A method to estimate the frequency of chromosomal rearrangements induced by CRISPR/Cas9 multiplexing in Drosophila
Authors: Bruce Reed; William Ng; Richard Do; Brittney Lato; Emily Baker; Mackenzie Vallee
Affiliation: University of Waterloo, Waterloo ON, CANADA
Keywords: g. CRISPR/Cas9; n. other (autosynaptic chromosome elements)
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. The frequency of chromosome rearrangements induced by CRISPR/Cas9 multiplexing, however, remains unknown in Drosophila. To estimate the frequency of chromosome rearrangements induced by multiplexing, we developed a self-selecting screening system using Drosophila stocks that carry an autosomal pericentric inversion in what is known as the autosynaptic form. All progeny of normal females crossed to males of these autosynaptic stocks are lethal due to excessive aneuploidy. If an inversion is induced within the female germline, however, and if this inversion 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 screening method, we were able to estimate the frequency of viable progeny from females having germline expression of active Cas9 together with ubiquitously expressed sgRNAs targeted to desired inversion breakpoints. Salivary gland polytene chromosome analysis, PCR, and sequencing confirmed the recovery of chromosome breakpoints induced between the two sgRNA target sites. Overall, we demonstrate that CRISPR/Cas9 multiplexing can induce chromosomal rearrangements in Drosophila. In general, the generation of a pericentric inversion within a female germline using CRISPR/Cas9 multiplexing varied depending on the choice of sgRNAs and occurred less than once for every 100 germlines tested. This frequency was also found to be comparable to using FLP/FRT site specific recombination. In using this particular system, we conclude that the induction of chromosomal rearrangements associated with CRISPR/Cas9 multiplexing is not a high frequency event.