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.

Keywords: Allele validation; CRISPR/Cas9; Conditional; Homologous recombination; Long single-stranded DNA; Mouse; Mutant.

Fig. 1

Generation of a Syt7 floxed allele. a Diagrammatic representation of the genomic sequence with the Syt7 critical exon highlighted, the corresponding template for lssDNA synthesis and the position of sgRNAs for in vivo delivery together with the primer locations used for reverse transcription and for genotyping. Note loxP sites in the lssDNA prevent reprocessing of repaired alleles by CRISPR-Cas9 complex. Diagram shows the process for the generation of lssDNA through in vitro transcription and reverse transcription. HA homology arm. b PCR products amplified from genomic DNA extracted from the 17 F₀ born from the microinjection session using Syt7-F1 and Syt7-R1 primers. 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). Sequence trace data derived from animals Syt7-4 and Syt7-8 are displayed in Additional file 2: Figure S1.

Fig. 2

Gckr^P446L point mutation. Different designs of reagents for genome editing employing (a) oligonucleotides or (b) a lssDNA donor. Donors were designed containing both coding (in red) and silent mutations (in black) that prevent reprocessing of engineered alleles in accordance with the selected sgRNAs. Guide sequences are named sgRNAs. The shared colour coding of guides and donors highlights reagents injected within the same mix

Fig. 3

Guide-it validation of the five sgRNAs synthesized for the generation of the Gckr^P446L point mutation. Cas9 protein is complexed with each sgRNA (B, D–G) and incubated with short double-stranded DNA fragments containing the protospacer target. Lanes A and C are controls and show the target template but no Cas9/sgRNA complex. The reactions are analyzed for cleavage by electrophoresis on agarose gel. L2 = 100 bp DNA molecular weight ladder (thick bands are 1000 and 500 bp). Protospacer sequences are detailed in Additional file 1: Table S1

Fig. 4

Screening by Sanger sequencing of animals for the generation of the Gckr^P446L point mutation with (a) oligonucleotides (F₀ individual ssO-Gckr^P446L-54) or (b) lssDNA donors (F₀ individuals lss-Gckr^P446L-11 and lss-Gckr^P446L-10 and F₁ individual lss-Gckr^P446L-11.1f). The figure shows Sanger sequencing chromatograms of an amplicon generated with primers anchored external to the intended site of donor sequence integration as detailed in Additional file 15: Figure S14. a ssODN donors only yielded introduction of the intended silent mutations, while (b) lssDNA yielded the desired mutation in some individuals (F₀ 11 transmitting to 11.f) and only the silent mutations in others (F₀ 10). Note that founders appeared homozygous (ssO-Gckr^P446L-54, lss-Gckr^P446L-11 and lss-Gckr^P446L-10) when analyzed by Sanger sequencing, but also could contain deletion alleles in trans, as suggested by copy counting (lss-Gckr^P446L-11 in Table 4). A summary of the microinjection session outcomes is detailed in Table 3, and raw sequencing data are provided in Additional file 16