For a list of plasmids, we generated for this study, see Additional file 3 : Table S2. The original plasmids we used for this project were obtained from Addgene: pCFD3 (#49410), pCFD5 (#73914), pACG:eCFP (#32597), pDsRed-attP (#51019), Ac5-Stable2-Neo (#32426), pC0056-LwaCas13a-msfGFP-NES (#105815), pC0040-LwaCas13a crRNA backbone (#103851), pC0046-EF1a-PspCas13b-NES-HIV (#103862), pC0043-PspCas13b crRNA backbone (#103854), pC0054-CMV-dPspCas13b-longlinker-ADAR2DD (E488Q/T375G) (103870), pXR001: EF1-CasRX-2A-eGFP (#109049), pXR004: CasRX pre-gRNA cloning backbone (#109054), pBID-UASc (#35200), [10 (link), 12 (link), 23 (link), 35 (link), 46 (link), 50 (link), 107 (link)–110 (link)]. We also obtained plasmids from the Drosophila Genetic Resource Center (DGRC): pAFW (#1111), pAHW (#1095), act-PhiC31-integrase (#1368). We also used plasmids we previously generated, enDmC, to generate some constructs for this study [8 (link)]. pMT-Gal4-puro plasmid was a kind gift from Christoph Metzendorf (University of Uppsala). All fragments used for the cloning step were amplified via PCR using Q5 high-fidelity DNA polymerase (NEB #M0491S) (Additional file 4 : Table S3) and fused together via Gibson assembly reaction [111 (link)].
Pdsred attp
Manufactured by Addgene
PDsRed-attP is a plasmid that contains the DsRed gene, which encodes a red fluorescent protein. The plasmid also includes an attP site, which is a DNA sequence recognized by the phage integrase enzyme and can be used for site-specific recombination.
Automatically generated - may contain errors
Lab products found in correlation
Gibson assembly master mix, by New England Biolabs (3 mentions)
Pcfd4, by Addgene (2 mentions)
Pu6 bbsi chirna, by Addgene (2 mentions)
Pbs hsp70 cas9, by Addgene (2 mentions)
Ac5 stable2 neo, by Addgene (1 mentions)
Pcfd5, by Addgene (1 mentions)
Pc0046 ef1a pspcas13b nes hiv, by Addgene (1 mentions)
Pc0043 pspcas13b crrna backbone, by Addgene (1 mentions)
Pcfd3, by Addgene (1 mentions)
Pc0040 lwacas13a crrna backbone, by Addgene (1 mentions)
Pcfd3 du6 3grna, by Addgene (1 mentions)
Vas cas9, by Bloomington Drosophila Stock Center (1 mentions)
6 protocols using pdsred attp
1
Comprehensive Plasmid Database for Genetic Manipulation
2
CRISPR/Cas9-Mediated IR52a Deletions
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IR52a deletions were generated using CRISPR/Cas9 homologous recombination. Guide RNAs (gRNAs) were designed using the flyCRISPR Optimal Target Finder [78 (link)]. gRNA plasmid was constructed using pCFD4 (Addgene 49411), following the protocol described in [79 (link)]. Gibson Assembly was performed using Gibson Assembly Master Mix (New England BioLabs, E2611S). gRNA plasmid and a donor plasmid (pDsRed-attP, Addgene 51019) with homologous arms and a DsRed marker was injected into embryos by Bestgene, Inc. (Chino Hills, CA). DsRed positive alleles were then backcrossed to our control w1118 Canton-S line for five generations. The primers used in the construction are shown in S7C Fig .
3
CRISPR Deletion of the Bombardier Gene
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The bombardier gene (CG18067) was deleted using CRISPR/Cas9 technology according to established protocols (26 (link)). Briefly, a pair of gRNAs designed to delete the region 2R: 20,534,248–20,536,154 were cloned into pU6-BbsI-chiRNA (Addgene plasmid #45946). Homology arms (1,017 bp left and 1,022 bp right) were cloned into pDsRed-attP (Addgene plasmid #51019). The plasmid pBS-Hsp70-Cas9 (Addgene plasmid #46294) was used as the Cas9 source. Constructs were injected into w1118 embryos. F1 progeny were screened for DsRed eyes and homozygous lines were established. See Supplemental Table 1 for gRNA and homology arm primer sequences.
4
Generating Odorant Receptor Knockouts
5
Generating fru Promoter Deletion Mutants
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CRISPR-Cas9 genome editing was used to remove P1 promoter (first exon) sequences in the fru locus generating line fruΔP1. Two fragments with homology to the fru genomic regions on either side of the P1 promoter were cloned using Gibson Assembly Master Mix (New England Biolabs) with primers for 5′ Fragment: G1_5f and G1_5r and 3′ Fragment: G1_3f and G1_3r (Table S1 ), into the targeting vector pDsRed-attP (a gift from Melissa Harrison & Kate O'Connor-Giles & Jill Wildonger; Addgene plasmid # 51019) digested with XhoI and NotI. gRNA expressing constructs pCFD3-Fru5_1 and pCFD3-Fru3_3 were generated in the vector pCFD3-dU6:3gRNA (a gift from Simon Bullock; Addgene plasmid #49410) against target regions Fru5_1 and Fru3_3. Constructs were co-injected into the strain vas-Cas9.RFP- (Bloomington stock #55821), progeny were screened for DsRed+ expression in the eye. Seven lines were identified, all of which mapped to the 3rd chromosome. PCR and sequencing were used to confirm the deletion of P1 sequences. DsRed was removed from two independently isolated lines (fruΔP1.1 and fruΔP1.2) by crossing to a Cre recombinase constitutively expressing line (Bloomington stock #851).
6
Genome Editing in Drosophila Using CRISPR
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