Rapid Screening for CRISPR-Directed Editing of the Drosophila Genome Using white Coconversion

Abstract

Adoption of a streamlined version of the bacterial clustered regular interspersed short palindromic repeat (CRISPR)/Cas9 defense system has accelerated targeted genome engineering. The Streptococcus pyogenes Cas9 protein, directed by a simplified, CRISPR-like single-guide RNA, catalyzes a double-stranded DNA break at a specific genomic site; subsequent repair by end joining can introduce mutagenic insertions or deletions, while repair by homologous recombination using an exogenous DNA template can incorporate new sequences at the target locus. However, the efficiency of Cas9-directed mutagenesis is low in Drosophila melanogaster. Here, we describe a strategy that reduces the time and effort required to identify flies with targeted genomic changes. The strategy uses editing of the white gene, evidenced by altered eye color, to predict successful editing of an unrelated gene-of-interest. The red eyes of wild-type flies are readily distinguished from white-eyed (end-joining-mediated loss of White function) or brown-eyed (recombination-mediated conversion to the whitecoffee allele) mutant flies. When single injected G0 flies produce individual G1 broods, flies carrying edits at a gene-of-interest were readily found in broods in which all G1 offspring carried white mutations. Thus, visual assessment of eye color substitutes for wholesale PCR screening of large numbers of G1 offspring. We find that end-joining-mediated mutations often show signatures of microhomology-mediated repair and that recombination-based mutations frequently involve donor plasmid integration at the target locus. Finally, we show that gap repair induced by two guide RNAs more reliably converts the intervening target sequence, whereas the use of Lig4169 mutants to suppress end joining does not improve recombination efficacy.

The RNA-binding ATPase, Armitage, Couples piRNA Amplification in Nuage to Phased piRNA Production on Mitochondria

“…In cultured ovarian somatic cells, tagged, overexpressed Zuc localizes to mitochondria through its N-terminal mitochondrial localization signal peptide (Saito et al, 2010;Handler et al, 2013). We examined the localization of endogenous Zuc in the germline using a fly strain in which endogenous Zuc bears a C-terminal 3×FLAG tag (Ge et al, 2016). Homozygous flies expressing only Zuc-3×FLAG showed no detectable defects in fertility or piRNA biogenesis, and the tagged Zuc, expressed from its native chromosomal location, was readily detected using anti-FLAG antibody.…”

“…armi ∆1 removes the armi, CycJ, and CG14971 coding sequences. We therefore used trans-heterozygous armi 72 Other stocks: Wild-type and the H169Y mutant endogenous Zuc, tagged with 3×FLAG strains have been described previously (Ge et al, 2016). minotaur z3-5967 (Vagin et al, 2013) was crossed in trans to Df(3R)ED6280 (Bloomington #29667) to obtain minotaur mutants.…”

ovoDco-selection: a method for enriching CRISPR/Cas9-edited alleles inDrosophila

“…Despite the enormous power of CRISPR/Cas9 for genome editing, screening for successful genome-editing events remains a time-consuming and laborious technical bottleneck in all organisms and in cell culture. In response to this challenge, a number of techniques have been developed to enrich and/or select for desired CRISPR events, collectively referred to as "CRISPR co-selection" (aka "co-CRISPR" or "CRISPR coconversion") (Kim et al 2014;Arribere et al 2014;Liao et al 2015;Shy et al 2016;Ge et al 2016;Agudelo et al 2017). CRISPR co-selection is based on the observation that when two independent short guide RNAs (sgRNAs) and Cas9 protein are introduced to a population of cells simultaneously, CRISPR events tend to cooccur at both loci within individual cells at a higher-than-random frequency.…”

Efficient Screening of CRISPR/Cas9-Induced Events in Drosophila Using a Co-CRISPR Strategy

“…A very high frequency of the marker phenotype would diminish its usefulness in identifying broods with a co-CRISPR event, and a poor frequency of conversion would likely lead to a failure in detecting CRISPR mutations in the gene of interest. During the preparation of our manuscript, a study in Drosophila was published using the white ( w ) gene as a co-CRISPR marker (Ge et al 2016). The advantages of the w gene as a co-CRISPR marker are similar to the e gene used in our study.…”