Double Selection Enhances the Efficiency of Target-AID and Cas9-Based Genome Editing in Yeast. Issue 10 (1st October 2018)

• Record Type: Journal Article
• Authors: Després, Philippe C; Dubé, Alexandre K; Nielly-Thibault, Lou; Yachie, Nozomu; Landry, Christian R
• Is Part Of: G3. Volume 8:Issue 10(2018)
• Page Start: 3163
• Page End: 3171
• Publication Date: 2018-10-01
• Subjects: Yeast Genome editing -- CRISPR-Cas9 -- Target-AID Base editing -- Loss of function screens -- Chromosomal fusions; Genetics -- Research -- Periodicals; Genomics -- Periodicals; Genetics; Genomics; Genes; Genetics -- Research; Genomics; Electronic journals; Periodical; Periodicals; Fulltext; Internet Resources; Periodicals; 572.8
• DOI: 10.1534/g3.118.200461
• Languages: English
• ISSNs: 2160-1836

Abstract

Abstract: CRISPR-Cas9 loss of function (LOF) and base editing screens are powerful tools in genetics and genomics. Yeast is one of the main models in these fields, but has only recently started to adopt this new toolkit for high throughput experiments. We developed a double selection strategy based on co-selection that increases LOF mutation rates using the Target-AID base editor. We constructed the pDYSCKO vector, which is amenable to high throughput double selection experiments, and show that the improvement in Target-AID efficiency generalizes across loci. Using modeling, we show that this improvement in efficiency provides the required increased in detection power to measure the fitness effects of thousands of mutations in typical yeast pooled screens. We show that double selection can also improve Cas9 mediated LOF rates, but that this multiplex genome editing causes programmable chromosomal translocations at high frequency. This suggests that multiplex LOF editing should be performed with caution and that base-editors could be preferable tools for some screens in yeast. Base editing using double selection is simple and straightforward and provides an alternative to homology directed repair based high throughput variant strain construction methods.