Research Report

A Method for Producing Multiple-gene Mutations in a Gene Family that Utilizes sgRNA-target Mismatch at the Site Directly 5 of the PAM  

Yan Zhang* , Miaomiao Qin* , Chunbo Miao , Shenkui Liu
State Key Laboratory of Subtropical Silviculture, School of Forestry and Biotechnology, Zhejiang A&F University, Hangzhou, 311300
*Contributed equally to this work
Author    Correspondence author
Molecular Soil Biology, 2020, Vol. 11, No. 4   doi: 10.5376/msb.2020.11.0004
Received: 12 Jul., 2020    Accepted: 23 Jul., 2020    Published: 26 Aug., 2020
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This is an open access article published under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Preferred citation for this article:

Zhang Y., Qin M.M., Miao C.B., and Liu S.K., 2020, A method for producing multiple-gene mutations in a gene family that utilizes sgRNA-target mismatch at the site directly 5’ of the PAM, Molecular Soil Biology, 11(4): 1-7 (doi: 10.5376/msb.2020.11.0004)


The CRISPR-Cas9 system uses a small single-guide RNA (sgRNA) to direct the endonuclease Cas9 to recognize and cleave the target DNA. Previous studies in bacteria and mammal cells showed that perfect base pairing between sgRNA and target sites within 10–12 bp directly 5’ of the protospacer adjacent motif (PAM) determined the Streptococcus pyogenes Cas9 (SpCas9, the most widely used Cas9) specificity, whereas mismatches in the other region can be tolerated. Here, through gene editing of rice DST and MIR156 genes, we found that CRISPR-SpCas9 with a mismatch at 20th base (counting from the distal of PAM) of the target sites could still lead to high-frequency gene editing in rice. In rice, these results enrich the rules of target site selection for SpCas9 and will be conducive to the emergence of multiple mutations in the gene family.

CRISPR-Cas9; SgRNA; Gene family; Multiple-gene mutations
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