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. 2020 Sep 2;8:524676. doi: 10.3389/fbioe.2020.524676

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Copyright © 2020 Hao, Suo, Lin, Chen, Zhou, Liu, Cui and Zhou.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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Deletion of larger gene clusters mediated by CCB-CIGE system derived from CRISPR-Cpf1. (A) Scheme showing the composition of the CIGE system for the deletion of larger gene clusters. (B) CIGE-mediated disruption of the sacA gene in the Bacillus subtilis 168 strain. The editing efficiency was 20/20. (C) Identification of bac operon deletion mutants. bac operon is composed of seven genes (bacABCDEFG), about 6688 bp. Ten transformants were selected and verified by colony PCR. M and ck represent the 2-kb DNA ladder and wild-type B. subtilis 168, respectively. (D) Confirmation of pps operon deletion using colony PCR. Lane M, the 5-kb DNA marker from Takara. Lane ck1 and ck2, PCR amplification with the B. subtilis 168 and B. subtilis comK genomic DNA (gDNA) using external primers, respectively. Lane 1-10, PCR amplification with the mutant gDNA using external primer. The editing efficiency was 8/10.