A Method for Rapid Selection of Randomly Induced Mutations in a Gene of Interest Using CRISPR/Cas9 Mediated Activation of Gene Expression. Issue 6 (1st June 2020)

• Record Type: Journal Article
• Authors: Ng, William A; Ma, Andrew; Chen, Molly; Reed, Bruce H
• Is Part Of: G3. Volume 10:Issue 6(2020)
• Page Start: 1893
• Page End: 1901
• Publication Date: 2020-06-01
• Subjects: CRISPR/Cas9 -- gene overexpression -- mutant screen -- hindsight/RREB-1; Genetics -- Research -- Periodicals; Genomics -- Periodicals; Genetics; Genomics; Genes; Genetics -- Research; Genomics; Electronic journals; Periodical; Periodicals; Fulltext; Internet Resources; Periodicals; 572.8
• DOI: 10.1534/g3.120.401299
• Languages: English
• ISSNs: 2160-1836

Abstract

Abstract: We have developed a CRISPR/Cas9 based method for isolating randomly induced recessive lethal mutations in a gene of interest (GOI) by selection within the F1 progeny of a single genetic cross. Our method takes advantage of the ability to overexpress a GOI using CRISPR/Cas9 mediated activation of gene expression. In essence, the screening strategy is based upon the idea that if overexpression of a wild type allele can generate a phenotype, then overexpression of a newly induced loss-of-function allele will lack this phenotype. As a proof-of-principle, we used this method to select EMS induced mutations of the Drosophila gene hindsight ( hnt ). From approximately 45, 000 F1 progeny we recovered 8 new EMS induced loss-of-function hnt alleles that we characterized as an allelic series of hypomorphic mutations. This new method can, in theory, be used to recover randomly induced point mutants in a GOI and can be applied to any circumstance where CRISPR/Cas9 mediated activation of gene expression is associated with lethality or a visible phenotype.