scholarly journals Genome editing in the nematode Caenorhabditis briggsae using the CRISPR/Cas9 system

2020 ◽  
Vol 5 (1) ◽  
Author(s):  
Elizabeth Culp ◽  
Cory Richman ◽  
Devika Sharanya ◽  
Nikita Jhaveri ◽  
Wouter van den Berg ◽  
...  
Keyword(s):  
Dna Sequence ◽  
Gene Function ◽  
Somatic Mutations ◽  
Caenorhabditis Briggsae ◽  
Loss Of Function ◽  
End Joining ◽  
C Elegans ◽  
Conserved Genes ◽  
Foreign Dna ◽  
Non Homologous End Joining

Abstract The CRISPR/Cas system has recently emerged as a powerful tool to engineer the genome of an organism. The system is adopted from bacteria where it confers immunity against invading foreign DNA. This work reports the first successful use of the CRISPR/Cas system in Caenorhabditis briggsae (a cousin of the well-known nematode C. elegans), to generate mutations via non-homologous end joining. We recovered deletion alleles of several conserved genes by microinjecting plasmids that express Cas9 endonuclease and an engineered CRISPR RNA corresponding to the DNA sequence to be cleaved. Evidence for somatic mutations and off-target mutations are also reported. Our approach allows for the generation of loss-of-function mutations in C. briggsae genes thereby facilitating a comparative study of gene function.

scholarly journals Genome Editing in Caenorhabditis briggsae using the CRISPR/Cas9 System

2015 ◽  
Author(s):  
Elizabeth Culp ◽  
Cory Richman ◽  
Devika Sharanya ◽  
Bhagwati Gupta
Keyword(s):  
Genome Editing ◽  
Comparative Studies ◽  
Somatic Mutations ◽  
Sister Species ◽  
Efficient Technique ◽  
Caenorhabditis Briggsae ◽  
End Joining ◽  
C Elegans ◽  
Non Homologous End Joining

The CRISPR/Cas9 system is an efficient technique for generating targeted alterations in an organism's genome. Here we describe a methodology for using the CRISPR/Cas9 system to generate mutations via non-homologous end joining in the nematode Caenorhabditis briggsae, a sister species of C. elegans. Evidence for somatic mutations and off-target mutations are also reported. The use of the CRISPR/Cas9 system in C. briggsae will greatly facilitate comparative studies to C. elegans.

Haploinsufficiency in non-homologous end joining factor 1 induces ovarian dysfunction in humans and mice

2021 ◽  
pp. jmedgenet-2020-107398
Author(s):  
Guoqing Li ◽  
Xi Yang ◽  
Lingbo Wang ◽  
Yuncheng Pan ◽  
Siyuan Chen ◽  
...  
Keyword(s):  
Ovarian Function ◽  
In Vitro Assays ◽  
Chinese Family ◽  
Common Disease ◽  
Loss Of Function ◽  
End Joining ◽  
Dsb Repair ◽  
Repair Capacity ◽  
Non Homologous End Joining

BackgroundPremature ovarian insufficiency (POI) is a common disease in women that leads to a reduced reproductive lifespan. The aetiology of POI is genetically heterogeneous, with certain double-strand break (DSB) repair genes being implicated in POI. Although non-homologous end joining (NHEJ) is an efficient DSB repair pathway, the functional relationship between this pathway and POI remains unknown.Methods and resultsWe conducted whole-exome sequencing in a Chinese family and identified a rare heterozygous loss-of-function variant in non-homologous end joining factor 1 (NHEJ1): c.532C>T (p.R178*), which co-segregated with POI and irregular menstruation. The amount of NHEJ1 protein in the proband was half of the normal level, indicating a link between NHEJ1 haploinsufficiency and POI. Furthermore, another rare heterozygous NHEJ1 variant c.500A>G (p.Y167C) was identified in one of 100 sporadic POI cases. Both variants were predicted to be deleterious by multiple in silico tools. In vitro assays showed that knock-down of NHEJ1 in human KGN ovarian cells impaired DNA repair capacity. We also generated a knock-in mouse model with a heterozygous Nhej1 variant equivalent to NHEJ1 p.R178* in familial patients. Compared with wild-type mice, heterozygous Nhej1-mutated female mice required a longer time to first birth, and displayed reduced numbers of primordial and growing follicles. Moreover, these mice exhibited higher sensitivity to DSB-inducing drugs. All these phenotypes are analogous to the progressive loss of ovarian function observed in POI.ConclusionsOur observations in both humans and mice suggest that NHEJ1 haploinsufficiency is associated with non-syndromic POI, providing novel insights into genetic counselling and clinical prevention of POI.

scholarly journals Efficient Generation of Knock-In Zebrafish Models for Inherited Disorders Using CRISPR-Cas9 Ribonucleoprotein Complexes

2021 ◽  
Vol 22 (17) ◽  
pp. 9429
Author(s):  
Erik de Vrieze ◽  
Suzanne E. de Bruijn ◽  
Janine Reurink ◽  
Sanne Broekman ◽  
Vince van de Riet ◽  
...  
Keyword(s):  
Dna Repair ◽  
Cost Effective ◽  
Patient Specific ◽  
Loss Of Function ◽  
Repair Pathway ◽  
End Joining ◽  
Inherited Disorders ◽  
Ribonucleoprotein Complexes ◽  
Cost Effective Method ◽  
Non Homologous End Joining

CRISPR-Cas9-based genome-editing is a highly efficient and cost-effective method to generate zebrafish loss-of-function alleles. However, introducing patient-specific variants into the zebrafish genome with CRISPR-Cas9 remains challenging. Targeting options can be limited by the predetermined genetic context, and the efficiency of the homology-directed DNA repair pathway is relatively low. Here, we illustrate our efficient approach to develop knock-in zebrafish models using two previously variants associated with hereditary sensory deficits. We employ sgRNA-Cas9 ribonucleoprotein (RNP) complexes that are micro-injected into the first cell of fertilized zebrafish eggs together with an asymmetric, single-stranded DNA template containing the variant of interest. The introduction of knock-in events was confirmed by massive parallel sequencing of genomic DNA extracted from a pool of injected embryos. Simultaneous morpholino-induced blocking of a key component of the non-homologous end joining DNA repair pathway, Ku70, improved the knock-in efficiency for one of the targets. Our use of RNP complexes provides an improved knock-in efficiency as compared to previously published studies. Correct knock-in events were identified in 3–8% of alleles, and 30–45% of injected animals had the target variant in their germline. The detailed technical and procedural insights described here provide a valuable framework for the efficient development of knock-in zebrafish models.

Uncover Genetic Interactions in Caenorhabditis elegans by RNA Interference

2005 ◽  
Vol 25 (5-6) ◽  
pp. 299-307 ◽  
Author(s):  
Angelo Fortunato ◽  
Andrew G. Fraser
Keyword(s):  
Rna Interference ◽  
Caenorhabditis Elegans ◽  
Gene Function ◽  
Genetic Interactions ◽  
Loss Of Function ◽  
Future Directions ◽  
C Elegans ◽  
Genomics Tools ◽  
Almost All ◽  
Nematode Worm

RNA-mediated interference (RNAi) has emerged recently as one of the most powerful functional genomics tools. RNAi has been particularly effective in the nematode worm C. elegans where RNAi has been used to analyse the loss-of-function phenotypes of almost all predicted genes. In this review, we illustrate how RNAi has been used to analyse gene function in C. elegans as well as pointing to some future directions for using RNAi to examine genetic interactions in a systematic manner.

scholarly journals Genome-wide microhomologies enable precise template-free editing of biologically relevant deletion mutations

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Janin Grajcarek ◽  
Jean Monlong ◽  
Yoko Nishinaka-Arai ◽  
Michiko Nakamura ◽  
Miki Nagai ◽  
...  
Keyword(s):  
Loss Of Function ◽  
End Joining ◽  
Protein Coding ◽  
Strand Breaks ◽  
Deletion Mutations ◽  
Biologically Relevant ◽  
Genome Wide ◽  
Template Free ◽  
Non Homologous End Joining ◽  
Induced Pluripotent

Abstract The functional effect of a gene edit by designer nucleases depends on the DNA repair outcome at the targeted locus. While non-homologous end joining (NHEJ) repair results in various mutations, microhomology-mediated end joining (MMEJ) creates precise deletions based on the alignment of flanking microhomologies (µHs). Recently, the sequence context surrounding nuclease-induced double strand breaks (DSBs) has been shown to predict repair outcomes, for which µH plays an important role. Here, we survey naturally occurring human deletion variants and identify that 11 million or 57% are flanked by µHs, covering 88% of protein-coding genes. These biologically relevant mutations are candidates for precise creation in a template-free manner by MMEJ repair. Using CRISPR-Cas9 in human induced pluripotent stem cells (hiPSCs), we efficiently create pathogenic deletion mutations for demonstrable disease models with both gain- and loss-of-function phenotypes. We anticipate this dataset and gene editing strategy to enable functional genetic studies and drug screening.

scholarly journals Loss of BRCA1 or BRCA2 markedly increases the rate of base substitution mutagenesis and has distinct effects on genomic deletions

Oncogene ◽  
2016 ◽  
Vol 36 (6) ◽  
pp. 746-755 ◽  
Author(s):  
J Zámborszky ◽  
B Szikriszt ◽  
J Z Gervai ◽  
O Pipek ◽  
Á Póti ◽  
...  
Keyword(s):  
Large Scale ◽  
Dna Lesions ◽  
High Rate ◽  
Base Substitution ◽  
Loss Of Function ◽  
End Joining ◽  
Genomic Deletions ◽  
Non Homologous End Joining ◽  
Risk Of Cancer ◽  
Mutant Cells

Abstract Loss-of-function mutations in the BRCA1 and BRCA2 genes increase the risk of cancer. Owing to their function in homologous recombination repair, much research has focused on the unstable genomic phenotype of BRCA1/2 mutant cells manifest mainly as large-scale rearrangements. We used whole-genome sequencing of multiple isogenic chicken DT40 cell clones to precisely determine the consequences of BRCA1/2 loss on all types of genomic mutagenesis. Spontaneous base substitution mutation rates increased sevenfold upon the disruption of either BRCA1 or BRCA2, and the arising mutation spectra showed strong and specific correlation with a mutation signature associated with BRCA1/2 mutant tumours. To model endogenous alkylating damage, we determined the mutation spectrum caused by methyl methanesulfonate (MMS), and showed that MMS also induces more base substitution mutations in BRCA1/2-deficient cells. Spontaneously arising and MMS-induced insertion/deletion mutations and large rearrangements were also more common in BRCA1/2 mutant cells compared with the wild-type control. A difference in the short deletion phenotypes of BRCA1 and BRCA2 suggested distinct roles for the two proteins in the processing of DNA lesions, as BRCA2 mutants contained more short deletions, with a wider size distribution, which frequently showed microhomology near the breakpoints resembling repair by non-homologous end joining. An increased and prolonged gamma-H2AX signal in MMS-treated BRCA1/2 cells suggested an aberrant processing of stalled replication forks as the cause of increased mutagenesis. The high rate of base substitution mutagenesis demonstrated by our experiments is likely to significantly contribute to the oncogenic effect of the inactivation of BRCA1 or BRCA2.

scholarly journals CopyCatchers are versatile active genetic elements that detect and quantify inter-homolog somatic gene conversion

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Zhiqian Li ◽  
Nimi Marcel ◽  
Sushil Devkota ◽  
Ankush Auradkar ◽  
Stephen M. Hedrick ◽  
...  
Keyword(s):  
Gene Conversion ◽  
Somatic Mutations ◽  
Genetic Screens ◽  
End Joining ◽  
Genetic Elements ◽  
Homology Directed Repair ◽  
Somatic Gene ◽  
Non Homologous End Joining ◽  
Or Gene ◽  
Therapy Strategies

AbstractCRISPR-based active genetic elements, or gene-drives, copied via homology-directed repair (HDR) in the germline, are transmitted to progeny at super-Mendelian frequencies. Active genetic elements also can generate widespread somatic mutations, but the genetic basis for such phenotypes remains uncertain. It is generally assumed that such somatic mutations are generated by non-homologous end-joining (NHEJ), the predominant double stranded break repair pathway active in somatic cells. Here, we develop CopyCatcher systems in Drosophila to detect and quantify somatic gene conversion (SGC) events. CopyCatchers inserted into two independent genetic loci reveal unexpectedly high rates of SGC in the Drosophila eye and thoracic epidermis. Focused RNAi-based genetic screens identify several unanticipated loci altering SGC efficiency, one of which (c-MYC), when downregulated, promotes SGC mediated by both plasmid and homologous chromosome-templates in human HEK293T cells. Collectively, these studies suggest that CopyCatchers can serve as effective discovery platforms to inform potential gene therapy strategies.

scholarly journals MMEJ-based Precision Gene Editing for applications in Gene Therapy and Functional Genomics

2020 ◽  
Author(s):  
Gabriel Martínez-Gálvez ◽  
Armando Manduca ◽  
Stephen C. Ekker
Keyword(s):  
Gene Editing ◽  
Reverse Genetics ◽  
Embryonic Stem ◽  
Strand Break ◽  
Loss Of Function ◽  
End Joining ◽  
Dsb Repair ◽  
Therapeutic Applications ◽  
Predictive Algorithm ◽  
Non Homologous End Joining

ABSTRACTExperiments in gene editing commonly elicit error-prone non-homologous end joining for DNA double-strand break (DSB) repair. Microhomology-mediated end joining (MMEJ) can generate more predictable outcomes for functional genomic and somatic therapeutic applications. MENTHU is a computational tool that predicts nuclease-targetable sites likely to result in MMEJ-repaired, homogeneous genotypes (PreMAs) in zebrafish. We deployed MENTHU on 5,885 distinct Cas9-mediated DSBs in mouse embryonic stem cells, and compared the predictions to those by inDelphi, another DSB repair predictive algorithm. MENTHU correctly identified 46% of all PreMAs available, doubling the sensitivity of inDelphi. We also introduce MENTHU@4, an MENTHU update trained on this large dataset. We trained two MENTHU-based algorithms on this larger dataset and validated them against each other, MENTHU, and inDelphi. Finally, we estimated the frequency and distribution of SpCas9-targetable PreMAs in vertebrate coding regions to evaluate MMEJ-based targeting for gene discovery. 44 out of 54 genes (81%) contained at least one early out-of-frame PreMA and 48 out of 54 (89%) did so when also considering Cas12a. We suggest that MMEJ can be deployed at scale for reverse genetics screenings and with sufficient intra-gene density rates to be viable for nearly all loss-of-function based gene editing therapeutic applications.

scholarly journals NHJ-1 regulates canonical non-homologous end joining in Caenorhabditis elegans

2019 ◽  
Author(s):  
Aleksandar Vujin ◽  
Steven J. Jones ◽  
Monique Zetka
Keyword(s):  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Dapi Staining ◽  
Strand Breaks ◽  
C Elegans ◽  
Meiotic Dsbs ◽  
Ring Complex ◽  
Non Homologous End Joining

AbstractCanonical non-homologous end joining (cNHEJ) is a near-universally conserved pathway for the repair of DNA double-strand breaks (DSBs). While the cNHEJ pathway encompasses more than a dozen factors in vertebrates and is similarly complex in other eukaryotes, in the nematode C. elegans the entire known cNHEJ toolkit consists of two proteins that comprise the Ku ring complex, cku-70 and cku-80, and the terminal ligase lig-4. Here, we report the discovery of nhj-1 as the fourth cNHEJ factor in C. elegans. Observing a difference in the phenotypic response to ionizing radiation (IR) between two lines of the wild type N2 strain, we mapped the locus causative of IR-sensitivity to a candidate on chromosome V. Using CRISPR-Cas9 mutagenesis, we show that disrupting the nhj-1 sequence induces IR-sensitivity in an IR-resistant background. Double mutants of nhj-1 and the cNHEJ factors lig-4 or cku-80 do not exhibit additive IR-sensitivity, arguing that nhj-1 is a member of the cNHEJ pathway. Furthermore, like the loss of lig-4, the loss of nhj-1 in the com-1 genetic background, in which meiotic DSBs are repaired by cNHEJ instead of homologous recombination, increased the number of DAPI-staining bodies in diakinesis, consistent with increased chromosome fragmentation in the absence of cNHEJ repair. Finally, we show that NHJ-1 localizes to many somatic nuclei in the L1 larva, but not the primordial germline, which is in accord with a role in the predominantly somatically active cNHEJ. Although nhj-1 shares no sequence homology with other known eukaryotic cNHEJ factors and is taxonomically restricted to the Rhadbitid family, its discovery underscores the evolutionary plasticity of even highly conserved pathways, and may represent a springboard for further characterization of cNHEJ in C. elegans.

scholarly journals Splice donor site sgRNAs enhance CRISPR/Cas9-mediated knockout efficiency

2019 ◽  
Author(s):  
Ignacio García-Tuñón ◽  
Verónica Alonso-Pérez ◽  
Elena Vuelta ◽  
Sandra Pérez-Ramos ◽  
María Herrero ◽  
...  
Keyword(s):  
Donor Site ◽  
Null Alleles ◽  
Splice Donor Site ◽  
Loss Of Function ◽  
End Joining ◽  
Splice Donor ◽  
Null Result ◽  
Nucleotide Insertions ◽  
Non Homologous End Joining ◽  
Therapy Strategies

ABSTRACTCRISPR/Cas9 enables the generation of knockout cell lines and null zygotes by inducing site-specific double-stranded breaks. In most cases the DSB is repaired by non-homologous end joining, resulting in small nucleotide insertions or deletions that can be used to construct knockout alleles. However, these mutations do not produce the desired null result in all cases, but instead generate a similar, functionally active protein. This effect could limit the therapeutic efficiency of gene therapy strategies based on abrogating oncogene expression, and therefore needs to be considered carefully. If there is an acceptable degree of efficiency of CRISPR/Cas9 delivery to cells, the key step for success lies in the effectiveness of a specific sgRNA at knocking out the oncogene, when only one sgRNA can be used. This study shows that the null effect could be increased with an sgRNA targeting the splice donor site (SDS) of the chosen exon. Following this strategy, the generation of null alleles would be facilitated in two independent ways: the probability of producing a frameshift mutation and the probability of interrupting the canonical mechanism of pre-mRNA splicing. In these contexts, we propose to improve the loss-of-function yield driving the CRISPR system at the SDS of critical exons.

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