Additional file 1: of Correction of the auditory phenotype in C57BL/6N mice via CRISPR/Cas9-mediated homology directed repair

The following additional data are available with the online version of this paper. Figure S1. In vitro assessment of sgRNA efficacy. Table S1. The sequences of the oligonucleotides used in this study. Table S2. The sequences and locations of the predicted off-target sites for the two sgRNAs used in design 1. Table S3. Oligonucleotide sequences for Sanger sequencing of sgRNA_U1 and sgRNA_D1 predicted off-target sites (three or fewer mismatches). (DOCX 922 kb

Additional file 2: Figure S2. of Aneuploidy screening of embryonic stem cell clones by metaphase karyotyping and droplet digital polymerase chain reaction

Quality of karyotype of JM8-derived clones is equivalent among distributing repositories. The figure shows the percentages of euploid clones sourced from the two main distributors of JM8-derived cells and GLT rate obtained with them. The numbers of clones in each instance is shown. These numbers illustrate that materials obtained from different distributors are of similar quality in terms of karyotype and GLT ability. Data was analysed using the Fisher Exact test that and showed no evidence of difference of quality between the two distributors. (PDF 77 kb

Additional file 6: Figure S5. of Aneuploidy screening of embryonic stem cell clones by metaphase karyotyping and droplet digital polymerase chain reaction

Outcome of chromosome counting by ddPCR with genomic DNA extracted from ear biopsies. The figure shows copy numbers obtained using the karyotype screen assay panel on DNA extracted from euploid ES cells (C), trisomic ES cells (Ts), X0 ES cells (Ns Y) and female mouse ear clip (E), using the same lysis method. Vertical bars are Standard Errors. The data illustrate that the assays are able detect the expected copy numbers on gDNA extracted from tisssues (2 Chr 1, 8 and 11) and that the number of these chromosomes is lower in genomes extracted from tissue cultures. (PDF 63 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 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 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 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 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