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

Abstract

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 selfselecting 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 selfselection 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.

Transgenic tools for targeted chromosome rearrangements allow construction of balancer chromosomes in non-melanogaster Drosophila species

“…Similar efficient approaches have not been reported in Drosophila , and I have failed to recover inversions in D. melanogaster using this kind of approach to generate inversions on the order of 10 kb–1 Mbp. Recently, Ng and Reed reported successful isolation of a single inversion event in D. melanogaster using transgenic sources of Cas9 and gRNAs ( Ng and Reed 2019 ). The efficiency of this approach is not yet known.…”

Transgenic tools for targeted chromosome rearrangements allow construction of balancer chromosomes in non-melanogaster Drosophila species

“…Similar efficient approaches have not been reported in Drosophila, and I have failed to recover inversions in D. melanogaster using this kind of approach to generate inversions on the order of 10kb-1Mbp. Recently, Ng and Reed reported successful isolation of a single inversion event in D. melanogaster using transgenic sources of Cas9 and gRNAs (Ng and Reed 2019). The efficiency of this approach is not yet known.…”