#  Using TRiP-CRISPR lines 

 



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### 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](http://www.flyrnai.org/tools/grna_tracker/).

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.



 

 Mutants (TRiP-KO) Mosaics (TRiP-KO) Overexpression (TRiP-OE) 

## Mutants (TRiP-KO)

 

 

### 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 1](/sites/g/files/omnuum5366/files/fly/files/germline1.jpg)

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

 ![step 2](/sites/g/files/omnuum5366/files/fly/files/germline2.png)

 


### 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](/sites/g/files/omnuum5366/files/fly/files/germline_3.jpg)

 

### 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](/sites/g/files/omnuum5366/files/fly/files/germline_4.jpg)

 

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

 ![step 5](/sites/g/files/omnuum5366/files/fly/files/germline_5.jpg)

 

### 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.



 



 

 

 

## Mosaics (TRiP-KO)

 

 

### 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](/trip-crispr-toolbox-fly-stocks) 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 2: collect male or female F1 progeny containing tissue-specific-Gal4, UAS-Cas9 and sgRNA transgenes and analyze phenotype.

 ![step 2](/sites/g/files/omnuum5366/files/fly/files/mosaic2.jpg)

 

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## Overexpression (TRiP-OE)

 

 

### 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](/trip-crispr-toolbox-fly-stocks) 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](/sites/g/files/omnuum5366/files/fly/files/overexpress1.jpg)

 

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

 ![step 2](/sites/g/files/omnuum5366/files/fly/files/overexpress2.jpg)

 



 



 

 

 

 

 

### 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](/trip-crispr-toolbox-fly-stocks) and can be obtained from the BDSC



 

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](https://www.ncbi.nlm.nih.gov/pubmed/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](https://www.ncbi.nlm.nih.gov/pubmed/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](https://www.ncbi.nlm.nih.gov/pubmed/23287718)