Systematic analysis of CRISPR-Cas9 mismatch tolerance reveals low levels of off-target activity
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- 作者：Emily M. Anderson ; Amanda Haupt ; John A. Schiel ; Eldon Chou ; Hidevaldo B. Machado ; 沤aklina Strezoska ; Steve Lenger ; Shawn McClelland ; Amanda Birmingham ; Annaleen Vermeulen ; Anja van Brabant Smith ; anja.smith@ge.com" class="auth_mail" title="E-mail the corresponding author
- 关键词：CRISPR ; clustered regularly interspaced palindromic repeats ; Cas9 ; CRISPR associated protein 9 ; crRNA ; CRISPR RNA ; tracrRNA ; trans-activating CRISPR RNA ; PAM ; protospacer adjacent motif ; sgRNA ; single guide RNA ; NGS ; next generation sequencing ; PSC ; pluripotent stem cells ; ACE ; monoacetate orthoester ; siRNA ; small interfering RNA ; T7E1 ; T7 endonuclease I ; PCR ; polymerase chain reaction ; GFP ; green fluorescent protein ; EGFP ; enhanced green fluorescent protein ; Ubi-GFP ; ubiquitin-conjugated green
- 刊名：Journal of Biotechnology
- 出版年：2015
- 出版时间：10 October 2015
- 年：2015
- 卷：211
- 期：Complete
- 页码：56-65
- 全文大小：2067 K
文摘
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CRISPR–Cas9 with synthetic crRNA: tracrRNA is an efficient system for gene editing.
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We tested 100s of mismatched-to-target crRNAs in a high-throughput functional assay.
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96% (365/380) of tested 2-base mismatched crRNAs do not function.
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2-Base mismatched sequences do not function in the PAM-proximal region of the crRNA.
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∼7% of 2-base mismatch combinations in the PAM-distal 5′ end of the crRNA function.