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Crispr/Cas9-mediated cleavages facilitate homologous recombination during genetic engineering of a large chromosomal region
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  • Fan Zhang,
  • De Cheng,
  • Shuwen Wang,
  • Jiyue Zhu
Fan Zhang
Washington State University
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De Cheng
Washington State University
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Shuwen Wang
Washington State University
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Jiyue Zhu
Washington State University
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Abstract

Homologous recombination over large genomic regions is difficult to achieve due to low efficiencies. Here, we report the successful engineering of a humanized mTert allele, hmTert, in the mouse genome by replacing an 18.1-kb genomic region around the mTert gene with a recombinant fragment of over 45.5-kb, using homologous recombination facilitated by the Crispr/Cas9 technology, in mouse embryonic stem cells (mESCs). In our experiments, with specific sites of DNA double strand breaks (DSBs) by Crispr/Cas9 system, the homologous recombination efficiency was up to 11% and 16% in two mESC lines TC1 and v6.5, respectively. Overall, we obtained a total of 27 mESC clones with heterozygous hmTert/mTert alleles and 3 clones with homozygous hmTert alleles. DSBs induced by Crispr/Cas9 cleavages also caused high rates of genomic DNA deletions and mutations at small guide RNA (sgRNA) target sites. Our results indicated the Crispr/Cas9 system significantly increased the efficiency of homologous recombination-mediated gene editing over a large genomic region in mammal cells, but also inherently caused mutations at unedited target sites. Overall, this strategy provides an efficient and feasible way for manipulating large chromosomal regions.

Peer review status:ACCEPTED

31 Dec 2019Submitted to Biotechnology and Bioengineering
04 Jan 2020Submission Checks Completed
04 Jan 2020Assigned to Editor
11 Jan 2020Reviewer(s) Assigned
20 Feb 2020Review(s) Completed, Editorial Evaluation Pending
20 Feb 2020Editorial Decision: Revise Minor
26 Apr 20201st Revision Received
27 Apr 2020Submission Checks Completed
27 Apr 2020Assigned to Editor
12 May 2020Reviewer(s) Assigned
18 May 2020Review(s) Completed, Editorial Evaluation Pending
18 May 2020Editorial Decision: Accept