Ng, W. A. | Search bioRxiv

bioRxiv2019

A method to estimate the frequency of chromosomal rearrangements induced by CRISPR/Cas9 multiplexing in Drosophila.

Using CRISPR/Cas9 to simultaneously induce mutations in two or more target genes, commonly referred to as multiplexing, may result in chromosomal rearrangements such as inversions or translocations. While this may be undesirable in some contexts, the ability to recover chromosomal rearrangements targeted to specific sites in the genome is potentially a powerful tool. Before developing such tools, however, it is first important to measure the frequency with which chromosome rearrangements are induced by CRISPR/Cas9 multiplexing. To do this, we have developed a self-selecting screening system using a Drosophila line that carries an autosomal pericentric inversion in what is known as the autosynaptic form. All progeny of normal females crossed to males of this autosynaptic stock are lethal due to excessive aneuploidy. If an inversion is induced within the female germline, and if it is analogous to the inversion in the male autosynaptic line, then it is possible to recover progeny in which aneuploidy is reduced and viability is restored. Using this self-selection method, we screened 130 females and recovered one new autosynaptic element. Salivary gland polytene chromosome analysis, PCR, and sequencing confirmed the recovery of a breakpoint induced precisely between the two sgRNA target sites. Overall, we demonstrate that CRISPR/Cas9 multiplexing can induce chromosomal rearrangements in Drosophila. Also, in using this particular system, the recovery of chromosomal rearrangements was not a high frequency event.

genetics↗

bioRxiv2019

A method for rapid selection of randomly induced mutations in a gene of interest using CRISPR/Cas9 mediated activation of gene expression.

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.

genetics↗