Defining genome-wide CRISPR–Cas genome-editing nuclease activity with GUIDE-seq

J. Keith Joung, Martin J. Aryee, Nikolay L. Malinin, GaHyun Lee, Cicera R. Lazzarotto, Yichao Li, Zongli Zheng, Nhu T. Nguyen, Matthew Liebers, Ved V. Topkar, A. John Iafrate, Long P. Le, Shengdar Q. Tsai

Published: 2021-11-11 DOI: 10.1038/s41596-021-00626-x

off-target sites

high-throughput sequencing

GUIDE-seq

genome-editing

nuclease activity

AI 解读

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Genome-wide unbiased identification of double-stranded breaks enabled by sequencing (GUIDE-seq) is a sensitive, unbiased, genome-wide method for defining the activity of genome-editing nucleases in living cells. GUIDE-seq is based on the principle of effi
Introduction
Materials
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Anticipated results
References
ACKNOWLEDGMENTS

Maeder, M. L. & Gersbach, C. A. Genome-editing technologies for gene and cell therapy. Mol. Ther. 24, 430–446 (2016).
Wang, H., Russa, M. L. & Qi, L. S. CRISPR/Cas9 in genome editing and beyond. Annu. Rev. Biochem. 85, 1–38 (2015).
Anderson, K. R. et al. CRISPR off-target analysis in genetically engineered rats and mice. Nat. Methods https://doi.org/10.1038/s41592-018-0011-5 (2018).
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).
Xie, K. & Yang, Y. RNA-guided genome editing in plants using a CRISPR–Cas system. Mol. Plant 6, 1975–1983 (2013).
Cheng, Y. & Tsai, S. Q. Illuminating the genome-wide activity of genome editors for safe and effective therapeutics. Genome Biol. 19, 226 (2018).
Putney, S., Benkovic, S. & Schimmel, P. A DNA fragment with an alpha-phosphorothioate nucleotide at one end is asymmetrically blocked from digestion by exonuclease III and can be replicated in vivo. Proc. Natl Acad. Sci. 78, 7350–7354 (1981).
Lazzarotto, C. R. et al. CHANGE-seq reveals genetic and epigenetic effects on CRISPR–Cas9 genome-wide activity. Nat. Biotechnol. 38, 1317–1327 (2020).
Zheng, Z. et al. Anchored multiplex PCR for targeted next-generation sequencing. Nat. Med. 20, 1479–1484 (2014).
Tsai, S. Q., Topkar, V. V., Joung, K. J. & Aryee, M. J. Open-source guideseq software for analysis of GUIDE-seq data. Nat. Biotechnol. 34, 483–483 (2016).
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).
Wienert, B. et al. Unbiased detection of CRISPR off-targets in vivo using DISCOVER-Seq. Science 364, 286–289 (2019).
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).
Kim, D. et al. Digenome-seq: genome-wide profiling of CRISPR-Cas9 off-target effects in human cells. Nat. Methods 12, 237–243 (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).
Schmid-Burgk, J. L. et al. Highly parallel profiling of Cas9 variant specificity. Mol. Cell 78, 794–800.e8 (2020).
Crosetto, N. et al. Nucleotide-resolution DNA double-strand break mapping by next-generation sequencing. Nat. Methods 10, 361–365 (2013).
Wang, L. et al. Meganuclease targeting of PCSK9 in macaque liver leads to stable reduction in serum cholesterol. Nat. Biotechnol. 36, 717–725 (2018).
Miller, J. C. et al. Enhancing gene editing specificity by attenuating DNA cleavage kinetics. Nat. Biotechnol. 37, 945–952 (2019).
Kleinstiver, B. et al. Genome-wide specificities of CRISPR-Cas Cpf1 nucleases in human cells. Nat. Biotechnol. 34, 869.
Nobles, C. L. et al. iGUIDE: an improved pipeline for analyzing CRISPR cleavage specificity. Genome Biol. 20, 14 (2019).
Walton, R. T., Christie, K. A., Whittaker, M. N. & Kleinstiver, B. P. Unconstrained genome targeting with near-PAMless engineered CRISPR-Cas9 variants. Science https://doi.org/10.1126/science.aba8853 (2020).
Hendel, A. et al. Chemically modified guide RNAs enhance CRISPR-Cas genome editing in human primary cells. Nat. Biotechnol. 33, 985–989 (2015).
Fiume, M. et al. CRISPResso2 provides accurate and rapid genome editing sequence analysis. Nat. Biotechnol. 37, 224–226 (2019).

Defining genome-wide CRISPR–Cas genome-editing nuclease activity with GUIDE-seq

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