Journal Article

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

Mackenzie, Matthew, McCabe, Christopher V., Stewart, Michelle E., Allan, Alasdair J., Hutchison, Marie, Christou, Skevoulla, Anstee, Quentin M., Pike, Fran J., Volynski, Kirill E., Wells, Sara, King, Ruairidh, Codner, Gemma F., Simon, Michelle M., Mianné, Joffrey, Caulder, Adam, Teboul, Lydia, Agius, Loranne, Kullmann, Dimitri M., Loeffler, Jorik, Fell, Rachel, Kumar, Saumya, Joynson, Sam

2018

Overview

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.

• Publisher: BioMed Central,BioMed Central Ltd,Springer Nature B.V,BMC
• Subject: Allele validation / Alleles / Animal models / Animals / Biomedical and Life Sciences / Conditional / CRISPR / CRISPR-Cas Systems / CRISPR/Cas9 / Cutting / Data processing / Deoxyribonucleic acid / DNA / DNA, Single-Stranded / Editing / Feasibility studies / Gene Editing - methods / Gene mutations / Gene Targeting / Genetic engineering / Genome editing / Genomes / Homologous recombination / Life Sciences / Maintenance / Mice / Mice - genetics / Molecular chains / Molecular weight / Mouse / Mutagenesis / Mutant / Mutants / Mutation / Polymerase chain reaction / Proteins / Quality control / Reliability aspects / Reproducibility of Results / Research Article / Single-stranded DNA / Stem cells