CRISPR‐Cas9 Genome Editing in Drosophila

Abstract

The CRISPR-Cas9 system has transformed genome engineering of model organisms from possible to practical. CRISPR-Cas9 can be readily programmed to generate sequence-specific double-strand breaks that disrupt targeted loci when repaired by error-prone non-homologous end joining or to catalyze precise genome modification through homology-directed repair (HDR). Here we describe a streamlined approach for rapid and highly efficient engineering of the Drosophila genome via CRISPR-Cas9-mediated HDR. In this approach, transgenic flies expressing Cas9 are injected with plasmids to express guide RNAs (gRNAs) and positively marked donor templates. We detail target site selection; gRNA plasmid generation; donor template design and construction; and the generation, identification and molecular confirmation of engineered lines. We also present alternative approaches and highlight key considerations for experimental design. The approach outlined here can be used to rapidly and reliably generate a variety of engineered modifications, including genomic deletions and replacements, precise sequence edits, and incorporation of protein tags.

Big Lessons from Tiny Flies:<em> Drosophila Melanogaster </em>as a Model to Explore Dysfunction of Dopaminergic and Serotonergic Neurotransmitter Systems

“…The life cycle of Drosophila melanogaster consists of four stages: egg, larva, pupa and fly. The duration of the life cycle is temperature dependent and it is completed in about ten days when the flies are maintained at 25 o C. Drosophila lends itself to large scale genetic screens [3][4][5], cell specific transgene expression [6][7][8] and precise genome editing (reviewed in [9,10]). The availability of this large genetic tool box makes Drosophila an ideal model organism to study conserved biological processes.…”

Tools and strategies for scarless allele replacement inDrosophilausing CRISPR/Cas9

“…Most notably, the CRISPR/Cas9 RNAguided nuclease system has been developed and optimized for a variety of applications from basic gene disruption to knock-ins, endogenous tagging of proteins, and modulation of gene expression. [1][2][3][4][5] In the CRISPR/ Cas9 system, adapted from Streptococcus pyogenes (henceforth shortened as "CRISPR"), the Cas9 endonuclease complexes with a single-guide RNA (sgRNA), which is designed by the experimenter to match a »20bp target sequence, and causes doublestrand cleavage 3 nucleotides 5 0 of the 3 0 end of the target sequence. 5 The selection of sgRNA target sites is flexible, but a protospacer-adjacent motif (PAM) (the trinucleotide 5 0 -NGG-3 0 ) is required immediately 3 0 of the 20bp target sequence.…”

“…5 After the doublestrand break is induced at the target site, the cell's native DNA repair machinery can (i) rejoin the 2 ends of the break through non-homologous end joining (NHEJ), an error-prone process that frequently results in small insertions or deletions, or (ii) close the gap using homology-directed repair (HDR), in which a DNA molecule with sequence homologous to the break site is used as a repair template. 3 The repair template used for double-strand break repair by HDR can be a homologous chromosome or an exogenous donor molecule with homology to both sides of the break site.…”

“…1A). 3,4,6,7 dsDNA plasmids can be much larger than ssODNs, allowing modifications to be made at a greater distance from the cleavage site as well as allowing larger sequences to be inserted at the site of repair. 4,6 To modify larger regions of sequence, 2 target sites can be used to cut out the region to be modified.…”

“…As an alternative, Drosophila researchers often choose to incorporate a visible transformation marker for phenotypic screening. 3 When gene deletion, disruption or tagging are desired, a selectable marker may be permanently integrated at the targeted locus, but if the goal is to determine the effects of precise nucleotide changes, any transformation markers used should be removed prior to phenotypic analysis. Recombinase-mediated cassette exchange (RMCE) has been used to remove and replace such a marker gene in Drosophila…”