Simultaneous Gene Excision and Integration by Dual-Guide CRISPR-Cas9.

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.