Using TRiP-CRISPR lines

TRiP-CRISPR (TRiP-OE and TRiP-KO) constructs are injected into the following stocks

  • y v; attP2 (genotype: y[1] v[1]; P{y[+t7.7]=CaryP}attP2)
  • y v; attP40 (genotype: y[1] v[1]; P{y[+t7.7]=CaryP}attP40)

TRiP-CRISPR stock phenotype

  • wildtype cuticle color, yellow+ (y+) indicates that the attP site is present
  • wildtype eye color, vermillion+ (v+) indicates that the sgRNA is present
  • loss of scutellar bristles, scute- (sc-) indicates the presence of the y sc v  X chromosome
  • note that the TRiP no longer injects into sc mutant embryos, but the transformants are sometimes crossed to y sc v flies when they are balanced

The genotype of each TRiP-CRISPR stock can be found in the DRSC/TRiP sgRNA Stock Tracking System.

Please note that TRiP-CRISPR stocks do not contain a mini-white marker. For the TRiP-CRISPR stocks that have recessive sc on the X chromosome, the allele of sc used is not very penetrant if grown at 18C, but is fully penetrant at 25C.

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 genestep 1

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

 

step 2

 

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 3

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 4

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

step 5

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.

Here we describe the fly crossing scheme for making mosaics in somatic tissue with TRiP-CRISPR Knockout (TRiP-KO) stocks

 

Step 1: cross tissue specific-Gal4 + UAS-Cas9 stock to individual TRiP-KO stock.

  • TRiP-KO insertions are marked with vermillion+ (v+)
  • TRiP-KO insertions are abbreviated TKO.GS followed by the specific sgRNA stock ID number
  • most UAS-Cas9 and Gal4 insertions are marked with white+ (w+)
  • attP sites are marked with yellow+ (y+)
  • see the TRiP-CRISPR Toolbox page for Gal4>Cas9 stocks available from the TRiP
  • In the example below we describe a cross with the wing-specific nubbin-Gal4 line

step 1

Step 2: collect male or female F1 progeny containing tissue-specific-Gal4, UAS-Cas9 and sgRNA transgenes and analyze phenotype.

 

step 2

 

Here we describe the fly crossing scheme for overexpressing genes with TRiP-CRISPR Overexpression (TRiP-OE) stocks

 

Step 1: cross tissue specific-Gal4 + UAS-dCas9-VPR stock to individual TRiP-OE stock.

  • TRiP-OE insertions are marked with vermillion+ (v+)
  • TRiP-OE insertions are abbreviated TOE.GS followed by the specific sgRNA stock ID number
  • UAS-dCas9-VPR and most Gal4 insertions are marked with white+ (w+)
  • attP sites are marked with yellow+ (y+)
  • see the TRiP-CRISPR Toolbox page for Gal4>dCas9-VPR stocks available from the TRiP
  • In the example below we describe a cross with the wing-specific nubbin-Gal4 line

step 1

Step 2: collect male or female F1 progeny containing tissue-specific-Gal4, UAS-dCas9-VPR and sgRNA transgenes and analyze phenotype.

step 2

Additional Notes


Lethality

  • 5-10% of the TRiP stocks are homozygous lethal
  • lethal stocks are maintained over balancers
  • we attribute lethality to naturally occurring second site lethals on the 2nd or 3rd chromosomes
  • non-lethal but unhealthy stocks are also maintained over balancers to protect the integrity of the stock
  • if the balancer is present in all flies, then 50% of the progeny will not carry the sgRNA transgene
  • second site lethals can be recombined away from the attP insertion site if necessary
  • when examining embryos, larvae or pupae, switch balancer to one containing marker present at the relevant stage of development

Cas9 expression

  • any GAL4>Cas9 or promoter-Cas9 line can be used with a TRiP-CRISPR Overexpression or Knockout stock
  • TRiP-CRISPR stocks ubiquitously express sgRNAs, but should give no phenotype without concurrent Cas9 expression 
  • a variety of Gal4>Cas9 drivers are listed in the TRiP-CRISPR Toolbox and can be obtained from the BDSC

Relevant Publications

Andrew R. Bassett, Charlotte Tibbit, Chris P. Ponting, and Ji-Long Liu. 2013. "Highly Efficient Targeted Mutagenesis of Drosophila with the CRISPR/Cas9 System." Cell Rep. 2013 Jul 11;4(1):220-8. PMID:23827738

Haoyi Wang, Hui Yang, Chikdu S. Shivalila Meelad M. Dawlaty, Albert W. Cheng, Feng Zhang, and Rudolf Jaenisch. 2013. "One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering." Cell. 2013 May 9;153(4):910-8. PMID:23643243

Le Cong, F. Ann Ran, David Cox, Shuailiang Lin, Robert Barretto, Naomi Habib, Patrick D. Hsu, Xuebing Wu, Wenyan Jiang, Luciano A. Marraffini, and Feng Zhang. 2013. "Multiplex Genome Engineering Using CRISPR/Cas Systems." Science. 2013 Feb 15;339(6121):819-23.  PMID:23287718