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Ng, W.A., Ma, A., Chen, M., Reed, B.H. (2020). A Method for Rapid Selection of Randomly Induced Mutations in a Gene of Interest Using CRISPR/Cas9 Mediated Activation of Gene Expression.  G3 (Bethesda) 10(6): 1893--1901.
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Research paper

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.

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PMC7263667 (PMC) (EuropePMC)
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    G3 (Bethesda)
    G3 : genes - genomes - genetics
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