Prediction-based highly sensitive CRISPR off-target validation using target-specific DNA enrichment

被引：0
作者：
Seung-Hun Kang
Wi-jae Lee
Ju-Hyun An
Jong-Hee Lee
Young-Hyun Kim
Hanseop Kim
Yeounsun Oh
Young-Ho Park
Yeung Bae Jin
Bong-Hyun Jun
Junho K. Hur
Sun-Uk Kim
Seung Hwan Lee
机构：
[1] Kyung Hee University,Department of Medicine, Graduate School
[2] Korea Research Institute of Bioscience and Biotechnology (KRIBB),Futuristic Animal Resource & Research Center (FARRC)
[3] Konkuk University,Department of Bioscience and Biotechnology
[4] Korea University of Science and Technology (UST),Department of Functional Genomics, KRIBB School of Bioscience
[5] Korea Research Institute of Bioscience and Biotechnology (KRIBB),National Primate Research Center (NPRC)
[6] Kyungpook National University,School of Life Sciences and Biotechnology, BK21 Plus KNU Creative BioResearch Group
[7] Korea University,Division of Biotechnology, College of Life Science and Biotechnology
[8] Kyung Hee University,Department of Pathology, College of Medicine
[9] Kyung Hee University,Department of Biomedical Science, Graduate School
[10] Hanyang University,Department of Medical Genetics, College of Medicine
来源：
Nature Communications | / 11卷
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摘要：
CRISPR effectors, which comprise a CRISPR-Cas protein and a guide (g)RNA derived from the bacterial immune system, are widely used for target-specific genome editing. When the gRNA recognizes genomic loci with sequences that are similar to the target, deleterious mutations can occur. Off-target mutations with a frequency below 0.5% remain mostly undetected by current genome-wide off-target detection techniques. Here we report a method to effectively detect extremely small amounts of mutated DNA based on predicted off-target-specific amplification. In this study, we used various genome editors to induce intracellular genome mutations, and the CRISPR amplification method detected off-target mutations at a significantly higher rate (1.6~984 fold increase) than an existing targeted amplicon sequencing method. In the near future, CRISPR amplification in combination with genome-wide off-target detection methods will allow detection of genome editor-induced off-target mutations with high sensitivity and in a non-biased manner.
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  • [1] Ma H(2017)Correction of a pathogenic gene mutation in human embryos Nature 548 413-419
  • [2] Jiang W(2013)RNA-guided editing of bacterial genomes using CRISPR-Cas systems Nat. Biotechnol. 31 233-239
  • [3] Bikard D(2016)Genome-wide specificities of CRISPR-Cas Cpf1 nucleases in human cells Nat. Biotechnol. 34 869-874
  • [4] Cox D(2013)High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells Nat. Biotechnol. 31 822-826
  • [5] Zhang F(2015)GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases Nat. Biotechnol. 33 187-197
  • [6] Marraffini LA(2013)High-throughput profiling of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity Nat. Biotechnol. 31 839-843
  • [7] Kleinstiver BP(2016)Genome-wide analysis reveals specificities of Cpf1 endonucleases in human cells Nat. Biotechnol. 34 863-868
  • [8] Fu Y(2019)Unbiased detection of CRISPR off-targets in vivo using DISCOVER-Seq Science 364 286-289
  • [9] Tsai SQ(2014)Improving CRISPR-Cas nuclease specificity using truncated guide RNAs Nat. Biotechnol. 32 279-284
  • [10] Pattanayak V(2016)High-fidelity CRISPR–Cas9 nucleases with no detectable genome-wide off-target effects Nature 529 490-88
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