Screening for Genomic Modifications: A Method to Identify CRISPR-Generated Mutants in Drosophila

- When injected embryos develop into G0 adults, cross all individual flies to suitable balancer stocks to expand potential F1 CRISPR edited lines and be able to track the inheritance of the genomic modification. Then, cross randomly chosen F1 single males to suitable balancer females to be able to recover offspring if an F1 male is positive for the mutation. Once the cross has taken, transfer the F1 male to a tube for screening.

To extract genomic DNA, squish the fly with a pipette tip containing extraction buffer. Once the tissue is broken down, eject the liquid. The extraction buffer contains proteinase K, an enzyme that digests proteins in the sample and is inactivated by high heat.

Now that DNA is accessible, proceed to PCR screening. For example, use primers designed to amplify a region that includes the new sequence. Only in the presence of the modification will the second primer bind and a PCR product be amplified. In the example, we will see flies being bred and screened by PCR for insertion of a transactivation sequence replacing the first exon of the branchless gene.

- When nos-Cas9 embryos previously injected with both the guide RNA expression plasmid and the replacement