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. 2022 Feb 7;9:803252. doi: 10.3389/fcell.2021.803252

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Copyright © 2022 Feng, Wang, Li, Xie, Liu, Li, Li, Wang, Hu, Yang and Guo.

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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Optimization strategies of different steps of the CRISPR/Cas system. (A) The sgRNA sequence was optimally designed and modified by truncation or addition of 3’ or 5’ end of sgRNA, DNA-RNA chimera, etc. (B) Cas9 protein was optimized for concentration, temperature, and time, as well as application of variants. (C) Optimization of the RNP complex was conducted for proportion, function conditions, and transformation methods. (D) Donor DNA was optimized for design of the DNA template, proximity to CRISPR components, and choice of high-efficient delivery system. (E) DNA repair pathway was optimized with inhibition of the NHEJ pathway, enhancement of the HDR pathway, and modification of other pathways.