Additional file 18: of Application of long single-stranded DNA donors in genome editing: generation and validation of mouse mutants

Figure S16. Generation of a point mutation in Rims1 with ssODN donors. (a) The table details the F0 animals obtained for generation of Rims1 mutant with ssODN donors. The ID and outcome of sequencing the region of interest, as well as the conclusion for each individual are shown. (b) PCR amplification of region of interest with Rims1-F1 and Rims1-R1 primers (241 bp) from biopsies taken from the F0 animals. Sequences of Rims1-ODN-151 mosaic and of sub-cloned amplicons are shown in Additional file 3: Figure S2u and v, demonstrating the presence of the desired mutation in this animal that was therefore mated. (c) PCR amplification of region of interest with Rims1-F1 and Rims1-R1 primers (241 bp) from biopsies taken from Rims1-ODN-151’s offspring. Animal IDs are shown. + is positive control amplified from an unrelated WT animal. L1 = 1 kb DNA molecular weight (thick bands are 3 kb); L2 = 100 bp DNA molecular weight ladder (thick bands are 1000 and 500 bp). (d) The table details the first litter obtained by mating Rims1-ODN-151 with a WT mouse. The ID, outcome of sequencing the region of interest and copy counting of the region of interest as well as the conclusion for each individual are shown. Sequencing of Rims1-ODN-151.1g is shown in Additional file 3: Figure S2w and illustrates the failure of transmission of the desired allele. (PNG 893 kb

Additional file 20: of Application of long single-stranded DNA donors in genome editing: generation and validation of mouse mutants

The file contains the raw sequencing data obtained from the founders generated for the Rims1R655H point mutation. (ZIP 21747 kb

Additional file 17: of Application of long single-stranded DNA donors in genome editing: generation and validation of mouse mutants

Figure S15. Design of a Rims1R655H point mutation. The figure illustrates the changes designed at the nucleotide and proteomic levels with the mutagenesis strategy employing (a) oligonucleotides and (b) lssDNA. Coding sequences are translated into protein sequences above annotated exon. Note that the region containing Rims1 is not entirely accurate in the GRCm38 assembly. We have re-sequenced this region prior to designing of the mutant (primers shown in Additional file 1: Table S1). (PNG 521 kb

Additional file 15: of Application of long single-stranded DNA donors in genome editing: generation and validation of mouse mutants

Figure S14. Design of a GckrP446L point mutation. Figure illustrates the changes designed at the nucleotide and proteomic levels with the mutagenesis strategy employing (a) oligonucleotides and (b) lssDNA. Coding sequences are highlighted in pink, engineered P446L change is highlighted in black with yellow text, silent mutations are highlighted in grey and sgRNA sequences are highlighted in green. Primers external to the donors employed for mutant analysis are also shown in blue and detailed in Additional file 1: Table S1. (PNG 1857 kb

Additional file 14: of Application of long single-stranded DNA donors in genome editing: generation and validation of mouse mutants

Figure S13. Unexpected outcome of CRISPR/Cas9-aided mutagenesis. The figure illustrates an example of a rearranged allele obtained from the co-injection of CRISPR/Cas9 reagents and lssDNA to generate a conditional Ikzf2 allele. Panel (a) shows the design of the lssDNA donor compared to the WT sequence. HA homology arm, BP breakpoint (genomic sequence removed in the intended floxed allele). Panel (b) shows sequencing of an F1 (Ikzf2–2.1e) that bears a recombined allele where the critical region and a loxP site are lost (allele with major representation) and a WT allele (with minor representation). (PNG 309 kb

Additional file 13: of Application of long single-stranded DNA donors in genome editing: generation and validation of mouse mutants

Figure S12. Examples of unexpected point mutations in the F0 animals obtained from the co-injection of CRISPR/Cas9 reagents and lssDNA in 6430573F11Rik (a) and Cx3cl1 (b and c) projects. Blue 5′ homology arm; orange universal sequences for diagnostics; green critical region with exon in capitals; red loxP sites; grey 3′ homology arm. Unexpected point mutations are detected by Sanger sequencing of amplicons generated with primers external to the donor; (a) shows one intronic SNP in floxed critical region, (b) shows two intronic nucleotide changes (black arrows, grey highlight) and one coding nucleotide change (red arrow, pink highlight) which was found associated with (c) SNP in 3’ loxP site. Mutations are highlighted on the sequence alignment (a) and seen on the sequence chromatograms (b and c). (PNG 1332 kb

Additional file 10: of Application of long single-stranded DNA donors in genome editing: generation and validation of mouse mutants

Figure S9. Analysis of the 6430573F11Rik project. PCR amplification of genomic DNA of (a) F0 animals, (f) 6430573F11Rik-11’s offspring or (i) 6430573F11Rik-28’s offspring with (a, f) 6430573F11Rik-F3 and 6430573F11Rik-R2 (1721-bp amplicon) and (b, f) LoxPF and LoxPR (999-bp amplicon). Sequencing of PCR amplicons from (c) 6430573F11Rik-11 and (g) 6430573F11Rik-11.1a with 6430573F11Rik-F3 and 6430573F11Rik-R2. LoxPs are in blue. ID, outcome of PCR analysis and conclusion for (d) each F0 animal and (e) the first litter obtained by mating 6430573F11Rik-11 with a WT mouse. Two founders were mated for cKO GLT. *Mated; ⁑no evidence of loxP in 6430573F11Rik amplicon, suggesting donor integrated randomly (6430573F11Rik-28 sequence trace in Additional file 3: Figure S2q). (g) Only WT sequence is found, indicating random donor insertion. (f, i) Animal IDs are shown. + is positive control from unrelated WT and conditional floxed animal for 6430573F11Rik and LoxP PCR, respectively. L1 = 1 kb DNA molecular weight ladder (thick band is 3 kb). (h) First litter obtained by mating 6430573F11Rik-28 with a WT mouse. ID, outcome of sequencing and copy counting of the region of interest and the conclusion for each individual. (j) Sequencing of amplicons obtained with 6430573F11Rik-F3 and 6430573F11Rik-R2 and 6430573F11Rik-28.1a. Only WT sequence is found, indicating random donor insertion. Sequencing of deletion allele in founder 6430573F11Rik-6, summary of analysis of F1 animals derived from 6430573F11Rik-6 and transmitted deletion allele are shown in Additional file 3: Figure S2r, s and t. (PNG 1011 kb

Additional file 12: of Application of long single-stranded DNA donors in genome editing: generation and validation of mouse mutants

Figure S11. Analysis of the Inpp5k project. The figure shows the PCR amplification of the genomic region of interest with (a) Inpp5k-F1 and Inpp5k-R1 primers (1705-bp amplicon) and (b) LoxPF and LoxPR primers (1194-bp amplicon) from biopsies taken from the F0 animals. Animal IDs are shown. + is positive control amplified from an unrelated WT and conditional floxed animal for the Inpp5k and LoxP PCR, respectively. L1 = 1 kb DNA molecular weight ladder (thick band is 3 kb). (c) Sequencing chromatogram of PCR amplicons obtained from Inpp5k-7 with Inpp5k-F1 and Inpp5k-R1. LoxP sequence is highlighted in blue. (d) The table details the F0 animals obtained. The ID, outcome of PCR analysis of the region of interest and the conclusion for each individual are shown. Two founders are mated for cKO allele transmission (LoxP PCR positive and sequence of complex mosaic). *Mated as loxP presence confirmed by sequencing of Inpp5k PCR amplicon. (e) First litter obtained by mating Inpp5k-7 and Inpp5k-8 with a WT mouse. The ID, outcome of sequencing the region of interest and the conclusion for each individual are shown. PCR amplification of region of interest with (f) Inpp5k-F1 and Inpp5k-R1 primers (1705-bp amplicon) and (g) LoxPF and LoxPR primers (1194-bp amplicon) from biopsies taken from Inpp5k-7’s and Inpp5k-8’s offspring. Animal IDs are shown. + is positive control amplified from an unrelated WT and conditional floxed animal for the Inpp5k and LoxP PCR, respectively. L1 = 1 kb DNA molecular weight ladder (thick band is 3 kb). Panels illustrate the sequencing data from amplicons obtained from Inpp5k-7.1b (h, i, j) and Inpp5k-8.1c (k, l) genomic DNA with (h, i, k) Inpp5k-F1 and (j, l) Inpp5k-R1 primers. (PNG 877 kb

Additional file 7: of Application of long single-stranded DNA donors in genome editing: generation and validation of mouse mutants

Figure S6. Analysis of the Usp45 project. The figure shows the PCR amplification of the genomic region of interest with (a) Usp45-F1 and Usp45-R3 primers (1440-bp amplicon) and (b) LoxPF and LoxPR primers (741-bp amplicon) from biopsies taken from the F0 animals. (c) The panels show the Usp45 PCR amplicon generated from the Usp45-18 can be sequenced with LoxPF and LoxPR primers, demonstrating the presence of loxP on locus. (d) The table details the F0 animals obtained. The ID and outcome of PCR analysis of the region of interest as well as the conclusion for each individual are shown. Usp45-18 was mated for cKO allele transmission. (e) The table details three litters obtained by mating Usp45-18 with a WT mouse. The ID, outcome of sequencing the region of interest and the conclusion for each individual are shown. PCR amplification of region of interest with Usp45-F1 and Usp45-R3 primers (1440-bp amplicon (f) and LoxPF and LoxPR primers (741-bp amplicon (g) from biopsies taken from Usp45-18’s offspring. Animal IDs are shown. + is positive control amplified from an unrelated WT (a, f). L1 = 1 kb DNA molecular weight ladder (thick band is 3 kb). Sequencing data obtained from Usp45-18.1a and Usp45-18.1b are shown in Additional file 3: Figure S2l and m. (a) Litter 3 died prior to biopsy age. (b) Deletion affecting the region recognized by the TaqMan assay. (c) Litter died prior to biopsy age. (d) Copy number counting of mutated sequence. n.d. = not determined. Further data are displayed in Additional file 3: Figure S2. (PNG 618 kb

Additional file 19: of Application of long single-stranded DNA donors in genome editing: generation and validation of mouse mutants

Figure S17. Generation of a point mutation in Rims1 with a lssDNA donors. (a) PCR amplification of region of interest with Rims1-F2 and Rims1-R2 primers (647 bp) from biopsies taken from the F0 animals. Animal IDs are shown. + is positive control amplified from an unrelated WT animal. L1 = 1 kb DNA molecular weight ladder (thick band is 3 kb). (b) Sequencing of amplicon obtained from the Rims1-lss-2, Rims1-lss-20, Rims1-lss-21 and Rims1-lss-36 animals: point mutation is observed (blue highlight) when sequencing the Rims1-F2 primer. (c) The table details the F0 animals obtained for generation of Rims1 mutant with lssDNA donors. The ID, outcome of sequencing the region of interest and the conclusion for each individual are shown. (d) The table details the first litter obtained by mating Rims1-lss-36 with a WT mouse. The ID, outcome of sequencing the region of interest, copy counting of the region of interest and conclusion for each individual are shown. (e) PCR amplification of region of interest with Rims1-F3 and Rims1-R3 primers (647 bp) from biopsies taken from Rims-lss-36’s offspring. Animal IDs are shown. + is a positive control amplified from an unrelated WT animal. L2 = 100 bp DNA molecular weight ladder (thick bands are 1000 and 500 bp). (f) Sequencing of amplicon obtained from Rims1-lss-36.1a, legitimate repair observed (blue highlight) when sequencing both directions (Rims1-F3 and Rims1-R3 primers). (g) Alignment of Rims1-lss-36-1a offspring, legitimate repair aligned against WT allele. R655H coding change highlighted in red. Grey background with red text highlights silent mutations introduced by long donor. (PNG 1287 kb