Journal: STAR Protocols

Article Title: CRISPR-Cas9-mediated insertion of a short artificial intron for the generation of conditional alleles in mice

doi: 10.1016/j.xpro.2023.102116

Figure Lengend Snippet: Targeting strategy to engineer the Scyl1 AIv4 allele (A) Schematic representation of the Scyl1 gene, the sgRNA target site within exon 3 of Scyl1 and the HDR template containing sequences encoding the AIv4 cassette flanked by homology arms of 58 and 60 nucleotides. A black arrowhead indicates the location of the Cas9 cleavage site in exon 3. Grey boxes indicate exons. Gray lines indicate introns. The yellow box flanked by two back triangles represents the AIv4 cassette. The black arrow represents the translation start site. Yellow and red arrows represent the genotyping primers S1E154D-F1 and S1E154D-R2. (B) Sanger sequencing chromatogram of the TOPO-TA cloned PCR fragment obtained from a founder animal. 5′ and 3′ junctions are indicated by black arrowheads. The splice donor site is indicated in green. loxP sites are shown in black. The branch point is shown in blue. The bipartite polypyrimidine tract is shown in yellow. The splice acceptor site is shown in red.

Article Snippet: For all genotyping procedures, Qiagen Taq Polymerase (Qiagen, 201205) is used.

Techniques: Sequencing, Clone Assay

Journal: STAR Protocols

Article Title: CRISPR-Cas9-mediated insertion of a short artificial intron for the generation of conditional alleles in mice

doi: 10.1016/j.xpro.2023.102116

Figure Lengend Snippet: Representative PCR based genotyping and TOPO® TA Cloning® results obtained from each breeding step (A) Schematic representation of the Scyl1 gene highlighting the location and banding pattern of PCR genotyping primers for the characterization of founder animals and routine genotyping. S1E154D-F1 (red) and S1E154D-R1 (yellow) were used for genotyping F0, F1, and F2 mice. Using this primer pair, amplicons of 667 bp, 868 bp, and 721 bp are obtained for wild-type, Scyl1 AIv4 , and Scyl1 AIv4Δ alleles, respectively. For routine genotyping, a second primer pair, Scyl1_AIv4_F51 (blue) and Scyl1_AIv4_R52 (green), were designed to more easily distinguish the wild-type allele from the Scyl1 AIv4Δ allele. Using this second set of primers, amplicons of 304 bp, 505 bp, and 358 bp are obtained for wild-type, Scyl1 AIv4 , and Scyl1 AIv4Δ alleles, respectively. (B) PCR genotyping of Scyl1 +/+ , Scyl1 +/AIv4 , F1 or F2 animals obtained from F0 or F1 outbreeds to wild-type C57BL/6J mice. Amplicons of 667 bp correspond to the wild-type allele, whereas amplicons of 868 bp correspond to the Scyl1 AIv4 allele. (C) Representative image of digested plasmids obtained from TOPO® TA Cloning® of PCR products obtained from founder animals. The ∼868 bp upper band likely contains a PCR product corresponding to the AIv4 allele, while the shorter fragments likely correspond to the wild-type allele or contain indels. This is further confirmed by Sanger sequencing. (D) PCR genotyping of potential off-target cleavage sites in F1 animals. Three potential off-target sites were identified, as described in the “ ” section of this manuscript. Primer pairs were designed to amplify each site: Scyl1_AIv4_OT1-F1 and Scyl1_AIv4_OT1-R2 generate a 504 bp fragment corresponding to off-target site 1, Scyl1_AIv4_OT2-F1 and Scyl1_AIv4_OT2-R2 generate a 563 bp fragment corresponding to off-target site 2, and Scyl1_AIv4_OT3-F1 and Scyl1_AIv4_OT3-R2 generate a 474 bp fragment corresponding to off-target site 3. Direct Sanger sequencing of these PCR products was used to confirm that no off-target editing occurred at these sites. (E) PCR genotyping of Scyl1 +/+ , Scyl1 +/AIv4 , and Scyl1 AIv4/AIv4 F3 animals obtained from F2 outbreeds. Amplicons of 304 bp correspond to the wild-type allele, whereas amplicons of 505 bp correspond to the AIv4 allele. (F) PCR genotyping of mice obtained from CMV-Cre+ mice crossed with Scyl1 AIv4/AIv4 mice. Amplicons of 304, 358, and 505 bp correspond to the wild-type, Scyl1 AIv4Δ , and Scyl1 AIv4 alleles, respectively. In the lower panel, CMV-Cre+ mice are identified by a separate PCR reaction: CMV-Cre+ mice produce a 470 bp amplicon, whereas wild-type mice do not produce a PCR product. (G) PCR genotyping of Scyl1 + , Scyl1 AIv4 , and Scyl1 AIv4Δ alleles. Amplicons of 304, 358, and 505 bp correspond to the wild-type, Scyl1 AIv4 , and Scyl1 AIv4Δ alleles, respectively. A separate PCR reaction identifies CMV-Cre+ mice, which produce a 470 bp amplicon, whereas wild-type mice do not produce a PCR product.

Article Snippet: For all genotyping procedures, Qiagen Taq Polymerase (Qiagen, 201205) is used.

Techniques: TA Cloning, Sequencing, Amplification