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Background: Recent developments in CRISPR/Cas9 genome-editing tools have facilitated the introduction of precise alleles, including genetic intervals spanning several kilobases, directly into the embryo. However, the introduction of donor templates, via homology directed repair, can be erroneous or incomplete and these techniques often produce mosaic founder animals. Thus, newly generated alleles must be verified at the sequence level across the targeted locus. Screening for the presence of the desired mutant allele using traditional sequencing methods can be challenging due to the size of the interval to be sequenced, together with the mosaic nature of founders. Methodology/Principal findings: In order to help disentangle the genetic complexity of these animals, we tested the application of Oxford Nanopore Technologies long-read sequencing at the targeted locus and found that the achievable depth of sequencing is sufficient to offset the sequencing error rate associated with the technology used to validate targeted regions of interest. We have assembled an analysis workflow that facilitates interrogating the entire length of a targeted segment in a single read, to confirm that the intended mutant sequence is present in both heterozygous animals and mosaic founders. We used this workflow to compare the output of PCR-based and Cas9 capture-based targeted sequencing for validation of edited alleles. Conclusion: Targeted long-read sequencing supports in-depth characterisation of all experimental models that aim to produce knock-in or conditional alleles, including those that contain a mix of genome-edited alleles. PCR- or Cas9 capture-based modalities bring different advantages to the analysis. Author summary: In this study we investigated the use of long-read sequencing for the analysis of genome editing experiments. We found that Oxford Nanopore Technology sequencing is a superior alternative to Sanger sequencing as it yields a more rapid and comprehensive characterisation of the genotype of both mosaic animals and their progeny. We compared PCR-based and nCATs targeted sequencing modalities and found they have different advantages and applicabilities. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

• The structure of alleles obtained by homologous recombination must be checked. • Southern blot recognises segmental rearrangements and random/multiple integrations. • This protocol employs probes for standard cassettes used in genome engineering. • This method employs non-radioactive chemistry for broad applicability. The widespread availability of recombineered vectors and gene targeted embryonic stem cells from large-scale repositories facilitates the generation of mouse models for functional genetic studies. Southern blotting validates the structure of these targeted alleles produced by homologous recombination, as well as indicating any additional integrations of the vector into the genome. Traditionally this technique employs radioactively-labelled probes; however, there are many laboratories that are restricted in their use of radioactivity. Here, we present a widely applicable protocol for Southern blot analysis using cold probes and alternative procedures employing radioactive probes. Furthermore, the probes are designed to recognise standardised regions of gene-targeting cassettes and so represent universally applicable reagents for assessing allelic integrity. [ABSTRACT FROM AUTHOR]