利用整合酶缺陷型慢病毒载体无偏检测 CRISPR-Cas9 和 TALENs 的脱靶切割。
Unbiased detection of off-target cleavage by CRISPR-Cas9 and TALENs using integrase-defective lentiviral vectors.
机构信息
Department of Virology, Beckman Research Institute of City of Hope, Duarte, California, USA.
1] Department of Virology, Beckman Research Institute of City of Hope, Duarte, California, USA. [2] Bone Marrow Transplantation Center, The First Affiliated Hospital, Zhejiang University, Hangzhou, Zhejiang, China.
出版信息
Nat Biotechnol. 2015 Feb;33(2):175-8. doi: 10.1038/nbt.3127. Epub 2015 Jan 19.
The utility of CRISPR-Cas9 and TALENs for genome editing may be compromised by their off-target activity. We show that integrase-defective lentiviral vectors (IDLVs) can detect such off-target cleavage with a frequency as low as 1%. In the case of Cas9, we find frequent off-target sites with a one-base bulge or up to 13 mismatches between the single guide RNA (sgRNA) and its genomic target, which refines sgRNA design.
CRISPR-Cas9 和 TALENs 用于基因组编辑的实用性可能因其脱靶活性而受到影响。我们表明，整合酶缺陷型慢病毒载体 (IDLVs) 可以以低至 1%的频率检测到这种脱靶切割。在 Cas9 的情况下，我们发现 sgRNA 与其基因组靶标之间存在频繁的脱靶位点，具有一个碱基凸起或多达 13 个错配，这需要改进 sgRNA 设计。
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