Mosaic Mutations Research Articles

Genetic analyses of a secondary poroma and trichoblastoma in a HRAS-mutated sebaceous nevus.

A sebaceous nevus is a congenital skin hamartoma caused by postzygotic HRAS or KRAS mosaic mutations. With age, affected individuals may develop secondary tumors within a sebaceous nevus. RAS mutations are harbored from the onset of sebaceous nevus, and further mutations can be expected to be required in order to explain the initiation of secondary tumors. However, genetic analyses of the secondary tumors have not been conducted. Herein, we describe the rare coexistence of a poroma and a trichoblastoma arising in a sebaceous nevus. This is the first report of an investigation of multiple genes in a secondary tumor in an SN. First, HRAS c.37G>C, which is the common mutation in sebaceous nevus, was detected in all three lesions (sebaceous nevus, poroma, and trichoblastoma). Next, to elucidate the potential second-hit mutations in the secondary poroma and trichoblastoma, we applied a panel sequencing for skin cancers that was newly developed in our institution. Our comparison of the mutational profile of 95 skin cancer-related genes in each of the three lesions newly revealed TP53 p.R158P in the poroma and NOTCH2 p.G329S in the trichoblastoma. TP53 p.R158P has been determined as a pathogenic mutation in other tumors, and NOTCH2 p.G329S was a novel mutation. We identified two novel mutations that may have contributed to the pathogenesis of the secondary tumor's development. The roles of the mutations remain unclear.

Detection of Brain Somatic Mutations in Cerebrospinal Fluid from Refractory Epilepsy Patients.

Brain mosaic mutations are a major cause of refractory focal epilepsies with cortical malformations such as focal cortical dysplasia, hemimegalencephaly, malformation of cortical development with oligodendroglial hyperplasia in epilepsy, and ganglioglioma. Here, we collected cerebrospinal fluid (CSF) during epilepsy surgery to search for somatic variants in cell-free DNA (cfDNA) using targeted droplet digital polymerase chain reaction. In 3 of 12 epileptic patients with known somatic mutations previously identified in brain tissue, we here provide evidence that brain mosaicism can be detected in the CSF-derived cfDNA. These findings suggest future opportunities for detecting the mutant allele driving epilepsy in CSF. ANN NEUROL 2021;89:1248-1252.

WDR45 Gene and Its Role in Pediatric Epilepsies

AbstractWD repeat domain 45 (WDR45) gene has been increasingly found in patients with developmental delay (DD) and epilepsy. Previously, WDR45 de novo mutations were reported in sporadic adult and pediatric patients presenting iron accumulation, while heterozygous mutations were associated with β-propeller protein-associated neurodegeneration (BPAN), a subtype of neurodegeneration with brain iron accumulation disorders, characterized by extrapyramidal movement disorders and abnormal accumulation of iron in the basal ganglia. Overall, people harboring WDR45 mutations have moderate to severe DD and different types of seizures. The phenotype of adult patients is characterized by extrapyramidal movement, dystonia, parkinsonism, language impairment, and involvement of the substantia nigra and in the globus pallidus at brain magnetic resonance imaging. Importantly, there are no findings of brain iron accumulation in brain in BPAN patients in the first decade of life, thus suggesting a progressive course of the disease. Comparatively, the main phenotype of pediatric patients is epilepsy with early onset, most of which present infantile spasms and arrest or regression of psychomotor development. The phenotype of patients with WDR45 mutations is variable, being different if caused by somatic mosaicism or germline mutations, and presenting with a different spectrum of manifestations in males and females. The treatment of affected individuals is symptomatic. Regarding the seizures, specific, gene-based approaches with specific antiepileptic drugs are not currently available. The early diagnosis of BPAN could be useful in some aspects, such as providing families a supportive treatment to their affected children.

A late-onset male Fabry disease patient with somatic mosaicism of a classical GLA mutation: a case report

Fabry disease (FD) is an X-linked lysosomal storage disorder caused by mutations in the α-galactosidase A gene (GLA). Male patients of FD develop early sign and symptoms in childhood or adolescence. However, "de novo somatic mosaicism" is rare and might be developed a relatively mild phenotype despite carrying a classic type. A 34-year-old male patient visited with foamy urine. Renal biopsy findings were consistent with FD. Leukocyte α-galactosidase activity was markedly reduced at 5.3 nmol/hr/mg (normal range, 25-126). Sequence analysis of the patient's GLA gene identified mosaicism for the mutation GLA[NM_000169.2] c.820=/G>C. This mutation results in a substitution of the amino acid in position 274 from glycine to arginine. However, no family members showed FD-related symptoms, and the daughter of the patient was also tested for paternity and was identified as a real biological daughter, but DNA sequence analysis for FD showed no mutations. Based on these results, we diagnosed the patients as de novo mutation with somatic mosaicism. Next generation sequencing turned out that 58% of the readings had the mutated allele in buccal cells, 84% in blood, and 85% in urine, when 100% should be expected in a hemizygous affected male confirming the presence of somatic mosaicisms. The patient has been on treatment for enzyme replacement therapy (agalsidase-β, 1.0 mg/kg biweekly) for past 9 years and has maintained normal renal function (serum creatinine 1.0 mg/dL) with mild albuminuria (123 mg/g Cr). Therefore, this case suggests somatic mosaicism is one of important phenotype modifiers.

Multiple versus solitary giant cell lesions of the jaw: Similar or distinct entities?

The majority of giant cell lesions of the jaw present as a solitary focus of disease in bones of the maxillofacial skeleton. Less frequently they occur as multifocal lesions. This raises the clinical dilemma if these should be considered distinct entities and therefore each need a specific therapeutic approach. Solitary giant cell lesions of the jaw present with a great diversity of symptoms. Recent molecular analysis revealed that these are associated with somatic gain-of-function mutations in KRAS, FGFR1 or TRPV4 in a large component of the mononuclear stromal cells which all act on the RAS/MAPK pathway. For multifocal lesions, a small group of neoplastic multifocal giant cell lesions of the jaw remain after ruling out hyperparathyroidism. Strikingly, most of these patients are diagnosed with jaw lesions before the age of 20years, thus before the completion of dental and jaw development. These multifocal lesions are often accompanied by a diagnosis or strong clinical suspicion of a syndrome. Many of the frequently reported syndromes belong to the so-called RASopathies, with germline or mosaic mutations leading to downstream upregulation of the RAS/MAPK pathway. The other frequently reported syndrome is cherubism, with gain-of-function mutations in the SH3BP2 gene leading through assumed and unknown signaling to an autoinflammatory bone disorder with hyperactive osteoclasts and defective osteoblastogenesis. Based on this extensive literature review, a RAS/MAPK pathway activation is hypothesized in all giant cell lesions of the jaw. The different interaction between and contribution of deregulated signaling in individual cell lineages and crosstalk with other pathways among the different germline- and non-germline-based alterations causing giant cell lesions of the jaw can be explanatory for the characteristic clinical features. As such, this might also aid in the understanding of the age-dependent symptomatology of syndrome associated giant cell lesions of the jaw; hopefully guiding ideal timing when installing treatment strategies in the future.

Molecular Diagnosis of Neurofibromatosis by Multigene Panel Testing.

Neurofibromatosis (NF) is an autosomal genetic disorder for which early and definite clinical diagnoses are difficult. To identify the diagnosis, five affected probands with suspected NF from unrelated families were included in this study. Molecular analysis was performed using multigene panel testing and Sanger sequencing. Ultradeep sequencing was used to analyze the mutation rate in the tissues from the proband with mosaic mutations. Three different pathogenic variants of the NF1 gene were found in three probands who mainly complained of café-au-lait macules (CALMs), including one frameshift variant c.5072_5073insTATAACTGTAACTCCTGGGTCAGGGAGTACACCAA:p.Tyr1692Ilefs in exon 37, one missense variant c.3826C > T:p.Arg1276Ter in exon 28, and one splicing variant c.4110 + 1G > T at the first base downstream of the 3′-end of exon 30. One NF1 gene mosaic variant was found in a proband who complained of cutaneous neurofibroma with the frameshift variant c.495_498del:p.Thr165fs in exon 5, and ultradeep sequencing showed the highest mutation rate of 10.81% in cutaneous neurofibromas. A frameshift variant, c.36_39del:p.Ser12fs in exon 1 of the NF2 gene, was found in a proband who presented with skin plaques and intracranial neurogenic tumors. All of these pathogenic variants were heterozygous, one was not reported, and one not in Chinese before. This study expands the pathogenic variant spectrum of NF and demonstrates the clinical diagnosis.

Lipofection-Mediated Introduction of CRISPR/Cas9 System into Porcine Oocytes and Embryos.

Simple SummaryLiposome-mediated gene transfer has become an alternative method for establishing a gene targeting framework, and the production of mutant animals may be feasible even in laboratories without specialized equipment. However, whether blastocyst genome editing can be performed by treatment with lipofection reagent, guide RNA, and Cas9, without performing electroporation or microinjection, remains unclear. In this study, we demonstrated that lipofection treatment successfully induced mutation into zygotes during in vitro fertilization and in embryos at the 2- and 4-cell stages. Although liposome-mediated gene editing is a feasible system for use with zona-pellucida-free oocytes/embryos, several challenges must be overcome.Liposome-mediated gene transfer has become an alternative method for establishing a gene targeting framework, and the production of mutant animals may be feasible even in laboratories without specialized equipment. However, how this system functions in mammalian oocytes and embryos remains unclear. The present study was conducted to clarify whether blastocyst genome editing can be performed by treatment with lipofection reagent, guide RNA, and Cas9 for 5 h without using electroporation or microinjection. A mosaic mutation was observed in blastocysts derived from zona pellucida (ZP)-free oocytes following lipofection treatment, regardless of the target genes. When lipofection treatment was performed after in vitro fertilization (IVF), no significant differences in the mutation rates or mutation efficiency were found between blastocysts derived from embryos treated at 24 and 29 h from the start of IVF. Only blastocysts from embryos exposed to lipofection treatment at 29 h after IVF contained biallelic mutant. Furthermore, there were no significant differences in the mutation rates or mutation efficiency between blastocysts derived from embryos at the 2- and 4-cell stages. This suggests that lipofection-mediated gene editing can be performed in ZP-free oocytes and ZP-free embryos; however, other factors affecting the system efficiency should be further investigated.

In vitro Electroporation in the Presence of CRISPR/Cas9 Reagents as a Safe and Effective Method for Producing Biallelic Knock-Out Porcine Embryos

The production of genetically modified (GM) pigs is considered valuable in biomedical research for the development of model animals for various diseases and pigs with resistance against viral infection. The porcine genome may be modified using several methods, such as somatic cell nuclear transfer (SCNT) using GM cells as the SCNT donor, direct injection of the transgene or the genome editing components (GEC) into fertilized eggs referred to as zygotes, the in vitro electroporation (EP) of the zygotes in the presence of GECs, viral infection using retroviruses, injection of the GECs into the SCNT-treated embryos, and the in vitro EP of the SCNT-treated embryos in the presence of GECs. In our previous study, we administered a cytoplasmic injection of CRISPR/Cas9-based GEC into parthenogenetically-activated porcine oocytes (referred to as parthenotes) and observed that these oocytes comprised a mixture of genome-edited and genome-unedited cells, referred to as the “mosaic”. In contrast, when in vitro EP of the SCNT-treated embryos in the presence of GEC was performed, bi-allelic knock out (KO) of the target gene was detected in most oocytes (82%; 9/11). The production of bi-allelic KO piglets is particularly beneficial for investigating GM domestic animals as it does not require further breeding trials to obtain bi-allelic KO individuals, which would otherwise be a time-consuming and laborious task. In this context, the present study was aimed to confirm the efficiency of in vitro EP in producing bi-allelic KO porcine embryos without multiple breeding trials, for which parthenotes were subjected to EP in the presence of a ribonucleoprotein containing Cas9 protein and single-guide RNA (targeted toward GGTA1). The treated embryos were cultured until they transformed into blastocysts. The genomic DNA isolated from these blastocysts was used for molecular biology analysis to detect the possible insertion and deletion of sequences (indels) at the GGTA1 locus. Among the 32 blastocysts obtained, 21 (66%) were observed to be the bi-allelic KO ones. The remaining embryos either had a normal phenotype (25%; 8/32) or mosaic mutations (9%; 3/32). These findings confirm the efficiency of in vitro EP in producing bi-allelic KO porcine embryos.

In vitro Electroporation in the Presence of CRISPR/Cas9 Reagents as a Safe and Effective Method for Producing Biallelic Knock-Out Porcine Embryos

The production of genetically modified (GM) pigs is considered valuable in biomedical research for the development of model animals for various diseases and pigs with resistance against viral infection. The porcine genome may be modified using several methods, such as somatic cell nuclear transfer (SCNT) using GM cells as the SCNT donor, direct injection of the transgene or the genome editing components (GEC) into fertilized eggs referred to as zygotes, the in vitro electroporation (EP) of the zygotes in the presence of GECs, viral infection using retroviruses, injection of the GECs into the SCNT-treated embryos, and the in vitro EP of the SCNT-treated embryos in the presence of GECs. In our previous study, we administered a cytoplasmic injection of CRISPR/Cas9-based GEC into parthenogenetically-activated porcine oocytes (referred to as parthenotes) and observed that these oocytes comprised a mixture of genome-edited and genome-unedited cells, referred to as the “mosaic”. In contrast, when in vitro EP of the SCNT-treated embryos in the presence of GEC was performed, bi-allelic knock out (KO) of the target gene was detected in most oocytes (82%; 9/11). The production of bi-allelic KO piglets is particularly beneficial for investigating GM domestic animals as it does not require further breeding trials to obtain bi-allelic KO individuals, which would otherwise be a time-consuming and laborious task. In this context, the present study was aimed to confirm the efficiency of in vitro EP in producing bi-allelic KO porcine embryos without multiple breeding trials, for which parthenotes were subjected to EP in the presence of a ribonucleoprotein containing Cas9 protein and single-guide RNA (targeted toward GGTA1). The treated embryos were cultured until they transformed into blastocysts. The genomic DNA isolated from these blastocysts was used for molecular biology analysis to detect the possible insertion and deletion of sequences (indels) at the GGTA1 locus. Among the 32 blastocysts obtained, 21 (66%) were observed to be the bi-allelic KO ones. The remaining embryos either had a normal phenotype (25%; 8/32) or mosaic mutations (9%; 3/32). These findings confirm the efficiency of in vitro EP in producing bi-allelic KO porcine embryos.

MIPP-Seq: ultra-sensitive rapid detection and validation of low-frequency mosaic mutations

BackgroundMosaic mutations contribute to numerous human disorders. As such, the identification and precise quantification of mosaic mutations is essential for a wide range of research applications, clinical diagnoses, and early detection of cancers. Currently, the low-throughput nature of single allele assays (e.g., allele-specific ddPCR) commonly used for genotyping known mutations at very low alternate allelic fractions (AAFs) have limited the integration of low-level mosaic analyses into clinical and research applications. The growing importance of mosaic mutations requires a more rapid, low-cost solution for mutation detection and validation.MethodsTo overcome these limitations, we developed Multiple Independent Primer PCR Sequencing (MIPP-Seq) which combines the power of ultra-deep sequencing and truly independent assays. The accuracy of MIPP-seq to quantifiable detect and measure extremely low allelic fractions was assessed using a combination of SNVs, insertions, and deletions at known allelic fractions in blood and brain derived DNA samples.ResultsThe Independent amplicon analyses of MIPP-Seq markedly reduce the impact of allelic dropout, amplification bias, PCR-induced, and sequencing artifacts. Using low DNA inputs of either 25 ng or 50 ng of DNA, MIPP-Seq provides sensitive and quantitative assessments of AAFs as low as 0.025% for SNVs, insertion, and deletions.ConclusionsMIPP-Seq provides an ultra-sensitive, low-cost approach for detecting and validating known and novel mutations in a highly scalable system with broad utility spanning both research and clinical diagnostic testing applications. The scalability of MIPP-Seq allows for multiplexing mutations and samples, which dramatically reduce costs of variant validation when compared to methods like ddPCR. By leveraging the power of individual analyses of multiple unique and independent reactions, MIPP-Seq can validate and precisely quantitate extremely low AAFs across multiple tissues and mutational categories including both indels and SNVs. Furthermore, using Illumina sequencing technology, MIPP-seq provides a robust method for accurate detection of novel mutations at an extremely low AAF.

Optimization and Validation of Multimodular, Long-Range PCR–Based Next-Generation Sequencing Assays for Comprehensive Detection of Mutation in Tuberous Sclerosis Complex

The genetic diagnosis of tuberous sclerosis complex is difficult because of its broad spectrum of mutations. In addition to point mutations in coding regions, intragenic or chromosomal-level large deletions, deep intronic splicing mutations, and mosaic mutations represent a significant proportion of the mutations. In this study, multimodular, long-range PCR-based next-generation sequencing assays were optimized and validated using >100 samples with known TSC1 and TSC2 variants. Multiplex, long-range PCR covering the entire genomic region of both genes detected all 138 known variants; however, it also yielded false-positive results. Intragenic large deletions were detected with accurate breakpoint sequences. Chromosomal-level deletions were estimated by discordant allele segregation in the family and confirmed by DNA microarray. Deep intronic mutations were verified using a combination of long-range DNA PCR and full-length mRNA sequencing. DNA samples were mixed to simulate mosaic mutations, and most variants were detected but could not be distinguished from equivalently detected false-positive results. Repeated false-positive results were classified, and the strategy of selecting the common variants detected in the duplicate analysis and eliminating known false-positive results improved the sensitivity (85.2%) and positive predictive value (96.6%) of a 10% mosaic simulation. Long-range PCRbased next-generation sequencing is a highly versatile genetic test; however, confirmation tests remain necessary for clinical use because false-positive results cannot be completely eliminated from single experiments.

Nevus comedonicus syndrome: A systematic review of the literature.

Nevus comedonicus (NC) syndrome is a condition first identified in 1978. The cause of NC syndrome has been recently proven to be a gain-of-function, mosaic postzygotic mutation of the NEK9 gene. A systematic review of the literature retrieved 43 well-established cases of NC syndrome reported so far. Three morphological variants of NC in NC syndrome emerged: (a) the more common, predominantly comedonal type; (b) "Selhorst type"; and (c) "atrophoderma vermiculatum" type. NC syndrome is mainly associated with ocular, skeletal, and neural abnormalities, most typically ipsilateral congenital cataract and malformations of fingers and toes.

First radiobiological characterization of the McCune–Albright syndrome: influence of the ATM protein and effect of statins + bisphosphonates treatment

Purpose MacCune–Albright syndrome (MAS) is a rare autosomal dominant osteo-hormonal disorder. MAS is characterized by a severe form of polyostotic fibrous dysplasia, ‘café-au-lait’ pigmentation of the skin and multiple endocrinopathies. MAS was shown to be caused by mosaic missense somatic mutations in the GNAS gene coding for the alpha-subunit of the stimulatory G-protein. MAS is also associated with radiation-induced malignant tumors, like osteosarcoma, fibrosarcoma and chondrosarcoma but their origin remains misunderstood. In parallel, bisphosphonates treatment was shown to improve the MAS patients’ outcome, notably by increasing bone density but, again, the molecular mechanisms supporting these observations remain misunderstood. Materials and methods Here, by using fibroblast and osteoblast cell lines derived from 2 MAS patients, the major radiobiological features of MAS were investigated. Notably, the clonogenic cell survival, the micronuclei and the γH2AX, pATM and MRE11 immunofluorescence assays were applied to MAS cells. Results It appears that cells from the 2 MAS patients are associated with a moderate but significant radiosensitivity, a delayed radiation-induced nucleoshuttling of the ATM kinase likely caused by its sequestration in cytoplasm, suggesting impaired DNA double-strand breaks (DSB) repair and signaling in both fibroblasts and osteoblasts. Such delay may be partially corrected by using bisphosphonates combined with statins, which renders cells more radioresistant. Conclusions Our findings represent the first radiobiological characterization of fibroblasts and osteoblasts providing from MAS patients. Although the number of studied cases is reduced, our findings suggest that the MAS cells tested belong to the group of syndromes associated with moderate but significant radiosensitivity. Further investigations are however required to secure the clinical transfer of the combination of bisphosphonates and statins, to reduce the disease progression and to better evaluate the potential risks linked to radiation exposure.

Spotted around the web: Antisense therapies, mosaic mutations, researcher productivity

Spotted around the web: Antisense therapies, mosaic mutations, researcher productivity

CLOVES syndrome caused by mosaic mutation in the PIK3CA gene identified in fibroblasts

CLOVES syndrome caused by mosaic mutation in the PIK3CA gene identified in fibroblasts

Light-based treatment of pediatric port-wine birthmarks.

Port-wine birthmarks (PWBs) are progressive vascular malformations with significant disfigurement and psychosocial morbidity; early light-based treatment has shown improved outcomes in the pediatric population. Somatic mosaic mutations underly the progressive nature of PWBs and explain the significant differences in response and heterogeneity of vessel architecture in the pediatric population when compared to the adult cohort. Here, we summarize a review of pediatric specific literature on the various light-based treatment modalities, including pulsed dye laser, near-infrared lasers, and intense pulsed light, providing the various indications, tips, advantages, and disadvantages for the pediatric dermatologist.

Sarcomeres regulate murine cardiomyocyte maturation through MRTF-SRF signaling

The paucity of knowledge about cardiomyocyte maturation is a major bottleneck in cardiac regenerative medicine. In development, cardiomyocyte maturation is characterized by orchestrated structural, transcriptional, and functional specializations that occur mainly at the perinatal stage. Sarcomeres are the key cytoskeletal structures that regulate the ultrastructural maturation of other organelles, but whether sarcomeres modulate the signal transduction pathways that are essential for cardiomyocyte maturation remains unclear. To address this question, here we generated mice with cardiomyocyte-specific, mosaic, and hypomorphic mutations of α-actinin-2 (Actn2) to study the cell-autonomous roles of sarcomeres in postnatal cardiomyocyte maturation. Actn2 mutation resulted in defective structural maturation of transverse-tubules and mitochondria. In addition, Actn2 mutation triggered transcriptional dysregulation, including abnormal expression of key sarcomeric and mitochondrial genes, and profound impairment of the normal progression of maturational gene expression. Mechanistically, the transcriptional changes in Actn2 mutant cardiomyocytes strongly correlated with those in cardiomyocytes deleted of serum response factor (SRF), a critical transcription factor that regulates cardiomyocyte maturation. Actn2 mutation increased the monomeric form of cardiac α-actin, which interacted with the SRF cofactor MRTFA and perturbed its nuclear localization. Overexpression of a dominant-negative MRTFA mutant was sufficient to recapitulate the morphological and transcriptional defects in Actn2 and Srf mutant cardiomyocytes. Together, these data indicate that Actn2-based sarcomere organization regulates structural and transcriptional maturation of cardiomyocytes through MRTF-SRF signaling.

Functional significance of germline EPAS1 variants.

Mosaic or somatic EPAS1 mutations are associated with a range of phenotypes including pheochromocytoma and/or paraganglioma (PPGL), polycythemia and somatostatinoma. The pathogenic potential of germline EPAS1 variants however is not well understood. We report a number of germline EPAS1 variants occurring in patients with PPGL, including a novel variant c.739C>A (p.Arg247Ser); a previously described variant c.1121T>A (p.Phe374Tyr); several rare variants, c.581A>G (p.His194Arg), c.2353C>A (p.Pro785Thr) and c.2365A>G (p.Ile789Val); a common variant c.2296A>C (p.Thr766Pro). We performed detailed functional studies to understand their pathogenic role in PPGL. In transient transfection studies, EPAS1/HIF-2α p.Arg247Ser, p.Phe374Tyr and p.Pro785Thr were all stable in normoxia. In co-immunoprecipitation assays, only the novel variant p.Arg247Ser showed diminished interaction with pVHL. A direct interaction between HIF-2α Arg247 and pVHL was confirmed in structural models. Transactivation was assessed by means of a HRE-containing reporter gene in transiently transfected cells, and significantly higher reporter activity was only observed with EPAS1/HIF-2α p.Phe374Tyr and p.Pro785Thr. In conclusion, three germline EPAS1 variants (c.739C>A (p.Arg247Ser), c.1121T>A (p.Phe374Tyr) and c.2353C>A (p.Pro785Thr)) all have some functional features in common with somatic activating mutations. Our findings suggest that these three germline variants are hypermorphic alleles that may act as modifiers to the expression of PPGLs.

Evaluation of mutation rates, mosaicism and off target mutations when injecting Cas9 mRNA or protein for genome editing of bovine embryos

The CRISPR/Cas9 genome editing tool has the potential to improve the livestock breeding industry by allowing for the introduction of desirable traits. Although an efficient and targeted tool, the CRISPR/Cas9 system can have some drawbacks, including off-target mutations and mosaicism, particularly when used in developing embryos. Here, we introduced genome editing reagents into single-cell bovine embryos to compare the effect of Cas9 mRNA and protein on the mutation efficiency, level of mosaicism, and evaluate potential off-target mutations utilizing next generation sequencing. We designed guide-RNAs targeting three loci (POLLED, H11, and ZFX) in the bovine genome and saw a significantly higher rate of mutation in embryos injected with Cas9 protein (84.2%) vs. Cas9 mRNA (68.5%). In addition, the level of mosaicism was higher in embryos injected with Cas9 mRNA (100%) compared to those injected with Cas9 protein (94.2%), with little to no unintended off-target mutations detected. This study demonstrated that the use of gRNA/Cas9 ribonucleoprotein complex resulted in a high editing efficiency at three different loci in bovine embryos and decreased levels of mosaicism relative to Cas9 mRNA. Additional optimization will be required to further reduce mosaicism to levels that make single-step embryo editing in cattle commercially feasible.

SCELLECTOR: ranking amplification bias in single cells using shallow sequencing

BackgroundThe study of mosaic mutation is important since it has been linked to cancer and various disorders. Single cell sequencing has become a powerful tool to study the genome of individual cells for the detection of mosaic mutations. The amount of DNA in a single cell needs to be amplified before sequencing and multiple displacement amplification (MDA) is widely used owing to its low error rate and long fragment length of amplified DNA.However, the phi29 polymerase used in MDA is sensitive to template fragmentation and presence of sites with DNA damage that can lead to biases such as allelic imbalance, uneven coverage and over representation of C to T mutations. It is therefore important to select cells with uniform amplification to decrease false positives and increase sensitivity for mosaic mutation detection.ResultsWe propose a method, Scellector (single cell selector), which uses haplotype information to detect amplification quality in shallow coverage sequencing data. We tested Scellector on single human neuronal cells, obtained in vitro and amplified by MDA. Qualities were estimated from shallow sequencing with coverage as low as 0.3× per cell and then confirmed using 30× deep coverage sequencing. The high concordance between shallow and high coverage data validated the method.ConclusionScellector can potentially be used to rank amplifications obtained from single cell platforms relying on a MDA-like amplification step, such as Chromium Single Cell profiling solution.