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

Codner et al. BMC Biology (2018) 16:70 https://doi.org/10.1186/s12915-018-0530-7 RESEARCH ARTICLE Open Access Application of long single-stranded DNA donors in genome editing: generation and validation of mouse mutants Gemma F. Codner1†, Joffrey Mianné1†, Adam Caulder1, Jorik Loeffler1, Rachel Fell1, Ruairidh King1, Alasdair J. Allan1, Matthew Mackenzie1, Fran J. Pike1, Christopher V. McCabe1, Skevoulla Christou1, Sam Joynson1, Marie Hutchison1, Michelle E. Stewart1, Saumya Kumar2, Michelle M. Simon2, Loranne Agius3, Quentin M. Anstee3, Kirill E. Volynski4, Dimitri M. Kullmann4, Sara Wells1 and Lydia Teboul1* 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, Conditional, CRISPR/Cas9, Homologous recombination, Mouse, Mutant, Long single-stranded DNA * Correspondence: † Gemma F. Codner and Joffrey Mianné contributed equally to this work. 1 The Mary Lyon Centre, MRC Harwell Institute, Didcot, Oxon OX11 0RD, UK Full list of author information is available at the end of the article © Teboul et al. 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Codner et al. BMC Biology (2018) 16:70 Background Classical gene targeting employing embryonic stem cells has long been the principal method to introduce complex alleles into the mouse genome [1]. More recently, microinjection of an RNA-guided engineered nuclease (RGEN) together with a single-stranded oligodeoxynucleotide (ssODN) has revolutionized our ability to direct mutations in vivo [2]. However, clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9)-aided knock-ins of larger cassettes or loxP sites directly into one-cell mouse embryos [3, 4] were breakthroughs that have remained technically very challenging [5]. Equally, CRISPR/Cas9 reagents and ssODNs have become widely used for the introduction of point mutations in one-cell embryos (see examples in [6–8]). However, particular locations within genomes, including sequences that are highly conserved and/or repeated, regions with a low number or absence of -NGG tri-nucleotides or sequences without active single guide RNA (sgRNA) close to the target can represent a barrier to the generation of specific mutants [9]. Miura and colleagues [10] first proposed long single-stranded DNA (lssDNA) molecules, larger than standard chemically synthesized oligonucleotides, as an efficient alternative donor template for RGEN-aided homologous recombination (HR). The authors recently extended the method to the creation of conditional alleles and tag insertions, showing the generation of sequence-perfect alleles [11]. We and others documented that CRISPR/Cas9-aided genome editing can give rise to unexpected allele rearrangements (“illegitimate repairs” [7], “KI + indels” [9, 12]); therefore, thorough validation of new models is essential to ensure reproducibility of the studies employing these models [12–15]. However, limited data are available on unexpected events arising from the use of lssDNA and the associated requirements for the quality control (QC) of new models. With our extensive experience in the generation of conditional alleles through large-scale mouse model production [16, 17], we have developed a strategy for validation of these alleles. Here, we have extended the application of lssDNA to the generation of more conditional knock-out (cKO) alleles directly in the embryo. We also produced point mutations where the desired nucleotide change is remote from active CRISPR cutting sites, which so far had proved technically challenging with the available protocols. Although not all attempts were successful, we confirm that new designs employing lssDNA indeed facilitated mutant production for cKOs and particular point mutations that had previously been challenging to generate. Furthermore, we show that novel point mutations and imperfect and/or off-target donor integration(s) can occur in the process of mutagenesis. This work emphasizes the importance of a Page 2 of 16 comprehensive strategy for the QC of new mutants. We conclude that the utilization of lssDNA donor templates shifts the challenge of mutagenesis from generation to the validation of new mutant models. Results Generation of a conditional knock-out allele Production of F0 animals Proof of principle for the RGEN-aided generati (...truncated)