CRISPR system stocks for site-specific mutagenesis
Updated November 30, 2016
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The CRISPR system allows investigators to target double-stranded DNA breaks to specific genomic sequences. A trans-activating RNA (tracrRNA) interacts with a nuclease (Cas9) to direct cleavage to a sequence complementary to an introduced RNA (CRISPR RNA).

The following stocks may be used to express Cas9 nuclease and/or tracrRNA maternally so that chimeric RNAs (also referred to as guide RNAs) or CRISPR RNAs (crRNAs) can be injected into fly embryos to generate new mutations without the need for helper plasmids, or so that they can be used in combination with chimeric or CRISPR RNAs expressed from transgenic constructs.

Detailed information about the CRISPR system in Drosophila is available from the flyCRISPR, FlyCas9 and CRISPR fly design websites.

The following stocks were generated by Kate O'Connor-Giles, Jill Wildonger, Melissa Harrison and colleagues. The vasa-Cas9 construct was described in Gratz et al. (2014) "Highly Specific and Efficient CRISPR/Cas9-Catalyzed Homology-Directed Repair in Drosophila" Genetics 196: 961-971.

PBac{U6-tracrRNA} and M{U6-tracrRNA} express tracrRNA ubiquitously under the control of snRNA:U6:96Ab regulatory sequences.

M{vas-Cas9} and PBac{vas-Cas9} express Cas9 nuclease in the ovary under control of vasa regulatory sequences.

PBac{vas-Cas9,U6-tracrRNA} and M{vas-Cas9,U6-tracrRNA} combine both vas-Cas9 and U6-tracrRNA elements into a single construct.

Stock # Genotype
Chromosomes
51321 w[1118]; PBac{y[+mDint2] w[GMR.PHb]=U6-tracrRNA}VK00037/CyO, P{Wee-P.ph0}2
1;2
51322 y[1] M{w[GMR.PHb]=U6-tracrRNA}ZH-2A w[1118]/FM7c, P{w[+mC]=GAL4-Kr.C}DC1, P{w[+mC]=UAS-GFP.S65T}DC5, sn[+]
1
51323 y[1] M{vas-Cas9}ZH-2A w[1118]/FM7c
1
51324 w[1118]; PBac{y[+mDint2]=vas-Cas9}VK00027
1;3
51325 w[1118]; PBac{y[+mDint2]=vas-Cas9,U6-tracrRNA}VK00027
1;3
51326 y[1] M{vas-Cas9,U6-tracrRNA}ZH-2A w[1118]
1
55821 y[1] M{vas-Cas9.RFP-}ZH-2A w[1118]/FM7a, P{w[+mC]=Tb[1]}FM7-A
1
56552
w[1118]; PBac{y[+mDint2]=vas-Cas9}VK00037/CyO, P{w[+mC]=Tb[1]}Cpr[CyO-A]
1;2

The following stock generated by Ying Peng, Yi Guo and colleagues expresses Cas9 nuclease in the ovary under control of vasa regulatory sequences. It is similar to stock 51323 above, but it is an independent construct. It was described in Sebo et al. (2013) "A simplified and efficient germline-specific CRISPR/Cas9 system for Drosophila genomic engineering" Fly 8: 1-6.

Stock # Genotype
Chromosomes
52669 y[1] M{vas-Cas9.S}ZH-2A w[1118]
1

The following stocks were generated by the CRISPR fly design project. Most were described in Port et al. (2014) "Optimized CRISPR/Cas tools for efficient germline and somatic genome engineering in Drosophila" PNAS 111: E2967-E2976. This paper also compares the properties and efficiencies of constructs from different labs.

M{Act5C-Cas9.P} expresses Cas9 nuclease ubiquitously under control of Act5C regulatory sequences.

M{nos-Cas9.P} expresses express Cas9 nuclease in the ovary under control of nanos regulatory sequences.

P{UAS-Cas9.P}, P{UAS-Cas9.C} and P{UAS-Cas9.P2} express Cas9 nuclease under UAS control. P{UAS-Cas9.P2} expresses at lower levels.

P{nos-FokI.Cas9} and P{Act5C-FokI.Cas9} express the FokI endonuclease domain fused to catalytically inactive Cas9 in the ovary in the pattern of the nos gene or ubiquitously in the pattern of the Act5C gene. FokI must dimerize to cleave DNA; consequently, the Fok1-dCas9 fusion protein leads to heritable genome edits only when recruited by two guide RNAs targeting sequences flanking the cleavage site in a defined spacing and orientation. The specificity of DNA cleavage by the fusion protein is better than that obtained with wild type Cas9.

Stock # Genotype
Chromosomes
54590 y[1] M{w[+mC]=Act5C-Cas9.P}ZH-2A w[*]
1
54591 y[1] M{w[+mC]=nos-Cas9.P}ZH-2A w[*]
1
54592 P{ry[+t7.2]=hsFLP}1, y[1] w[1118]; P{y[+t7.7] w[+mC]=UAS-Cas9.P}attP2
1;3
54593 P{ry[+t7.2]=hsFLP}1, y[1] w[1118]; P{y[+t7.7] w[+mC]=UAS-Cas9.P}attP2 P{w[+mC]=GAL4::VP16-nos.UTR}CG6325[MVD1]
1;3
54594 P{ry[+t7.2]=hsFLP}1, y[1] w[1118]; P{y[+t7.7] w[+mC]=UAS-Cas9.P}attP40
1;2
54595 w[1118]; P{y[+t7.7] w[+mC]=UAS-Cas9.C}attP2
1;3
54596 w[1118]; P{y[+t7.7] w[+mC]=UAS-Cas9.D10A}attP2
1;3
58983 P{ry[+t7.2]=hsFLP}12, y[1] w[*]; P{y[+t7.7] w[+mC]=nos-FokI.Cas9}attP40/CyO
1;2
58984 P{ry[+t7.2]=hsFLP}12, y[1] w[*]; P{y[+t7.7] w[+mC]=Act5C-FokI.Cas9}attP40/CyO
1;2
58985 P{ry[+t7.2]=hsFLP}12, y[1] w[*]; P{y[+t7.7] w[+mC]=UAS-Cas9.P2}attP40
1;2
58986 P{ry[+t7.2]=hsFLP}12, y[1] w[*]; P{y[+t7.7] w[+mC]=UAS-Cas9.P2}attP2/TM6B, Tb[1]
1;3

The following stock was generated by Frank Schnorrer and colleagues and described in Zhang et al. (2014) "A versatile two-step CRISPR- and RMCE-based strategy for efficient genome engineering in Drosophila" (early edition).

M{Act5C-Cas9.P.RFP-}ZH-2A is a derivative of M{Act5C-Cas9.P}ZH-2A (see stock 54590) from which the miniwhite and RFP markers have been removed by cre-mediated recombination.

The introduction of novel sequences at the site of Cas9 cleavage by homology-directed repair is more efficient in a Lig4 null background.

Stock # Genotype
Chromosomes
58492 y[1] M{Act5C-Cas9.P.RFP-}ZH-2A w[1118] Lig4[169]
1

In the following stock, M{vas-Cas9}ZH-2A is carried on a w[+] chromosome. When the w gene and another gene are targeted simultaneously for CRISPR knockout/knock-in, mutation of w is strongly correlated with mutation of the other gene. Consequently, the efficiency of such screens can be improved by enriching the candidate pool for w mutant progeny.

This screening strategy was described in Ge et al. (2016) "Rapid Screening for CRISPR-Directed Editing of the Drosophila Genome Using white Coconversion" G3 6: 3197-3206.

Stock #Genotype
Chromosomes
66554 y[1] M{vas-Cas9}ZH-2A
1