Homology-Integrated CRISPR–Cas (HI-CRISPR) System for One-Step Multigene Disruption in Saccharomyces cerevisiae

Abstract

One-step multiple gene disruption in the model organism Saccharomyces cerevisiae is a highly useful tool for both basic and applied research, but it remains a challenge. Here, we report a rapid, efficient, and potentially scalable strategy based on the type II Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR associated proteins (Cas) system to generate multiple gene disruptions simultaneously in S. cerevisiae. A 100 bp dsDNA mutagenizing homologous recombination donor is inserted between two direct repeats for each target gene in a CRISPR array consisting of multiple donor and guide sequence pairs. An ultrahigh copy number plasmid carrying iCas9, a variant of wild-type Cas9, trans-encoded RNA (tracrRNA), and a homology-integrated crRNA cassette is designed to greatly increase the gene disruption efficiency. As proof of concept, three genes, CAN1, ADE2, and LYP1, were simultaneously disrupted in 4 days with an efficiency ranging from 27 to 87%. Another three genes involved in an artificial hydrocortisone biosynthetic pathway, ATF2, GCY1, and YPR1, were simultaneously disrupted in 6 days with 100% efficiency. This homology-integrated CRISPR (HI-CRISPR) strategy represents a powerful tool for creating yeast strains with multiple gene knockouts.

High-throughput creation and functional profiling of eukaryotic DNA sequence variant libraries using CRISPR/Cas9

“…However, upon selecting for cells with the guide+donor, we found that the number of colonies with the desired genetic alteration were low (<5%), consistent with earlier attempts at in cis guide+donor delivery in yeast 5 . We sought to increase the percentage of correctly edited cells in order to enable efficient genome-scale measurements via NGS.…”

Development of a CRISPR-Cas9 Tool Kit for Comprehensive Engineering of Bacillus subtilis

“…SD from experiments performed in triplicate are shown. 14) and S. cerevisiae (23,24), all of which rely on multicopy vectors to deliver CRISPR-Cas9 machinery.…”

“…The CRISPR-induced double-stranded breaks (DSBs) enable selection of mutants that evade DNA cleavage via recombination of exogenous editing templates. Accordingly, the CRISPR-Cas9 system has proven to be an indispensable tool for genome editing in bacteria (9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22), yeasts (23,24) and higher eukaryotes (25)(26)(27)(28).…”

“…While CRISPR-Cas9 offers potential solutions to these technical issues in E. coli and S. cerevisiae (9,10,23,24), the protocols include multicopy plasmids which must be removed from the cell. In that regard, an ideal CRISPR-Cas9 system should facilitate multiple mutations, either in series or simultaneously, while imposing minimal physiological impact on the host.…”

Advancing biotechnology with CRISPR/Cas9: recent applications and patent landscape

“…1e). Several examples, such as the type III CRISPR-Csy4 [25,76] or natural CRISPR array [1,14] have been shown to efficiently generate multiple gRNAs from a single transcript (Fig. 1e).…”