Optimized gene editing technology for Drosophila melanogaster using germ line-specific Cas9

Abstract

The ability to engineer genomes in a specific, systematic, and costeffective way is critical for functional genomic studies. Recent advances using the CRISPR-associated single-guide RNA system (Cas9/sgRNA) illustrate the potential of this simple system for genome engineering in a number of organisms. Here we report an effective and inexpensive method for genome DNA editing in Drosophila melanogaster whereby plasmid DNAs encoding short sgRNAs under the control of the U6b promoter are injected into transgenic flies in which Cas9 is specifically expressed in the germ line via the nanos promoter. We evaluate the off-targets associated with the method and establish a Web-based resource, along with a searchable, genome-wide database of predicted sgRNAs appropriate for genome engineering in flies. Finally, we discuss the advantages of our method in comparison with other recently published approaches.nanos-Cas9 | HRMA M uch of our knowledge of the mechanisms underlying biological processes relies on genetic approaches, whereby gene activity is perturbed and the phenotypic consequences of perturbation are analyzed in detail. In recent years, several major advances have been made in the design of methods for specifically and efficiently perturbing genomes. Arguably, the most exciting advances rely on the ability to induce double-strand breaks (DSBs) by targeting a nuclease to a specific genomic sequence. Repair of DSBs by the error-prone nonhomologous endjoining (NHEJ) mechanism allows for the recovery of small deletions; moreover, repair of DSBs by homologous recombination (HR) in the presence of a donor template opens the door to a wide range of specifically engineered changes at the targeted site (1).Two nuclease-based systems, the zinc-finger nuclease (ZFN) and transcription activator-like effector nuclease (TALEN) systems, work effectively in a number of organisms (2-7). But because these approaches require the production of a construct encoding a unique DNA-binding protein fused to the nuclease domain, they can be both cumbersome and costly. In contrast, the recent approach based on the bacterial CRISPR-associated single-guide RNA (Cas9/sgRNA) system does not require production of specific fusion proteins for each targeted sequence (8-10).Cas9 was first identified in type II Streptococcus pyogenes as an RNA-guided defense system against invading viruses and plasmids (11-13). This adaptive immune-like system contains three components: CRISPR RNA (crRNA), trans-activating CRISPR RNA (tracrRNA), and Cas9. The tracrRNA triggers Cas9 nuclease activity and the crRNA guides Cas9 to cleave the specific foreign dsDNA sequence via base-pairing between the crRNA and the target DNA. Importantly, a single-guide RNA (sgRNA, also known as chiRNA), comprising the minimal crRNA and tracrRNA, can function similarly to the crRNA and tracrRNA, thereby providing a simplified method for genome editing (8)(9)(10)(14)(15)(16)(17)(18)(19)(20).Given the great promise of the Cas9/sgRNA method for genome engineering, we set out to test the sys...

Optimized CRISPR/Cas tools for efficient germline and somatic genome engineering in Drosophila

“…We generated two constructs in which Cas9 was under the control of the nos promoter, which is active in the male and female germ line (15), and the nos 3′ UTR, which targets protein synthesis to the germ cells (15). These constructs are similar to those used by two previous studies to provide Cas9 transgenically (8,10). We also generated two novel constructs: (i) act-cas9, in which Cas9 is fused to the ubiquitously expressed actin5C (act) promoter and the SV40 3′ UTR, and (ii) UAScas9, which has yeast upstream-activating sequences (UAS), allowing expression under control of the Gal4 transcriptional activator (16), and the p10 3′ UTR, which promotes efficient translation (17).…”

“…Kondo and Ueda (8) expressed both Cas9 and gRNA from transgenes stably integrated into the genome, but all other studies have used microinjection of expression plasmids or of in vitro-transcribed RNA into embryos to deliver one or both CRISPR/Cas components (5)(6)(7)(9)(10)(11). Much of the currently available evidence suggests that transgenic provision of Cas9 increases rates of germ-line transmission substantially (8,10,11). However, the influence of different regulatory sequences within cas9 transgenes on the rate of mutagenesis and on the location where mutations are generated within the organism has not been evaluated.…”

Dendritic Eph organizes dendrodendritic segregation in discrete olfactory map formation in Drosophila

“…51324). The DNA fragments for the guide RNAs were subcloned in the BbsI-digested U6b-sgRNA-short (a kind gift from N. Perrimon) (Ren et al 2013) vector. The following primers were annealed to generate the DNA fragments for guide RNAs: ephrin I95 -1 (5 ′ -CTTCGATGTACCAAAAAAGGAAGA-3 ′ and 5 ′ -AAACTCTTCCTTTTTTGGTACATC-3 ′ ), ephrin I95 -2 (5 ′ -CTT CGGAATCAAATGATATTAATT-3 ′ and 5 ′ -AAACAATTAATA TCATTTGATTCC-3 ′ ), Eph-myc-1 (5 ′ -CTTCGACGGTAATCA TATTTTGGA-3´and 5´-AAACTCCAAAATATGATTACCG TC-3´), and Eph-myc-2 (5´CTTCGTACGTAAGGTGCGG TATTC-3 ′ and 5 ′ -AAACGAATACCGCACCTTACGTAC-3 ′ ).…”

Tousled-like kinase mediated a new type of cell death pathway in Drosophila

“…The efficiency of the atg2 RNAi was confirmed (Supplementary Figure S3). Using the CRISPRassociated single-guide RNA system (Cas9/sgRNA), 41 we generated a large deletion (2164 bp) in the exon region of the atg2 gene. The homozygous deletion was pupal lethal.…”