Robust CRISPR/Cas9-Mediated Tissue-Specific Mutagenesis Reveals Gene Redundancy and Perdurance inDrosophila

Abstract

Tissue-specific loss-of-function (LOF) analysis is essential for characterizing gene function. Here, we present a simple, yet highly efficient, clustered regularly interspaced short palindromic repeats (CRISPR)-mediated tissue-restricted mutagenesis (CRISPR-TRiM) method for ablating gene function in Drosophila. This binary system consists of a tissue-specific Cas9 and a ubiquitously expressed multi-guide RNA (gRNA) transgene. We describe convenient toolkits for making enhancer-driven Cas9 lines and multi-gRNAs that are optimized for mutagenizing somatic cells. We demonstrate that insertions or deletions in coding sequences more reliably cause somatic mutations than DNA excisions induced by two gRNAs. We further show that enhancer-driven Cas9 is less cytotoxic yet results in more complete LOF than Gal4-driven Cas9 in larval sensory neurons. Finally, CRISPR-TRiM efficiently unmasks redundant soluble N-ethylmaleimide-sensitive factor attachment protein receptor gene functions in neurons and epidermal cells. Importantly, Cas9 transgenes expressed at different times in the neuronal lineage reveal the extent to which gene products persist in cells after tissue-specific gene knockout. These CRISPR tools can be applied to analyze tissue-specific gene function in many biological processes.

A large-scale resource for tissue-specific CRISPR mutagenesis inDrosophila

“…Toxicity mediated by high levels of Cas9 have been previously described by us and others in Drosophila and have also been observed in various other species (Port et al, 2014;Yang et al, 2018;Jiang et al, 2014;Poe et al, 2019). It has recently been reported that Cas9 transgenic lines directly fused to tissue-specific enhancers can mediate conditional mutagenesis with reduced toxicity (Poe et al, 2019). However, the UAS-uCas9 system reported here has several advantages, such as the ability to harness the thousands of publicly available tissue-specific Gal4 lines, the possibility to modulate Cas9 expression levels independent of enhancer strength, or the availability of the temperature-sensitive Gal4 repressor Gal80 for additional temporal control of gene editing.…”

“…The small amount of Cas9 that is needed for efficient gene editing allows expression of Cas9 at low levels, which avoids toxicity without sacrificing on-target activity. Toxicity mediated by high levels of Cas9 have been previously described by us and others in Drosophila and have also been observed in various other species (Port et al, 2014;Yang et al, 2018;Jiang et al, 2014;Poe et al, 2019). It has recently been reported that Cas9 transgenic lines directly fused to tissue-specific enhancers can mediate conditional mutagenesis with reduced toxicity (Poe et al, 2019).…”

“…The experiments described above were largely performed using UAS-cas9.P2 (Port and Bullock, 2016), which compared to UAS-cas9.P1 (Port et al, 2014), was designed to mediate lower expression levels to prevent toxicity previously observed when expressing Cas9 from the Gal4/UAS system (Port et al, 2014;Poe et al, 2019;Huynh et al, 2018). However, in combination with several Gal4 driver lines UAS-cas9.P2 still resulted in abnormal phenotypes or lethality.…”

DrosophilaTrpm mediates calcium influx during egg activation

“…We designed three sets of gRNAs targeting trpm (see Materials and Methods). We used the "Cas9-LEThAL" (24) method to evaluate the efficiency of our gRNA expression constructs. Among them, gRNA set 3 displayed the highest efficiency (Table.S1).…”

“…To confirm the efficiency of constructs expressing gRNAs, we employed the "Cas9-LEThAL" method (24) to validate the efficiency of our gRNA constructs. Males carrying gRNA constructs were crossed to act-Cas9, lig4-mutant females.…”