SMRT Gate: A method for validation of synthetic constructs on Pacific Biosciences sequencing platforms

Current DNA assembly methods are prone to sequence errors, requiring rigorous quality control (QC) to identify incorrect assemblies or synthesized constructs. Such errors can lead to misinterpretation of phenotypes. Because of this intrinsic problem, routine QC analysis is generally performed on three or more clones using a combination of restriction endonuclease assays, colony PCR, and Sanger sequencing. However, as new automation methods emerge that enable high-throughput assembly, QC using these techniques has become a major bottleneck. Here, we describe a quick and affordable methodology for the QC of synthetic constructs. Our method involves a one-pot digestion-ligation DNA assembly reaction, based on the Golden Gate assembly methodology, that is coupled with Pacific Biosciences’ Single Molecule, Real-Time (PacBio SMRT) sequencing technology. </jats:p

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

Abstract Background Recent advances in clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) genome editing have led to the use of long single-stranded DNA (lssDNA) molecules for generating conditional mutations. However, there is still limited available data on the efficiency and reliability of this method. Results We generated conditional mouse alleles using lssDNA donor templates and performed extensive characterization of the resulting mutations. We observed that the use of lssDNA molecules as donors efficiently yielded founders bearing the conditional allele, with seven out of nine projects giving rise to modified alleles. However, rearranged alleles including nucleotide changes, indels, local rearrangements and additional integrations were also frequently generated by this method. Specifically, we found that alleles containing unexpected point mutations were found in three of the nine projects analyzed. Alleles originating from illegitimate repairs or partial integration of the donor were detected in eight projects. Furthermore, additional integrations of donor molecules were identified in four out of the seven projects analyzed by copy counting. This highlighted the requirement for a thorough allele validation by polymerase chain reaction, sequencing and copy counting of the mice generated through this method. We also demonstrated the feasibility of using lssDNA donors to generate thus far problematic point mutations distant from active CRISPR cutting sites by targeting two distinct genes (Gckr and Rims1). We propose a strategy to perform extensive quality control and validation of both types of mouse models generated using lssDNA donors. Conclusion lssDNA donors reproducibly generate conditional alleles and can be used to introduce point mutations away from CRISPR/Cas9 cutting sites in mice. However, our work demonstrates that thorough quality control of new models is essential prior to reliably experimenting with mice generated by this method. These advances in genome editing techniques shift the challenge of mutagenesis from generation to the validation of new mutant models

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 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 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 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