High-throughput creation and functional profiling of DNA sequence variant libraries using CRISPR-Cas9 in yeast.

Nat Biotechnol
Authors
Keywords
Abstract

Construction and characterization of large genetic variant libraries is essential for understanding genome function, but remains challenging. Here, we introduce a Cas9-based approach for generating pools of mutants with defined genetic alterations (deletions, substitutions, and insertions) with an efficiency of 80-100% in yeast, along with methods for tracking their fitness en masse. We demonstrate the utility of our approach by characterizing the DNA helicase SGS1 with small tiling deletion mutants that span the length of the protein and a series of point mutations against highly conserved residues in the protein. In addition, we created a genome-wide library targeting 315 poorly characterized small open reading frames (smORFs,

Year of Publication
2018
Journal
Nat Biotechnol
Volume
36
Issue
6
Pages
540-546
Date Published
2018 07
ISSN
1546-1696
DOI
10.1038/nbt.4147
PubMed ID
29786095
PubMed Central ID
PMC5990468
Links
Grant list
P50 HG005550 / HG / NHGRI NIH HHS / United States
RM1 HG008525 / HG / NHGRI NIH HHS / United States
T32 CA009216 / CA / NCI NIH HHS / United States