Using TRiP-CRISPR lines

Here we describe the fly crossing scheme for making mutants in the germline with TRiP-CRISPR Knockout (TRiP-KO) stocks.

Step 1: cross nanos-Cas9 (nos-Cas9) stock to individual TRiP-KO stock.

• TRiP nos-Cas9 and TRiP-KO insertions are marked with vermillion+ (v+)
• TRiP-KO insertions are abbreviated TKO.GS followed by the specific sgRNA stock ID number
• attP sites are marked with yellow+ (y+)
• both attP40 (chromosome II) and attP2 (chromosome III) nos-Cas9 insertions are avaialble
• most TRiP-KO transgenes are inserted at attP40 (chromosome II)

• make sure that at least one of the nos-Cas9 or TRiP-KO insertions is on a different chromosome from your target gene

  

Step 2: collect at least 15 male F1 progeny containing both nos-Cas9 and sgRNA transgenes.

Step 3: cross male F1 progeny en masse to appropriate balancer strain for your target gene (e.g. for chromosome III targets, use y sc v; Dr/TM3 or similar).

Step 4: collect 10-100 male or female F2 progeny (some will be heterozygous mutants) and cross each individually to balancer stock.

• this step can be done in batches while you confirm mutants
• F2 animals can also be screened for mutations prior to establishing stocks by extracting genomic DNA from a single wing without killing the flies (see Housden et al. 2014)
• if the nos-Cas9 and sgRNA transgenes are on different chromosomes in the F1 animals (e.g. y sc v; P{TKO.GS#,v+}attP40{y+}/+; P{nos-Cas9,v+}attP2{y+}/+) then you can select F2 animals with yellow cuticle and vermillion eyes to eliminate the transgenes from the stock

Step 5: balance mutations and eliminate nos-Cas9 and sgRNA transgenes (if still present)

Step 6: screen for mutations by restriction profiling, endonuclease assays or high-resolution melt assays (HRMAs) (Bassett et al., 2013; Cong et al., 2013; Wang et al., 2013) and confirm sequence alterations by sequencing the target site. For more details on these techniques see Housden et al., 2014.