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CRISPR/Cas9 inhibits rather than induces non-targeted DNA cleavage more likely to cause off-target single-nucleotide variants
  • +8
  • Ze Zhang,
  • Yuanyuan Guo,
  • Rongjia Zhang,
  • Wuchen Yang,
  • Zhengqing Xie,
  • Weiwei Chen,
  • Hua Ling,
  • Gangqiang Cao,
  • Baoming Tian,
  • Fang Wei,
  • shi gongyao
Ze Zhang
Zhengzhou University

Corresponding Author:[email protected]

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Yuanyuan Guo
Zhengzhou University
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Rongjia Zhang
Zhengzhou University
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Wuchen Yang
Zhengzhou University
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Zhengqing Xie
Zhengzhou University
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Weiwei Chen
Zhengzhou University
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Hua Ling
Zhengzhou University
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Gangqiang Cao
Zhengzhou University
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Baoming Tian
Zhengzhou University
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Fang Wei
Zhengzhou University
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shi gongyao
Zhengzhou University
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Abstract

CRISPR/Cas9 gene targeting technology has become the most widely used gene editing technology in both plants and animals. However, substantial off-target effect remains as a major imperfection hindering its further application. Here, Nicotiana benthamiana leaf cell-free system was used to simulate in vivo environment. And the effects of different CRISPR/Cas9 components on DNA stability in cell-free system were studied to explore possible mechanisms causing CRISPR off-target. The results showed that overexpressing Cas9, nCas9 and dCas9 significantly inhibited DNA cleavage in the cell extracts. While overexpressing RNPs accelerated the target DNA cleavage but inhibited non-target DNA digestion in cell extracts, overexpressing nRNP and dRNP blocked the cleavage of either target or non-target sequences. Meanwhile, analysis of whole-genome sequencing data from mice and rice edited by different CRISPR tools revealed that the main off-target mutations were SNVs (single nucleotide variants), rather than Indels (insertions and deletions) that were readily induced by DNA double-strand breaks. The off-target sites did not match the conventionally predicted places but were PAM-rich sites preferred. Our study suggests that PAM-dependent binding without cleavage of CRISPR/Cas9 to non-target sequences may increase off-target mutation risks through impeding the necessary cleavage process for repairing spontaneous or environmentally induced non-targeted DNA mutations.