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The effects of length and sequence of gRNA on Cas13b and Cas13d activity in vitro and in vivo
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  • Yuhui Liu,
  • Jian Fei,
  • Ping Jing,
  • Yi Zhou,
  • Jingyu Zhang,
  • Jiahao Shi,
  • Mengjie Zhang,
  • Hua Yang
Yuhui Liu
Tongji University
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Jian Fei
Tongji University

Corresponding Author:jfei@tongji.edu.cn

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Ping Jing
Tongji University
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Yi Zhou
Tongji University
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Jingyu Zhang
Tongji University
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Jiahao Shi
Tongji University
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Mengjie Zhang
Tongji University
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Hua Yang
Tongji University
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Abstract

Cas13 are the only CRISPR/Cas systems found so far, which target RNA strand while preserving chromosomal integrity. Cas13b or Cas13d cleaves RNA by the crRNA guidance. However, the effect of the characteristics of the spacer sequences, such as the length and sequence preference, on the activity of Cas13b and Cas13d remains unclear. Our study shows that neither Cas13b nor Cas13d has a particular preference for the sequence composition of gRNA, including the sequence of crRNA and its flanking sites on target RNA. However, the crRNA, complementary to the middle part of the target RNA, seems to show higher cleavage efficiency for both Cas13b and Cas13d. As for the length of crRNAs, the most appropriate crRNA length for Cas13b is 22-25 nt and crRNA as short as 15 nt is still functional. Whereas, Cas13d requires longer crRNA, and 22-30 nt crRNA can achieve good effect. Both Cas13b and Cas13d show the ability to process precursor crRNAs. Our study suggests that Cas13b may have a stronger precursor processing ability than Cas13d. There are few in vivo studies on the application of Cas13b or Cas13d in mammals. With the methods of transgenic mice and hydrodynamic injection via tail vein, our study showed that both of them had high knock-down efficiency against target RNA in vivo. These results indicate that Cas13b and Cas13d have great potential for in vivo RNA operation and disease treatment without damaging genomic DNA.
01 Jan 2023Submitted to Biotechnology Journal
03 Jan 2023Submission Checks Completed
03 Jan 2023Assigned to Editor
12 Jan 2023Reviewer(s) Assigned
21 Feb 2023Review(s) Completed, Editorial Evaluation Pending
27 Feb 2023Editorial Decision: Revise Major
26 Mar 20231st Revision Received
30 Mar 2023Submission Checks Completed
30 Mar 2023Assigned to Editor
30 Mar 2023Reviewer(s) Assigned