Screening for Genomic Modifications: A Method to Identify CRISPR-Generated Mutants in Drosophila
筆記録
– When injected embryos develop into G0 adults, cross all individual flies に suitable balancer stocks に expand potential F1 CRISPR edited lines and be able に track the inheritance of the genomic modification. Then, cross randomly chosen F1 single males に suitable balancer females に be able に recover offspring if an F1 male is positive for the mutation. Once the cross has taken, transfer the F1 male に 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 で the sample and is inactivated by high heat.
Now that DNA is accessible, proceed に PCR screening. For example, use primers designed に amplify a region that includes the new sequence. Only で 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 donor develop into adults, cross G0 flies individually に balancer flies from a line appropriate for the targeted allele. Anesthetize the F1 offspring from each G0 cross on a carbon dioxide pad and randomly pick 10 に 20 males under a stereo microscope. Individually cross each selected male に a balancer female from a line appropriate for the targeted allele.
When the F2 larvae hatch, pick the F1 father from each cross. Extract genomic DNA by squishing each fly for 10 に 15 seconds with a pipette tip containing 50 microliters of squishing buffer without dispensing the buffer. Then, dispense the remaining buffer into the tube and mix well.
Incubate at 37 degrees Celsius for 20 に 30 minutes. Following the incubation, place the tubes で 95 degrees Celsius heat block for 1 に 2 minutes に inactivate the proteinase K. After spinning down for 5 minutes at 10,000 times g, store the preparation at 4 degrees Celsius for further PCR analysis.
Perform three-step PCR based screens using primers forward 1 and reverse 1 に screen for the existence of the insertion or replacement. Perform PCR using forward 2 and reverse 2 primers に verify the insertion or replacement from three prime region. Perform PCR using primers M13F and reverse 3 に check ends-in HDR.
– Keep the fly lines with the confirmed ends-out HDR and establish balance stocks from the F2 generation. Out cross the balancer flies again に remove any unintended mutations on other chromosomes.
