Acanthopanax trichodon Franch. et Savat.

原著和名: ミヤマウコギ科名: ウコギ科 = Araliaceae採集地: 千葉県 清澄山 (上総〜安房 清澄山)採集日: 1961/6/18採集者: 萩庭丈壽整理番号: JH042319国立科学博物館整理番号: TNS-VS-99231

Additional file 3: Figure S1. of Evaluation of off-target and on-target scoring algorithms and integration into the guide RNA selection tool CRISPOR

Overlap of off-target detection for the EMX1 and VEGFA guides tested by different assays. Off-targets are only shown if they were detected by at least a single study and with a frequency of 0.1 %. See Additional file 1: Tables S1 and Additional file 4: Table S2 for the modification frequencies and additional details on the off-targets for the guides EMX1 and VEGFA, respectively. Additional file 4: Table S2 also includes the data by Hsu et al. [7], who quantified cleavage at putative off-target loci predicted by the CRISPR Design website ( http://crispr.mit.edu/ ) with targeted deep sequencing, Tsai et al. [3], who isolated double-strand breaks with modified oligonucleotides followed by sequencing, Frock et al. [28], who detected translocations, and Kim et al. [33] and Kim et al. [27], who performed whole-genome sequencing to find CRISPR-induced modifications. For details on the different studies, see Additional file 1: Table S1. (PDF 17 kb

Mentha viridis L.

原著和名: ミドリハクカ科名: シソ科 = Labiatae採集地: 千葉県 千葉市 千葉大学 (下総 千葉市 千葉大学)採集日: 1967/8/13採集者: 萩庭丈壽整理番号: JH042898国立科学博物館整理番号: TNS-VS-99289

Ilex serrata Thunb.

原著和名: ウメモドキ科名: モチノキ科 = Aquifoliaceae採集地: 愛知県 豊橋市 岩崎町 (三河 豊橋市 岩崎)採集日: 1968/10/20採集者: 萩庭丈壽整理番号: JH042317国立科学博物館整理番号: TNS-VS-99231

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 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

Additional file 6: of Application of long single-stranded DNA donors in genome editing: generation and validation of mouse mutants

Figure S5. Analysis of the Syt4 project. PCR amplification of the genomic region of interest with (a) Syt4-F2 and Syt4-R1 primers (2088-bp amplicon) and (b) Syt4-LoxPF and Syt4-LoxPR primers (1395-bp amplicon) from F0 animal biopsies. (c) Sequencing of PCR amplicon obtained from founder Syt4-29 with Syt4-F2 and Syt4-R1. LoxP sequences are highlighted in blue. (d) ID, PCR analysis of the region of interest and conclusion for each F0 individual are shown. *Syt4-29 was mated for cKO allele transmission. **Syt4-37 was identified as having a random insertion of the donor, as sequencing of the Syt4 PCR amplicon obtained from Syt4-37 shows no loxP, suggesting a random integration of the donor, Additional file 3: Figure S2j. (e) Details of the first litter obtained by mating Syt4-29 with a WT mouse. ID, outcome of sequencing and copy counting of the region of interest and the conclusion for each individual are shown. PCR amplification of region of interest with Syt4-F2 and Syt4-R1 primers (2088-bp amplicon (f) and LoxPF and LoxPR primers (1395-bp amplicon (g) from biopsies taken from founder Syt4-29’s offspring. (h) Sequencing data obtained from Syt4-29.1a. (a, b, f, g) 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). L2 = 100 bp DNA molecular weight ladder (thick bands are 1000 and 500 bp). Sequencing data showing a correct conditional allele are shown in Additional file 3: Figure S2k. Sequencing data showing the transmission of a deletion allele by founder Syt4-17 are shown in Additional file 3: Figure S2e, f and g. Sequencing data illustrating the possible insertion of loxP in Syt-28 and the transmission of an illegitimate repair are shown in Additional file 3: Figure S2i and j. (PNG 1045 kb

Additional file 8: of Application of long single-stranded DNA donors in genome editing: generation and validation of mouse mutants

Figure S7. Analysis of the Rapgef5 project. PCR amplification of the genomic region of interest with (a) Rapgef5-F1 and Rapgef5-R1 primers (1365-bp amplicon) and (b) LoxPF and LoxPR primers (724-bp amplicon) from biopsies taken from the F0 animals. (a, b) Animal IDs are shown. + is positive control amplified from an unrelated (a) WT, (b) conditional floxed animal. L1 = 1 kb DNA molecular weight ladder (thick band is 3 kb). (c) Panel shows the sequencing of PCR amplicon obtained from the Rapgef5-14 with Rapgef5-F1 and Rapgef5-R1 primers. LoxP sequences are highlighted in blue. (d) The table details the F0 animals obtained. The ID and outcome of PCR analysis of the region of interest and the conclusion for each individual are shown. Founder Rapgef5-14 died without offspring. Sequencing data showing the deletion allele identified in Rapgef5-3 are shown in Additional file 3: Figure S2n. (PNG 587 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