Founder Animals Research Articles

Germline transmission of MSTN knockout cattle via CRISPR-Cas9

Although the production of several founder animals (F0) for gene editing in livestock has been reported in cattle, very few studies have assessed germline transmission to the next generation due to the long sexual maturation and gestation periods. The present study aimed to assess the germline transmission of MSTN mutations (-12bps deletion) in MSTN mutant F0 male and female cattle. For this purpose, oocytes and semen were collected after the sexual maturation of MSTN cattle, and embryos produced by in vitro fertilization were analyzed. In addition, the embryos were subjected to additional gene (PRNP) editing using electroporation. Embryos produced by in vitro fertilization with MSTN male and female cattle were transferred to a surrogate, and one calf was successfully born. MSTN heterozygous mutation was shown by sequencing of the F1 calf, which had no health issues. As a further experiment, using electroporation, additional gene-edited embryos fertilized with the MSTN male sperm showed a high mutation rate of PRNP (86.2 ± 3.4%). These data demonstrate that the cattle produced through gene editing matured without health issues and had transmitted MSTN mutation from the germ cells. Also, additional mutation of embryos fertilized with the MSTN male sperm could enable further mutagenesis using electroporation.

Pedigree-Based Description of Three Traditional Hungarian Horse Breeds.

Simple SummaryThe most important purpose of animal conservation programs is to maintain genetic variability. The Furioso-North Star, the Gidran, and the Nonius are indigenous Hungarian horse breeds from the Mezőhegyes Stud. In the last century, the role of the horses was changed, the technical innovations and motorization replaced them, so the population size and the genetic variability of these breeds were reduced. Nowadays these breeds are endangered. The aims of this study were to give information about the current breeding population and support breeder associations during their gene conservation work. The pedigree quality, generation intervals, probability of gene origin, and inbreeding were evaluated. We found that breeds had a large bottleneck effect during breeding history. The level of inbreeding was measured with different methods, such as Ballou’s, Wright’s, and Kalinowski’s coefficient. Most of the current inbreeding coefficient was the result of previously fixed alleles for each breed. Effective population size was also estimated, and the status of the breeds was found to be not critical according to FAO criteria.The Mezőhegyes Stud was founded in 1784 where three different horse breeds were developed: the Furioso-North Star, the Gidran, and the Nonius. These breeds were based on the same mare population, but each breed had different utilization purposes. Our aim was to analyze the pedigree information of these three indigenous breeds. The genealogical information was traced back from the actual breeding population back to the founder animals, and the final database contained more than 47,000 horses. The reference populations were defined as the registered breeding animals in 2019. The complete generation equivalent was 16.45 for the Gidran breed, 15.18 for Furioso-North Star, and 12.64 for Nonius, respectively. Due to the utilization of English Thoroughbred during the breeding history, the average maximum generations were close to 36 generations for each breed. The average relatedness was approximately 4%. The average Wright’s inbreeding coefficient was the highest for the Nonius breed (5.59%). Kalinowski’s decomposition of inbreeding showed that inbreeding is originated mainly from the past; the current fixation of alleles was higher for the Nonius horse breed. There was a reasonable bottleneck effect for each breed. The estimated effective population sizes suggest that there is no problem with the maintaining of Mezőhegyes horse breeds.

Consideration of genetic variation and evolutionary history in future conservation of Indian one-horned rhinoceros (Rhinoceros unicornis)

BackgroundThe extant members of the Asian rhinos have experienced severe population and range declines since Pleistocene through a combination of natural and anthropogenic factors. The one-horned rhino is the only Asian species recovered from such conditions but most of the extant populations are reaching carrying capacity. India currently harbours ~ 83% of the global wild one-horned rhino populations distributed across seven protected areas. Recent assessments recommend reintroduction-based conservation approaches for the species, and implementation of such efforts would greatly benefit from detailed genetic assessments and evolutionary history of these populations. Using mitochondrial data, we investigated the phylogeography, divergence and demographic history of one-horned rhinos across its Indian range.ResultsWe report the first complete mitogenome from all the extant Indian wild one-horned rhino populations (n = 16 individuals). Further, we identified all polymorphic sites and assessed rhino phylogeography (2531 bp mtDNA, n = 111 individuals) across India. Results showed 30 haplotypes distributed as three distinct genetic clades (Fst value 0.68–1) corresponding to the states of Assam (n = 28 haplotypes), West Bengal and Uttar Pradesh (both monomorphic). The reintroduced population of Uttar Pradesh showed maternal signatures of Chitwan National Park, Nepal. Mitochondrial phylogenomics suggests one-horned rhino diverged from its recent common ancestors ~ 950 Kya and different populations (Assam, West Bengal and Uttar Pradesh/Nepal) coalesce at ~ 190–50 Kya, corroborating with the paleobiogeography history of the Indian subcontinent. Further, the demography analyses indicated historical decline in female effective population size ~ 300–200 Kya followed by increasing trends during ~ 110–60 Kya.ConclusionThe phylogeography and phylogenomic outcomes suggest recognition of three ‘Evolutionary Significant Units (ESUs)’ in Indian rhino. With ongoing genetic isolation of the current populations, future management efforts should focus on identifying genetically variable founder animals and consider periodic supplementation events while planning future rhino reintroduction programs in India. Such well-informed, multidisciplinary approach will be the only way to ensure evolutionary, ecological and demographic stability of the species across its range.

A scalable, clinically severe pig model for Duchenne muscular dystrophy.

ABSTRACTLarge-animal models for Duchenne muscular dystrophy (DMD) are crucial for the evaluation of diagnostic procedures and treatment strategies. Pigs cloned from male cells lacking DMD exon 52 (DMDΔ52) exhibit molecular, clinical and pathological hallmarks of DMD, but die before sexual maturity and cannot be propagated by breeding. Therefore, we generated female DMD+/− carriers. A single founder animal had 11 litters with 29 DMDY/−, 34 DMD+/− as well as 36 male and 29 female wild-type offspring. Breeding with F1 and F2 DMD+/− carriers resulted in an additional 114 DMDY/− piglets. With intensive neonatal management, the majority survived for 3-4 months, providing statistically relevant cohorts for experimental studies. Pathological investigations and proteome studies of skeletal muscles and myocardium confirmed the resemblance to human disease mechanisms. Importantly, DMDY/− pigs displayed progressive myocardial fibrosis and increased expression of connexin-43, associated with significantly reduced left ventricular ejection fraction, at 3 months. Furthermore, behavioral tests provided evidence for impaired cognitive ability. Our breeding cohort of DMDΔ52 pigs and standardized tissue repositories provide important resources for studying DMD disease mechanisms and for testing novel treatment strategies.

ناهمگنی اثر همخونی جزئی حاصل از حیوانات بنیان‌گذار بر صفات وزن تولد و وزن از شیرگیری بره‌های بلوچی

Uneven distribution of recessive alleles among founder animals genomes leads to the heterogeneity of inbreeding depression among their descendants. In this study, pedigree information, birth weight, and weaning weight records of 4032 Baluchi sheep collected between 1989 to 2017 at the Baluchi sheep breeding station were used to assess the heterogeneity of inbreeding depression between founders of the population. The Wright inbreeding coefficient of the inbred animals was decomposed to the partial inbreeding of the four founder animals with the greatest contribution to the inbreeding of the population. Wright inbreeding coefficients and partial inbreeding coefficients of the founder animals were included as a covariate in the animal model, in two separate analyses. The regression coefficients of birth weight and weaning weight from Wright inbreeding were estimated to be -6.4 and -61.9 g, respectively. The effect of partial inbreeding from different founders on the studied traits was heterogeneous. The regression coefficient of birth weight on partial inbreeding of founder B was -79 g, while partial inbreeding from founder D caused an increase of 121 g in this trait. In weaning weight, partial inbreeding of founder D caused an increase of 83 g in this trait, while partial inbreeding of other founders did not affect this trait. The observed heterogeneity in the effects of partial inbreeding of the different animals can confirm the hypothesis that a few recessive alleles with major effects are contributing to the inbreeding depression of these traits. According to the results of this study, the heterogeneity of the effect of inbreeding of the founder animals should be considered in the genetic evaluation model of this breed.

Screening and validation of genome-edited animals.

The emergence of an array of genome-editing tools in recent years has facilitated the introduction of genetic modifications directly into the embryo, increasing the ease, efficiency and catalogue of alleles accessible to researchers across a range of species. Bypassing the requirement for a selection cassette and resulting in a broad range of outcomes besides the desired allele, genome editing has altered the allele validation process both temporally and technically. Whereas traditional gene targeting relies upon selection and allows allele validation at the embryonic stem cell modification stage, screening for the presence of the intended allele now occurs in the (frequently mosaic) founder animals. Final confirmation of the edited allele can only take place at the subsequent G1 generation and the validation strategy must differentiate the desired allele from a range of unintended outcomes. Here we present some of the challenges posed by gene editing, strategies for validation and considerations for animal colony management.

25th ANNIVERSARY OF CLONING BY SOMATIC CELL NUCLEAR TRANSFER: Generation of genetically engineered livestock using somatic cell nuclear transfer.

Genetic engineering (GE) of livestock initially has been accomplished primarily using pronuclear microinjection into zygotes (1985-1996). The applications of the technology were limited due to low integration efficiency, aberrant transgene expression resulting from random integration and the presence of genetic mosaicism in transgenic founder animals. Despite enormous efforts to established embryonic stem cells (ESCs) for domestic species, the ESC GE technology does not exist for livestock. Development of somatic cell nuclear transfer (SCNT) has bypassed the need in livestock ESCs and revolutionized the field of livestock transgenesis by offering the first cell-based platform for precise genetic manipulation in farm animals. For nearly two decades since the birth of Dolly (1996-2013), SCNT was the only method used for the generation of knockout and knockin livestock. Arrival of CRISPRS/Cas9 system, a new generation of gene-editing technology, gave us an ability to introduce precise genome modifications easily and efficiently. This technological advancement accelerated production of GE livestock by SCNT and reinstated zygote micromanipulation as an important GE approach. The primary advantage of the SCNT technology is the ability to confirm in vitro that the desired genetic modification is present in the somatic cells prior to animal production. The edited cells could also be tested for potential off-target mutations. Additionally, this method eliminates the risk of genetic mosaicism frequently observed following zygote micromanipulation. Despite its low efficiency, SCNT is a well-established procedure in numerous laboratories around the world and will continue to play an important role in the GE livestock field.

EQUINE SARCOIDS IN CAPTIVE WILD EQUIDS: DIAGNOSTIC AND CLINICAL MANAGEMENT OF 16 CASES-A POSSIBLE PREDISPOSITION OF THE EUROPEAN COHORT OF SOMALI WILD ASS (EQUUS AFRICANUS SOMALIENSIS)?

Equine sarcoids (ES) were diagnosed in 12 Somali wild asses (SWA) (Equus africanus somaliensis) from 10 different institutions of the SWA European Endangered Species Programme from 1976 to 2019. Samples of surgically excised masses, biopsies, or necropsy samples were submitted for histologic and virologic analysis. In addition, tissue samples from one onager (Equus hemionus onager), one kulan (Equus hemionus kulan), and two Hartmann's mountain zebras (HMZ) (Equus zebra hartmannae) were examined. Histology confirmed the diagnosis of ES exhibiting the typical microscopic features. Polymerase chain reaction detected bovine papillomavirus type 1 (BPV1) DNA in eight SWA samples and bovine papillomavirus type 2 (BPV2) DNA in one SWA sample. The onager, kulan, and one HMZ sample tested positive for BPV1. The other HMZ tested positive for BPV1 and BPV2. This is the first report of ES in an onager. Surgical excision was the treatment elected by most veterinarians. A follow-up survey of the cases over several years after clinical diagnosis and therapy revealed variable individual outcome with ES recurrence in four cases. Three SWA and the kulan were euthanized due to the severity of the lesions. Nine affected SWA were males with seven having a sarcoid located at the prepuce. Because a genetic disposition is a risk factor for the development of ES in horses, this may also be true for endangered wild equids with few founder animals in their studbook history. Innovative approaches regarding therapy and prevention of ES in wild equids are therefore highly encouraged.

Generation and expression analysis of BAC humanized mice carrying HLA-DP401 haplotype.

BackgroundHuman leukocyte antigen (HLA)‐DP is much less studied than other HLA class II antigens, that is, HLA‐DR and HLA‐DQ, etc. However, the accumulating data have suggested the important roles of DP‐restricted responses in the context of cancer, allergy, and infectious disease. Lack of animal models expressing these genes as authentic cis‐haplotypes blocks our understanding for the role of HLA‐DP haplotypes in immunity.MethodsTo explore the potential cis‐acting control elements involved in the transcriptional regulation of the HLA‐DPA1/DPB1 gene, we performed the expression analysis using bacterial artificial chromosome (BAC)‐based transgenic humanized mice in the C57BL/6 background, which carried the entire HLA‐DP401 gene locus. We further developed a mouse model of Staphylococcus aureus pneumonia in HLA‐DP401 humanized transgenic mice, and performed the analysis on the expression pattern of HLA‐DP401 and immunological responses in the model.ResultsIn this study, we screened and identified a BAC clone spanning the entire HLA‐DP gene locus. DNA from this clone was analyzed for integrity by pulsed‐field gel electrophoresis and then microinjected into fertilized mouse oocytes to produce transgenic founder animals. Nine sets of PCR primers for regional markers with an average distance of 15 kb between each primer were used to confirm the integrity of the transgene in the five transgenic lines carrying the HLA‐DPA1/DPB1 gene. Transgene copy numbers were determined by real‐time PCR analysis. HLA‐DP401 gene expression was analyzed at the mRNA and protein level. Although infection with S aureus Newman did not alter the percentage of immune cells in the spleen and thymus from the HLA‐DP401‐H2‐Aβ1 humanized mice. Increased expression of HLA‐DP401 was observed in the thymus of the humanized mice infected by S aureus.ConclusionsWe generated several BAC transgenic mice, and analyzed the expression of HLA‐DPA1/DPB1 in those mice. A model of S aureus‐induced pneumonia in the HLA‐DP401‐H2‐Aβ1−/− humanized mice was further developed, and S aureus infection upregulated the HLA‐DP401 expression in thymus of those humanized mice. These findings demonstrate the potential of those HLA‐DPA1/DPB1 transgenic humanized mice for developing animal models of infectious diseases and MHC‐associated immunological diseases.

Potential functional variants in AHR signaling pathways are associated with age at puberty in swine.

Puberty in female pigs is defined as age at first estrus and gilts that have an earlier age at puberty are more likely to have greater lifetime productivity. Because age at puberty is predictive for sow longevity and lifetime productivity, but not routinely measured in commercial herds, it would be beneficial to use genomic or marker-assisted selection to improve these traits. A GWAS at the US Meat Animal Research Center (USMARC) identified several loci associated with age at puberty in pigs. Candidate genes in these regions were scanned for potential functional variants using sequence information from the USMARC swine population founder animals and public databases. In total, 135 variants (SNP and insertion/deletions) in 39 genes were genotyped in 1284 phenotyped animals from a validation population sired by Landrace and Yorkshire industry semen using the Agena MassArray system. Twelve variants in eight genes were associated with age at puberty (P<0.005) with estimated additive SNP effects ranging from 1.6 to 5.3days. Nine of these variants were non-synonymous coding changes in AHR, CYP1A2, OR2M4, SDCCAG8, TBC1D1 and ZNF608, two variants were deletions of one and four codons in aryl hydrocarbon receptor, AHR, and the most significant SNP was near an acceptor splice site in the acetyl-CoA carboxylase alpha, ACACA. Several of the loci identified have a physiological and a genetic role in sexual maturation in humans and other animals and are involved in AHR-mediated pathways. Further functional validation of these variants could identify causative mutations that influence age at puberty in gilts and possibly sow lifetime productivity.

Development of PCR based approach to detect potential mosaicism in porcine embryos

Direct injection of genome editing tools such as CRISPR/Cas9 system into developing embryos has been widely used to generate genetically engineered pigs. The approach allows us to produce pigs carrying targeted modifications at high efficiency without having to apply somatic cell nuclear transfer. However, the targeted modifications during embryogenesis often result in mosaicism, which causes issues in phenotyping founder animals and establishing a group of pigs carrying intended modifications. This study was aimed to establish a genomic PCR and sequencing system of a single blastomere in the four-cell embryos to detect potential mosaicism. We performed genomic PCR in four individual blastomeres from four-cell embryos. We successfully amplified target genomic region from single blastomeres of 4-cell stage embryo by PCR. Sanger sequencing of the PCR amplicons obtained from the blastomeres suggested that PCR-based genotyping of single blastomere was a feasible method to determine mutation type generated by genome editing technology such as CRISPR/Cas9 in early stage embryos. In conclusion, we successfully genotyped single blastomeres in a single 4-cell stage embryo to detect potential mosaicism in porcine embryos. Our approach offers a simple platform that can be used to screen the prevalence of mosaicism from designed CRISPR/Cas9 systems.

Population structure analysis of Yeonsan Ogye using microsatellite markers.

The Yeonsan Ogye (YO) chicken is a natural heritage of Korea, characterized by black feathers, skin, bones, eyes, and comb. The purebred of YO population has been reared under the natural mating system with no systematic selection and breeding plan. The purpose of this study was to identify the genetic diversity and find the optimal number of population sub-division using 12 polymorphic microsatellite (MS) markers to construct a pedigree-based breeding plan for the YO population. A total of 509 YO birds were used for this study. Genetic diversity and population structure analysis were conducted based on the MS marker genotype information. The overall average polymorphic information content value and expected heterozygosity of the population were 0.586, and 0.642, respectively. The K-mean cluster analysis based on the genetic distance result confirmed that the current YO population can be divided into three ancestry groups. Individuals in each group were evaluated based on their genetic distance to identify the potential candidates for a future breeding plan. This study concludes that a future breeding plan with known pedigree information of selected founder animals, which holds high genetic diversity, could be the best strategy to ensure the conservation of the Korean YO chicken population.

CRISPR Turbo Accelerated KnockOut (CRISPy TAKO) for Rapid in vivo Screening of Gene Function.

The development of CRISPR/Cas9 technology has vastly sped up the process of mammalian genome editing by introducing a bacterial system that can be exploited for reverse genetics-based research. However, generating homozygous functional knockout (KO) animals using traditional CRISPR/Cas9-mediated techniques requires three generations of animals. A founder animal with a desired mutation is crossed to produce heterozygous F1 offspring which are subsequently interbred to generate homozygous F2 KO animals. This study describes an adaptation of the CRISPR/Cas9-mediated method to develop a cohort of homozygous gene-targeted KO animals in one generation. A well-characterized ethanol-responsive gene, MyD88, was chosen as a candidate gene for generation of KO mice as proof-of-concept. Previous studies have reported changes in ethanol-related behavioral outcomes in MyD88 KO mice. One-cell mouse embryos were simultaneously electroporated with four gRNAs targeting a critical Exon of MyD88 along with Cas9 protein. DNA and RNA analysis of founder mice revealed a complex mix of genetic alterations, all of which were predicted to ablate MyD88 gene function. Behavioral testing confirmed the hypothesis that successful one-generation KO of MyD88 would reproduce the decreased ethanol-induced sedative/hypnotic effects and increased ethanol consumption in males that were observed in previous studies. This study additionally compared responses of Mock treatment control mice generated through electroporation to controls purchased from a vendor. No substantial behavioral changes were noted between control cohorts. Overall, the CRISPR/Cas9 KO protocol reported here, which we call CRISPR Turbo Accelerated KnockOut (CRISPy TAKO), will be useful for reverse genetic in vivo screens of gene function in whole animals.

A porcine model of phenylketonuria generated by CRISPR/Cas9 genome editing.

Phenylalanine hydroxylase–deficient (PAH-deficient) phenylketonuria (PKU) results in systemic hyperphenylalaninemia, leading to neurotoxicity with severe developmental disabilities. Dietary phenylalanine (Phe) restriction prevents the most deleterious effects of hyperphenylalaninemia, but adherence to diet is poor in adult and adolescent patients, resulting in characteristic neurobehavioral phenotypes. Thus, an urgent need exists for new treatments. Additionally, rodent models of PKU do not adequately reflect neurocognitive phenotypes, and thus there is a need for improved animal models. To this end, we have developed PAH-null pigs. After selection of optimal CRISPR/Cas9 genome-editing reagents by using an in vitro cell model, zygote injection of 2 sgRNAs and Cas9 mRNA demonstrated deletions in preimplantation embryos, with embryo transfer to a surrogate leading to 2 founder animals. One pig was heterozygous for a PAH exon 6 deletion allele, while the other was compound heterozygous for deletions of exon 6 and of exons 6–7. The affected pig exhibited hyperphenylalaninemia (2000–5000 μM) that was treatable by dietary Phe restriction, consistent with classical PKU, along with juvenile growth retardation, hypopigmentation, ventriculomegaly, and decreased brain gray matter volume. In conclusion, we have established a large-animal preclinical model of PKU to investigate pathophysiology and to assess new therapeutic interventions.

Genomic diversity and population structure of the Leonberger dog breed

BackgroundLeonberger is a giant dog breed formed in the 1850s in Germany. Its post-World War II popularity has resulted in a current global population of ~ 30,000 dogs. The breed has predispositions to neurodegenerative disorders and cancer, which is likely due in large part to limited genetic diversity. However, to date there is no scientific literature on the overall demography and genomic architecture of this breed.ResultsWe assessed extensive pedigree records, SNP array genotype data, and whole-genome sequences (WGS) on 142,072, 1203 and 39 Leonberger dogs, respectively. Pedigree analyses identified 22 founder animals and revealed an apparent popular sire effect. The average pedigree-based inbreeding coefficient of 0.29 and average kinship of 0.31 show a dramatic loss of genetic diversity. The observed average life span decreased over time from 9.4 years in 1989 to 7.7 years in 2004. A global health survey confirmed a high prevalence of cancer and neurological disorders. Analysis of SNP-based runs of homozygosity (ROH) identified 125,653 ROH with an average length of 5.88 Mb, and confirmed an average inbreeding coefficient of 0.28. Genome-wide filtering of the WGS data revealed 28 non-protein-changing variants that were present in all Leonberger individuals and a list of 22 potentially pathogenic variants for neurological disorders of which 50% occurred only in Leonbergers and 50% occurred rarely in other breeds. Furthermore, one of the two mtDNA haplogroups detected was present in one dog only.ConclusionsThe increasing size of the Leonberger population has been accompanied by a considerable loss of genetic diversity after the bottleneck that occurred in the 1940s due to the intensive use of popular sires resulting in high levels of inbreeding. This might explain the high prevalence of certain disorders; however, genomic data provide no evidence for fixed coding variants that explain these predispositions. The list of candidate causative variants for polyneuropathy needs to be further evaluated. Preserving the current genetic diversity is possible by increasing the number of individuals for breeding while restricting the number of litters per sire/dam. In addition, outcrossing would help optimize long-term genetic diversity and contribute to the sustainability and health of the population.

Assessment of between-founder heterogeneity in inbreeding depression for reproductive traits in Baluchi sheep

Magnitude of inbreeding depression could be different among descendants of various founder animals, when recessive genetic composition of a population is unevenly distributed among founder animal genomes. Reproductive records of Baluchi sheep for the litter variables size at birth (LSB), size at weaning (LSW), mean weight/lamb born (LMWLB), mean weight/lamb weaned (LMWLW), total weight at birth/ewe lambing (TLWB) and total weight at weaning/ewe lambing (TLWW) were used to examine heterogeneity in inbreeding depression between founder animals. Pedigree-based inbreeding coefficients were proportioned into components coming from founder animals and Mendellian contribution from non-founder animals. Two approaches were used to assess effects of inbreeding: overall inbreeding coefficients or partial inbreeding of the four main founder animals as possible covariates included in statistical models. Among the traits evaluated, there were effects on LMWLB, LMWLW and TLWW of inbreeding with there being a −8, -48, and −95 g decrease per 1% increase in inbreeding, respectively. Linear regressions of traits on partial inbreeding coefficients due to founder animals were of different magnitudes and ranged between -0.12 and +0.128. Heterogeneous contribution of founder animals to inbreeding depression occurred for LSB, LMWLB, and TLWW. These results indicate there was uneven distribution of recessive genetic composition among genomes of founder animals or differences in selection pressures on unfavorable alleles between different founder lines. The observed variation in founder-specific inbreeding depression indicates a small number of alleles with major effects are contributing to inbreeding depression.

A simple and practical workflow for genotyping of CRISPR-Cas9-based knockout phenotypes using multiplexed amplicon sequencing.

Founder animals carrying high proportions of somatic mutation induced by CRISPR-Cas9 enable a rapid and scalable strategy for the functional screening of numerous target genes in vivo. In this functional screening, genotyping using pooled amplicons with next-generation sequencing is the most suitable approach for large-scale management of multiple samples and accurate evaluation of the efficiency of Cas9-induced somatic mutations at target sites. Here, we present a simple workflow for genotyping of multiple CRISPR-Cas9-based knockout founders by pooled amplicon sequencing. Using custom barcoded primers, pooled amplicons from multiple individuals can be run in a single-indexed library on the Illumina MiSeq platform. Additionally, a user-friendly web tool, CLiCKAR, is available to simultaneously perform demultiplexing of pooled sequence data and evaluation of somatic mutation in each phenotype. CLiCKAR provides users with practical reports regarding the positions of insertions/deletions, as well as the frameshift ratio and tables containing mutation sequences, and read counts of each phenotype, with just a few clicks by the implementation of demultiplexing for pooled sample data and calculation of the frameshift ratio. This genotyping workflow can be harnessed to evaluate genotype-phenotype correlations in CRISPR-Cas9-based loss-of-function screening of numerous target genes in various organisms.

Suppressor mutations in Mecp2-null mice implicate the DNA damage response in Rett syndrome pathology.

Mutations in X-linked methyl-CpG-binding protein 2 (MECP2) cause Rett syndrome (RTT). To identify functional pathways that could inform therapeutic entry points, we carried out a genetic screen for secondary mutations that improved phenotypes in Mecp2/Y mice after mutagenesis with N-ethyl-N-nitrosourea (ENU). Here, we report the isolation of 106 founder animals that show suppression of Mecp2-null traits from screening 3177 Mecp2/Y genomes. Whole-exome sequencing, genetic crosses, and association analysis identified 22 candidate genes. Additional lesions in these candidate genes or pathway components associate variant alleles with phenotypic improvement in 30 lines. A network analysis shows that 63% of the genes cluster into the functional categories of transcriptional repression, chromatin modification, or DNA repair, delineating a pathway relationship with MECP2. Many mutations lie in genes that modulate synaptic signaling or lipid homeostasis. Mutations in genes that function in the DNA damage response (DDR) also improve phenotypes in Mecp2/Y mice. Association analysis was successful in resolving combinatorial effects of multiple loci. One line, which carries a suppressor mutation in a gene required for cholesterol synthesis, Sqle, carries a second mutation in retinoblastoma binding protein 8, endonuclease (Rbbp8, also known as CtIP), which regulates a DDR choice in double-stranded break (DSB) repair. Cells from Mecp2/Y mice have increased DSBs, so this finding suggests that the balance between homology-directed repair and nonhomologous end joining is important for neuronal cells. In this and other lines, two suppressor mutations confer greater improvement than one alone, suggesting that combination therapies could be effective in RTT.

Efficient and specific generation of knockout mice using Campylobacter jejuni CRISPR/Cas9 system

The Streptococcus pyogenes CRISPR/Cas9 (SpCas9) system is now widely utilized to generate genome engineered mice; however, some studies raised issues related to off-target mutations with this system. Herein, we utilized the Campylobacter jejuni Cas9 (CjCas9) system to generate knockout mice. We designed sgRNAs targeting mouse Tyr or Foxn1 and microinjected into zygotes along with CjCas9 mRNA. We obtained newborn mice from the microinjected embryos and confirmed that 50% (Tyr) and 38.5% (Foxn1) of the newborn mice have biallelic mutation on the intended target sequences, indicating efficient genome targeting by CjCas9. In addition, we analyzed off-target mutations in founder mutant mice by targeted deep sequencing and whole genome sequencing. Both analyses revealed no off-target mutations at potential off-target sites predicted in silico and no unexpected random mutations in analyzed founder animals. In conclusion, the CjCas9 system can be utilized to generate genome edited mice in a precise manner.

Assessing the fertility of two mares cloned from the same founder animal.

Two cloned mares, produced from the same sample of skin fibroblasts, were bred during four breeding seasons from their second year of age, as embryo donors, in exactly the same conditions, using the same stallions for both cloned mares. The aim of this study was to test the embryo donor potential of cloned mares and to compare the results obtained from two cloned mares of the same mare with other embryo donor mares (n=31-39 per breeding season) at the same stud. For both cloned mares, 19 embryos were recovered by 43 collection attempts (44%) (7/22 for one; 12/21 for the other), 16 (84%) pregnancies (5/7 for one, 11/12 for the other) were obtained at day 14 post-ovulation (D14 ), and 12 (3/7 for one; 9/12 for the other) foals were born. One cloned mare was a less efficient donor mare than the other (p<.05), In control donor mares, 623 embryo collections were performed, with a recovery rate (80%-496/623) significantly higher than for cloned mares. The recovery rate in the subpopulation of 2-5-year-old control donor mares (same age of cloned mares) (89%-127/143) and The recovery rate in the subpopulation of 12 control mares bred with the seven same stallions as clones (55%-17/31), were both higher than for cloned mare (p<.05). The success rate of transfer was not different between embryos produced by cloned mares (84%-16/19) and those produced by control donor mares (79%-392/496). However, the foaling rate per embryo collection was significantly lower for cloned mares (28%-12/43) than for control donor mares (52% - 325/623) (p<.05).