape of CRISPR–Cas9 cleavage. Nat. Methods 14, 600–606 (2017).
Kim, D. & Kim, J. DIG-seq: a genome-wide CRISPR off-target profiling method using chromatin DNA. Genome Res. 28, 1894–1900 (2018).
Kim, D., Luk, K., Wolfe, S. A. & Kim, J. S. Evaluating and enhancing target specificity of gene-editing nucleases and deaminases. Annu. Rev. Biochem. 88, 191–220 (2019).
Tsai, S. Q. & Joung, J. K. Defining and improving the genome-wide specificities of CRISPR–Cas9 nucleases. Nat. Rev. Genet. 17, 300–312 (2016).
Koo, T., Lee, J. & Kim, J. S. Measuring and reducing off-target activities of programmable nucleases including CRISPR–Cas9. Mol. Cells 38, 475–481 (2015).
Bae, S., Park, J. & Kim, J. S. Cas-OFFinder: a fast and versatile algorithm that searches for potential off-target sites of Cas9 RNA-guided endonucleases. Bioinformatics 30, 1473–1475 (2014).
Singh, R., Kuscu, C., Quinlan, A., Qi, Y. & Adli, M. Cas9-chromatin binding information enables more accurate CRISPR off-target prediction. Nucleic Acids Res. 43, e118 (2015).
Xiao, A. et al. CasOT: a genome-wide Cas9/gRNA off-target searching tool. Bioinformatics 30, 1180–1182 (2014).
Montague, T. G., Cruz, J. M., Gagnon, J. A., Church, G. M. & Valen, E. CHOPCHOP: a CRISPR/Cas9 and TALEN web tool for genome editing. Nucleic Acids Res. 42, W401–W407 (2014).
Haeussler, M. et al. Evaluation of off-target and on-target scoring algorithms and integration into the guide RNA selection tool CRISPOR. Genome Biol. 17, 148 (2016).
Wang, X. et al. Unbiased detection of off-target cleavage by CRISPR–Cas9 and TALENs using integrase-defective lentiviral vectors. Nat. Biotechnol. 33, 175–178 (2015).
Frock, R. L. et al. Genome-wide detection of DNA double-stranded breaks induced by engineered nucleases. Nat. Biotechnol. 33, 179–186 (2015).
Ran, F. A. et al. In vivo genome editing using Staphylococcus aureus Cas9. Nature 520, 186–191 (2015).
Yan, W. X. et al. BLISS is a versatile and quantitative method for genome-wide profiling of DNA double-strand breaks. Nat. Commun. 8, 15058 (2017).
Wienert, B. et al. Unbiased detection of CRISPR off-targets in vivo using DISCOVER-Seq. Science 364, 286–289 (2019).
Robinson, J. T. et al. Integrative genomics viewer. Nat. Biotechnol. 29, 24–26 (2011).
Rees, H. A. et al. Improving the DNA specificity and applicability of base editing through protein engineering and protein delivery. Nat. Commun. 8, 15790 (2017).
Schmittgen, T. D. & Livak, K. J. Analyzing real-time PCR data by the comparative CT method. Nat. Protoc. 3, 1101–1108 (2008).

Identifying genome-wide off-target sites of CRISPR RNA–guided nucleases and deaminases with Digenome-seq

Supplementary information

Reporting Summary

推荐阅读