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.
Abstract The International Mouse Phenotyping Consortium (IMPC) systematically produces and phenotypes mouse lines with presumptive null mutations to provide insight into gene function. The IMPC now uses the programmable RNA-guided nuclease Cas9 for its increased capacity and flexibility to efficiently generate null alleles in the C57BL/6N strain. In addition to being a valuable novel and accessible research resource, the production of 3,313 knockout mouse lines using comparable protocols provides a rich dataset to analyze experimental and biological variables affecting in vivo gene engineering with Cas9. Mouse line production has two critical steps – generation of founders with the desired allele and germline transmission (GLT) of that allele from founders to offspring. A systematic evaluation of the variables impacting success rates identified gene essentiality as the primary factor influencing successful production of null alleles. Collectively, our findings provide best practice recommendations for using Cas9 to generate alleles in mouse essential genes, many of which are orthologs of genes linked to human disease.
Abstract An autosomal dominant GGGGCC repeat expansion in intron 1 of the C9orf72 gene is the most common genetic cause of both amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Here, we set out to engineer a gene targeted mouse model harbouring a pathogenic length humanised C9orf72 repeat expansion allele, in order to model pathological mechanisms in a physiological context. In human disease, pathogenic repeats typically range from the hundreds to thousands of units in length, representing a considerable challenge for cellular and in vivo model generation given the instability of GC rich and repetitive DNA sequences during molecular cloning. To overcome this challenge, we developed new methodology to synthetically and iteratively build pure GGGGCC repeats within a linear vector system, which we then seamlessly and scarlessly embedded within the native human genomic sequence. This created a gene targeting DNA vector for homologous recombination of the human sequence in mouse embryonic stem cells. We used this novel targeting vector to generate a new gene targeted mouse allele, C9orf72 h370 , that for the first time has mouse C9orf72 intron 1 scarlessly replaced with human intron 1 including a pure (GGGGCC) 370 hexanucleotide repeat expansion. We confirm that the mouse model expresses human intron 1-derived RNA and produces dipeptide repeat proteins derived from the GGGGCC repeat expansion. We now provide this model as a new freely available resource for the field. In addition, we demonstrate the utility of our cloning method for engineering diverse repeat expansion sequences for modelling other disorders, such as Fragile X Syndrome.
Karyotypic integrity is essential for the successful germline transmission of alleles mutated in embryonic stem (ES) cells. Classical methods for the identification of aneuploidy involve cytological analyses that are both time consuming and require rare expertise to identify mouse chromosomes. As part of the International Mouse Phenotyping Consortium, we gathered data from over 1,500 ES cell clones and found that the germline transmission (GLT) efficiency of clones is compromised when over 50 % of cells harbour chromosome number abnormalities. In JM8 cells, chromosomes 1, 8, 11 or Y displayed copy number variation most frequently, whilst the remainder generally remain unchanged. We developed protocols employing droplet digital polymerase chain reaction (ddPCR) to accurately quantify the copy number of these four chromosomes, allowing efficient triage of ES clones prior to microinjection. We verified that assessments of aneuploidy, and thus decisions regarding the suitability of clones for microinjection, were concordant between classical cytological and ddPCR-based methods. Finally, we improved the method to include assay multiplexing so that two unstable chromosomes are counted simultaneously (and independently) in one reaction, to enhance throughput and further reduce the cost. We validated a PCR-based method as an alternative to classical karyotype analysis. This technique enables laboratories that are non-specialist, or work with large numbers of clones, to precisely screen ES cells for the most common aneuploidies prior to microinjection to ensure the highest level of germline transmission potential. The application of this method allows early exclusion of aneuploid ES cell clones in the ES cell to mouse conversion process, thus improving the chances of obtaining germline transmission and reducing the number of animals used in failed microinjection attempts. This method can be applied to any other experiments that require accurate analysis of the genome for copy number variation (CNV).
Clinical presentation of congenital heart disease is heterogeneous, making identification of the disease-causing genes and their genetic pathways and mechanisms of action challenging. By using in vivo electrocardiography, transthoracic echocardiography and microcomputed tomography imaging to screen 3,894 single-gene-null mouse lines for structural and functional cardiac abnormalities, here we identify 705 lines with cardiac arrhythmia, myocardial hypertrophy and/or ventricular dilation. Among these 705 genes, 486 have not been previously associated with cardiac dysfunction in humans, and some of them represent variants of unknown relevance (VUR). Mice with mutations in Casz1, Dnajc18, Pde4dip, Rnf38 or Tmem161b genes show developmental cardiac structural abnormalities, with their human orthologs being categorized as VUR. Using UK Biobank data, we validate the importance of the DNAJC18 gene for cardiac homeostasis by showing that its loss of function is associated with altered left ventricular systolic function. Our results identify hundreds of previously unappreciated genes with potential function in congenital heart disease and suggest causal function of five VUR in congenital heart disease.
The application of CRISPR/Cas9 technology has revolutionised genetics by greatly enhancing the efficacy of genome editing in the early embryo. Furthermore, the system has enabled the generation of allele types previously incompatible with in vivo mutagenesis. Despite its versatility and ease of implementation, CRISPR/Cas9 editing outcome is unpredictable and can generate mosaic founders. Therefore, careful genotyping and characterisation of new mutants is proving essential. The literature presents a wide range of protocols for molecular characterisation, each representing different levels of investment. We present strategies and protocols for designing, producing and screening CRISPR/Cas9 edited founders and genotyping their offspring according to desired allele type (indel, point mutation and deletion).
Sporadic Creutzfeldt-Jakob disease (sCJD), the most common human prion disease, is thought to occur when the cellular prion protein (PrPC) spontaneously misfolds and assembles into prion fibrils, culminating in fatal neurodegeneration. In a genome-wide association study of sCJD, we recently identified risk variants in and around the gene STX6, with evidence to suggest a causal increase of STX6 expression in disease-relevant brain regions. STX6 encodes syntaxin-6, a SNARE protein primarily involved in early endosome to trans-Golgi network retrograde transport. Here we developed and characterised a mouse model with genetic depletion of Stx6 and investigated a causal role of Stx6 expression in mouse prion disease through a classical prion transmission study, assessing the impact of homozygous and heterozygous syntaxin-6 knockout on disease incubation periods and prion-related neuropathology. Following inoculation with RML prions, incubation periods in Stx6−/− and Stx6+/− mice differed by 12 days relative to wildtype. Similarly, in Stx6−/− mice, disease incubation periods following inoculation with ME7 prions also differed by 12 days. Histopathological analysis revealed a modest increase in astrogliosis in ME7-inoculated Stx6−/− animals and a variable effect of Stx6 expression on microglia activation, however no differences in neuronal loss, spongiform change or PrP deposition were observed at endpoint. Importantly, Stx6−/− mice are viable and fertile with no gross impairments on a range of neurological, biochemical, histological and skeletal structure tests. Our results provide some support for a pathological role of Stx6 expression in prion disease, which warrants further investigation in the context of prion disease but also other neurodegenerative diseases considering syntaxin-6 appears to have pleiotropic risk effects in progressive supranuclear palsy and Alzheimer's disease.
Abstract Recent developments in CRISPR/Cas9 genome editing tools have facilitated the introduction of more complex alleles, often spanning genetic intervals of several kilobases, directly into the embryo. These techniques often produce mosaic founder animals and the introduction of donor templates, via homologous directed repair, can be erroneous or incomplete. 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 desired edit(s) together with founder mosaicism. In order to help disentangle the genetic complexity of these animals, we tested the application of Oxford Nanopore long read sequencing of the targeted locus. Taking advantage of sequencing the entire length of the segment in each single read, we were able to determine whether the entire intended mutant sequence was present in both mosaic founders and their offspring.
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. 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. 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.
'%3e%3cpath fill='%23012169' d='M0 0v30h60V0z'/%3e%3cpath stroke='%23fff' stroke-width='6' d='m0 0 60 30m0-30L0 30'/%3e%3cpath stroke='%23C8102E' stroke-width='4' d='m0 0 60 30m0-30L0 30' clip-path='url(%23b)'/%3e%3cpath stroke='%23fff' stroke-width='10' d='M30 0v30M0 15h60'/%3e%3cpath stroke='%23C8102E' stroke-width='6' d='M30 0v30M0 15h60'/%3e%3c/g%3e%3c/svg%3e)
