Reagents

The following CRISPR/Cas9 fly lines are available through the Bloomington Drosophila Stock Center:

 

51321    w[1118]; PBac{y[+mDint2] w[GMR.PHb]=U6-tracrRNA}VK00037/CyO, P{Wee-P.ph0}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[+]

51323    y[1] M{vas-Cas9}ZH-2A w[1118]/FM7c

51324    w[1118]; PBac{y[+mDint2]=vas-Cas9}VK00027

51325    w[1118]; PBac{y[+mDint2]=vas-Cas9,U6-tracrRNA}VK00027

51326    y[1] M{vas-Cas9,U6-tracrRNA}ZH-2A w[1118]

 

The following plasmids are available through Addgene:

  • pBS-Hsp70-Cas9 (in pBluescript)
  • pHsp70-Cas9 (low copy number version used in Gratz et al., 2013)
  • pU6-BbsI-chiRNA (gRNA vector using the U6-2 promoter used in Gratz et al., 2013)
  • pHD-DsRed-attP (donor vector used in used in Gratz et al., 2014)
  • pHD-DsRed (donor vector used in used in Gratz et al., 2014)

 

The following plasmids are available through the DGRC:

  • pBS-Hsp70-Cas9 (in pBluescript)
  • pU6-2-gRNA (gRNA vector using the U6-2 promoter)
  • pU6-3-gRNA (gRNA vector using the U6-3 promoter)
  • pHD-DsRed-attP (donor vector with loxP flanked DsRed marker used in used in Gratz et al., 2014)
  • pHD-DsRed (donor vector with loxP flanked DsRed marker used in used in Gratz et al., 2014)
  • pHD-ScarlessDsRed (donor vector with a PBac DsRed cassette for scarless marker removal)
  • pHD-sfGFP-ScarlessDsRed (donor vector for scarless tagging with sfGFP)
  • pHD-3xFLAG-ScarlessDsRed (donor vector for scarless tagging with 3xFLAG)
  • pHD-2xHA-ScarlessDsRed (donor vector for scarless tagging with 2xHA)

 

phsp70-Cas9

Expresses Cas9 nuclease under the control of the Drosophila hsp70 promoter

phsp70-Cas9 sequence file (PDF)

 

 

pU6-BbsI-chiRNA

For rapid generation of chiRNAs under the control of the Drosophila snRNA:U6:96Ab promoter (Wakiyama et al., 2005; see Protocols).

pU6-BbsI-chiRNA sequence file (PDF)

 

 

pHD-DsRed-attP

For rapid cloning of dsDNA donor vectors marked by removable DsRed
This vector is designed to replace a target locus with an attP docking site

pHD-DsRed-attP sequence file (PDF)

pHD-DsRed-attP map file (PDF)

 

Our Cas9 and chiRNA expression vectors were modified from the mammalian expression constructs in Cong et al., 2012. We thank Feng Zhang for generously sharing reagents and insights. The Zhang lab CRISPR website describes their reagents in detail.