Department of Chemical and Biomolecular Engineering, Clemson University, Clemson, SC, USA.
Department of Chemical and Biomolecular Engineering, University of Delaware, Newark, DE, USA.
Methods Mol Biol. 2021;2307:69-83. doi: 10.1007/978-1-0716-1414-3_4.
Metabolic engineering frequently requires both gene knockouts and gene integration. CRISPR-Cas9 has been extensively used to create double-stranded DNA breaks that result in indel mutations; however, such mutations can revert or create toxic product. Gene integration can also be accomplished by CRISPR-Cas9 introduced double-stranded DNA breaks and a donor DNA cassette. Here we describe our protocol for combining an efficient gene knockout created by introducing DNA cuts with two guide RNAs with a gene to be integrated at the knockout site. Including guide RNA target sites flanking the homology regions around the gene to be integrated enables both homology-directed repair and homology-mediated end joining, resulting in few deletions and a significant proportion of correctly knocked out and integrated genes.
代谢工程经常需要基因敲除和基因整合。CRISPR-Cas9 已被广泛用于产生导致缺失突变的双链 DNA 断裂；然而，这种突变可能会回复或产生有毒产物。基因整合也可以通过 CRISPR-Cas9 引入的双链 DNA 断裂和供体 DNA 盒来完成。在这里，我们描述了一种将通过引入 DNA 切割与两个向导 RNA 产生的高效基因敲除与要整合到敲除位点的基因相结合的方法。在要整合的基因周围的同源区域两侧包含向导 RNA 靶标，可同时实现同源定向修复和同源介导的末端连接，导致缺失较少，正确敲除和整合的基因比例显著增加。
