RT Journal Article
SR Electronic
T1 A method for rapid selection of randomly induced mutations in a gene of interest using CRISPR/Cas9 mediated activation of gene expression
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 788372
DO 10.1101/788372
A1 Ng, William A.
A1 Ma, Andrew
A1 Chen, Molly
A1 Reed, Bruce H.
YR 2019
UL http://biorxiv.org/content/early/2019/10/11/788372.abstract
AB We have developed a CRISPR/Cas9 based method for isolating randomly induced recessive lethal mutations in a gene of interest (GOI) by phenotype 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. This method also depends on the use of CRISPR/Cas9 based mutagenesis to recover alleles of a GOI that are refractory to CRISPR/Cas9 mediated activation of gene expression but are otherwise wild type. 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.