scholarly journals Efficient replacement of long DNA fragments via non-homologous end joining at non-coding regions

2020 ◽  
Author(s):  
Shanye Gu ◽  
Jia Li ◽  
Siyuan Li ◽  
Jianbin Cao ◽  
Jiwen Bu ◽  
...  
Keyword(s):  
Genomic Dna ◽  
Genomic Sequence ◽  
Genetic Manipulation ◽  
Expression Patterns ◽  
Egfp Expression ◽  
End Joining ◽  
Replacement Method ◽  
Endogenous Genes ◽  
Non Homologous End Joining ◽  
Genomic Regions

Abstract Genomic DNA replacement for achieving sophisticated genetic manipulation is implemented currently through homogenous recombination/homology-dependent repair (HR/HDR). Here we report a novel DNA fragment replacement method that is mediated by non-homologous end joining (NHEJ)-dependent DNA repair at two sites of CRISPR/Cas9-induced double-strand breaks at non-coding genomic regions flanking the exons of targeted genes. We demonstrated this method by generating three conditional alleles of zebrafish. Functional assays proved that the genomic sequence between two inserted loxP sites was deleted by the Cre recombinase, and the phenotype after Cre-induced excision was comparable to previously reported mutants or morphants. We further extended the application by making two EGFP reporter lines, in which the EGFP expression patterns were consistent with those of endogenous genes. Furthermore, combining double-fluorescence expression donor vectors, we showed that the DNA replacement efficiency of this NHEJ-mediated approach was around 3 times larger than that of HR/HDR-mediated approach. Together, we provides a feasible strategy for genomic DNA replacement in zebrafish, which can be applicable for other organisms as well.

scholarly journals Efficient replacement of long DNA fragments via non-homologous end joining at non-doding regions

2020 ◽  
Author(s):  
Shan-Ye Gu ◽  
Jia Li ◽  
Jian-Bin Cao ◽  
Ji-Wen Bu ◽  
Yong-Gang Ren ◽  
...  
Keyword(s):  
Genomic Dna ◽  
Genomic Sequence ◽  
Genetic Manipulation ◽  
Double Strand Breaks ◽  
End Joining ◽  
Strand Breaks ◽  
Replacement Method ◽  
Dna Fragment ◽  
Non Homologous End Joining ◽  
Genomic Regions

AbstractGenomic DNA replacement for achieving sophisticated genetic manipulation is implemented currently through homogenous recombination/homology-dependent repair (HR/HDR). Here we report an efficient DNA fragment replacement method that is mediated by non-homologous end joining (NHEJ)-dependent DNA repair at two sites of CRISPR/Cas9-induced double-strand breaks at non-coding genomic regions flanking the exons of targeted genes. We demonstrated this method by generating three conditional alleles and two reporter lines of zebrafish. Functional assays of the conditional alleles proved that the genomic sequence between two inserted loxP sites was deleted by the Cre recombinase, and the phenotype after Cre-induced excision was comparable to previously reported mutants or morphants. Furthermore, combining double-fluorescence expression donor vectors, we showed that the efficiency of this NHEJ-mediated DNA replacement was around 3 times larger than that of HR/HDR-mediated approach. Our method provides a feasible strategy for genomic DNA replacement in zebrafish, which can be applicable for other organisms as well.

Targeted gene insertion and replacement in the basidiomycete Ganoderma lucidum by inactivation of non-homologous end joining using CRISPR/Cas9

2021 ◽  
Author(s):  
Jun-Liang Tu ◽  
Xin-Yuan Bai ◽  
Yong-Liang Xu ◽  
Na Li ◽  
Jun-Wei Xu
Keyword(s):  
Homologous Recombination ◽  
Functional Genomics ◽  
Ganoderma Lucidum ◽  
Gene Disruption ◽  
Target Gene ◽  
End Joining ◽  
Gene Insertion ◽  
Replacement Method ◽  
Low Efficiency ◽  
Non Homologous End Joining

Targeted gene insertion or replacement is a promising genome editing tool for molecular breeding and gene engineering. Although CRISPR/Cas9 works well for gene disruption and deletion in Ganoderma lucidum , targeted gene insertion and replacement remains a serious challenge due to the low efficiency of homologous recombination (HR) in these species. In this work, we demonstrate that the DNA double-strand breaks induced by Cas9 were mainly repaired via the non-homologous end joining pathway (NHEJ) at a frequency of 96.7%. To establish an efficient target gene insertion and replacement tool in Ganoderma , we first inactivated the NHEJ pathway via disruption of the Ku70 gene ( ku70 ) using a dual sgRNA-directed gene deletion method. Disruption of the ku70 significantly decreased NHEJ activity in G. lucidum . Moreover, ku70 disruption strains exhibited 96.3% and 93.1% frequencies of a targeted gene insertion and replacement when target DNA orotidine 5’-monophosphate decarboxylase gene ( ura3 ) with 1.5 kb 5’ and 3’ homologous flanking sequences were used as a donor template, compared to 3.3% and 0% for a control strain (Cas9 strain) at these targeted sites, respectively. Our results indicated that ku70 disruption strains were efficient recipients for targeted gene insertion and replacement. This tool will advance our understanding of functional genomics in G. lucidum . Importance Functional genomic studies have been hindered in Ganoderma by the absence of adequate genome engineering tools. Although CRISPR/Cas9 works well for gene disruption and deletion in G. lucidum , targeted gene insertion and replacement has remained a serious challenge due to the low efficiency of homologous recombination in these species, although such precise genome modifications including site mutations, site-specific integrations and allele or promoter replacements would be incredibly valuable. In this work, we inactivated the non-homologous end joining repair mechanism in G. lucidum by disrupting the ku70 using the CRISPR/Cas9 system. Moreover, we established a target gene insertion and replacement method in ku70 -disrupted G. lucidum that possessed high-efficiency gene targeting. This technology will advance our understanding of the functional genomics of G. lucidum.

scholarly journals CDCA7 and HELLS suppress DNA:RNA hybrid-associated DNA damage at pericentromeric repeats

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Motoko Unoki ◽  
Jafar Sharif ◽  
Yuichiro Saito ◽  
Guillaume Velasco ◽  
Claire Francastel ◽  
...  
Keyword(s):  
Dna Damage ◽  
Autosomal Recessive ◽  
Ectopic Expression ◽  
Autosomal Recessive Disorder ◽  
End Joining ◽  
Icf Syndrome ◽  
Centromeric Instability ◽  
Non Homologous End Joining ◽  
Genomic Regions ◽  
Mutant Cells

Abstract Immunodeficiency, centromeric instability, facial anomalies (ICF) syndrome is a rare autosomal recessive disorder that is caused by mutations in either DNMT3B, ZBTB24, CDCA7, HELLS, or yet unidentified gene(s). Previously, we reported that the CDCA7/HELLS chromatin remodeling complex facilitates non-homologous end-joining. Here, we show that the same complex is required for the accumulation of proteins on nascent DNA, including the DNMT1/UHRF1 maintenance DNA methylation complex as well as proteins involved in the resolution or prevention of R-loops composed of DNA:RNA hybrids and ssDNA. Consistent with the hypomethylation state of pericentromeric repeats, the transcription and formation of aberrant DNA:RNA hybrids at the repeats were increased in ICF mutant cells. Furthermore, the ectopic expression of RNASEH1 reduced the accumulation of DNA damage at a broad range of genomic regions including pericentromeric repeats in these cells. Hence, we propose that hypomethylation due to inefficient DNMT1/UHRF1 recruitment at pericentromeric repeats by defects in the CDCA7/HELLS complex could induce pericentromeric instability, which may explain a part of the molecular pathogenesis of ICF syndrome.

scholarly journals Mono-homologous linear DNA recombination by the non-homologous end-joining pathway as a novel and simple gene inactivation method: a proof of concept study inDietziasp. DQ12-45-1b

2018 ◽  
Author(s):  
Shelian Lu ◽  
Yong Nie ◽  
Meng Wang ◽  
Hong-Xiu Xu ◽  
Dong-Ling Ma ◽  
...  
Keyword(s):  
Genetic Manipulation ◽  
Dna Recombination ◽  
Proof Of Concept ◽  
End Joining ◽  
Circular Dna ◽  
Linear Dna ◽  
Genes Encoding ◽  
Non Homologous End Joining ◽  
Nhej Activity

ABSTRACTNon-homologous end-joining (NHEJ) is critical for genome stability because of its roles in double-strand break repair. Ku and ligase D (LigD) are the crucial proteins in this process, and strains expressing Ku and LigD can cyclize linear DNAin vivo.Herein, we established a proof-of-concept mono-homologous linear DNA recombination for gene inactivation or genome editing by which cyclization of linear DNAin vivoby NHEJ could be used to generate non-replicable circular DNA and could allow allelic exchanges between the circular DNA and the chromosome. We achieved this approach inDietziasp. DQ12-45-1b, which expresses Ku and LigD homologs and presents NHEJ activity. By transforming the strain with a linear DNA mono homolog to the sequence in chromosome, we mutated the genome. This method did not require the screening of suitable plasmids and was easy and time-effective. Bioinformatic analysis showed that more than 20% prokaryotic organisms contain Ku and LigD, suggesting the wide distribution of NHEJ activities. Moreover, theEscherichia colistrain also showed NHEJ activity when the Ku and LigD ofDietziasp. DQ12-45-1b were introduced and expressed in it. Therefore, this method may be a widely applicable genome editing tool for diverse prokaryotic organisms, especially for non-model microorganisms.IMPORTANCEThe non-model gram-positive bacteria lack efficient genetic manipulation systems, but they express genes encoding Ku and LigD. The NHEJ pathway inDietziasp. DQ12-45-1b was evaluated and was used to successfully knockout eleven genes in the genome. Since bioinformatic studies revealed that the putative genes encoding Ku and LigD ubiquitously exist in phylogenetically diverse bacteria and archaea, the mono-homologous linear DNA recombination by the NHEJ pathway could be a potentially applicable genetic manipulation method for diverse non-model prokaryotic organisms.

BrADseq for DNA v1

2021 ◽  
Author(s):  
S. Thomas Kelly ◽  
tsuneo.hakoyama not provided ◽  
Kie Kumaishi ◽  
Haruka Okuda-Yabukami ◽  
Sachi Kato ◽  
...  
Keyword(s):  
Genomic Dna ◽  
Genomic Sequence ◽  
Sequence Data ◽  
Gc Content ◽  
Handling Time ◽  
Cost Effective ◽  
Dna Amount ◽  
Dna Libraries ◽  
Genomic Regions ◽  
Next Generation Sequencing Ngs

The amount of input DNA available to prepare next-generation sequencing (NGS) libraries is often limited, which can lead to GC content bias and enrichment of specific genomic regions with currently available protocols. In this study, we used breath capture technology to incorporate sequencing adapters into DNA to develop a novel cost-effective protocol for the preparation of genomic DNA libraries. We performed a benchmarking experiment comparing our protocol with common commercially available kits for genomic DNA library preparation with input DNA amount in the range of 1 to 50 ng. Our protocol can generate high-quality genomic sequence data with a marked improvement in coverage breadth and low GC bias, in contrast to standard protocols. Further, our protocol reduces sample handling time and reagent costs, and requires comparatively fewer enzymatic steps relative to other protocols, making it suitable for a range of genomics applications.

scholarly journals Targeted Inter-Homologs Recombination in Arabidopsis Euchromatin and Heterochromatin

2021 ◽  
Vol 22 (22) ◽  
pp. 12096
Author(s):  
Shdema Filler-Hayut ◽  
Kiril Kniazev ◽  
Cathy Melamed-Bessudo ◽  
Avraham A. Levy
Keyword(s):  
Gene Conversion ◽  
Experimental System ◽  
Strand Break ◽  
Typical Feature ◽  
End Joining ◽  
Dna Double Strand Break ◽  
Short Palindromic Repeat ◽  
Non Homologous End Joining ◽  
Genomic Regions ◽  
Targeted Recombination

Homologous recombination (HR) typically occurs during meiosis between homologs, at a few unplanned locations along the chromosomes. In this study, we tested whether targeted recombination between homologous chromosomes can be achieved via Clustered Regulatory Interspaced Short Palindromic Repeat associated protein Cas9 (CRISPR-Cas9)-induced DNA double-strand break (DSB) repair in Arabidopsis thaliana. Our experimental system includes targets for DSB induction in euchromatic and heterochromatic genomic regions of hybrid F1 plants, in one or both parental chromosomes, using phenotypic and molecular markers to measure Non-Homologous End Joining and HR repair. We present a series of evidence showing that targeted DSBs can be repaired via HR using a homologous chromosome as the template in various chromatin contexts including in pericentric regions. Targeted crossover was rare, but gene conversion events were the most frequent outcome of HR and were found in both “hot and cold” regions. The length of the conversion tracts was variable, ranging from 5 to 7505 bp. In addition, a typical feature of these tracks was that they often were interrupted. Our findings pave the way for the use of targeted gene-conversion for precise breeding.

scholarly journals Robust and stable transcriptional repression in Giardia using CRISPRi

2018 ◽  
Author(s):  
SG McInally ◽  
KD Hagen ◽  
C Nosala ◽  
J Williams ◽  
K Nguyen ◽  
...  
Keyword(s):  
Transcriptional Repression ◽  
Diarrheal Disease ◽  
Model Systems ◽  
Structural Defects ◽  
End Joining ◽  
Morpholino Knockdown ◽  
Endogenous Genes ◽  
Localization Signal ◽  
Ventral Disc ◽  
Non Homologous End Joining

AbstractGiardia lamblia is a binucleate protistan parasite causing significant diarrheal disease worldwide. An inability to target Cas9 to both nuclei, combined with the lack of non-homologous end joining and markers for positive selection, has stalled the adaptation of CRISPR/Cas9-mediated genetic tools for this widespread parasite. CRISPR interference (CRISPRi) is a modification of the CRISPR/Cas9 system that directs catalytically inactive Cas9 (dCas9) to target loci for stable transcriptional repression. Using a Giardia nuclear localization signal to target dCas9 to both nuclei, we developed efficient and stable CRISPRi-mediated transcriptional repression of exogenous and endogenous genes in Giardia. Specifically, CRISPRi knockdown of kinesin-2a and kinesin-13 causes severe flagellar length defects that mirror defects with morpholino knockdown. Knockdown of the ventral disc MBP protein also causes severe structural defects that are highly prevalent and persist in the population more than five days longer than transient morpholino-based knockdown. By expressing two gRNAs in tandem to simultaneously knock down kinesin-13 and MBP, we created a stable dual knockdown strain with both flagellar length and disc defects. The efficiency and simplicity of CRISPRi in polyploid Giardia allows for rapid evaluation of knockdown phenotypes and highlights the utility of CRISPRi for emerging model systems.

scholarly journals CRISPR-Cas9-Based Mutagenesis of the Mucormycosis-Causing Fungus Lichtheimia corymbifera

2020 ◽  
Vol 21 (10) ◽  
pp. 3727
Author(s):  
Sandugash Ibragimova ◽  
Csilla Szebenyi ◽  
Rita Sinka ◽  
Elham I. Alzyoud ◽  
Mónika Homa ◽  
...  
Keyword(s):  
Genetic Manipulation ◽  
Pathogenic Fungus ◽  
Strand Break ◽  
Targeted Mutagenesis ◽  
End Joining ◽  
Targeted Gene Disruption ◽  
Lichtheimia Corymbifera ◽  
Non Homologous End Joining ◽  
Opportunistic Pathogenic

Lichtheimia corymbifera is considered as one of the most frequent agents of mucormycosis. The lack of efficient genetic manipulation tools hampers the characterization of the pathomechanisms and virulence factors of this opportunistic pathogenic fungus. Although such techniques have been described for certain species, the performance of targeted mutagenesis and the construction of stable transformants have remained a great challenge in Mucorales fungi. In the present study, a plasmid-free CRISPR-Cas9 system was applied to carry out a targeted gene disruption in L. corymbifera. The described method is based on the non-homologous end-joining repair of the double-strand break caused by the Cas9 enzyme. Using this method, short, one-to-five nucleotide long-targeted deletions could be induced in the orotidine 5′-phosphate decarboxylase gene (pyrG) and, as a result, uracil auxotrophic strains were constructed. These strains are applicable as recipient strains in future gene manipulation studies. As we know, this is the first genetic modification of this clinically relevant fungus.

scholarly journals Non-Homologous End-Joining Gene Profiling Reveals Distinct Expression Patterns Associated with Lymphoma and Multiple Myeloma

2010 ◽  
Vol 149 (2) ◽  
pp. 258-262 ◽  
Author(s):  
Philippa L. Roddam ◽  
James M. Allan ◽  
Ann M. Dring ◽  
Lisa J. Worrillow ◽  
Faith E. Davies ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Expression Patterns ◽  
Gene Profiling ◽  
End Joining ◽  
Non Homologous End Joining

scholarly journals Integrin α6β4 signaling switches DNA repair from homologous recombination to non-homologous end-joining pathway to sensitize breast cancer cells to cisplatin

2019 ◽  
Author(s):  
Min Chen ◽  
Brock Marrs ◽  
Lei Qi ◽  
Teresa Knifley ◽  
Stuart G. Jarrett ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Dna Repair ◽  
Homologous Recombination ◽  
Genetic Manipulation ◽  
Strand Break ◽  
End Joining ◽  
Apoptosis Resistance ◽  
Integrin Α6β4 ◽  
Cisplatin Sensitivity ◽  
Non Homologous End Joining

AbstractIntegrin α6β4 is highly expressed in triple negative breast cancer (TNBC) and drives aggressiveness by stimulating proliferation, angiogenesis, cell migration, invasion and metastasis. Signaling from this integrin stimulates DNA repair and apoptosis resistance, suggesting that it could contribute to therapeutic resistance. Upon testing this hypothesis, we found that integrin α6β4 signaling promoted a three-fold greater sensitivity to cisplatin but exhibited no difference in response to other chemotherapies tested. Mechanistic investigations revealed that integrin α6β4 stimulated quicker and higher amplitude of activation of ATM, Chk2, p53, and 53BP1, which required the integrin β4 signaling domain. Genetic manipulation of gene expression demonstrated that mutant p53 cooperated with integrin α6β4 for cisplatin sensitivity and was necessary for downstream phosphorylation of 53BP1 and enhanced ATM activation. Additionally, we discovered that integrin α6β4 preferentially activated DNA-PKc in response to cisplatin, which led to formation of DNA-PKc-p53 complexes and 53BP1 activation. As a result, integrin α6β4 shifted double strand break repair from homologous recombination (HR) to non-homologous end joining (NHEJ). In summary, we discovered a novel function of integrin α6β4 in switching DSB repair from HR to NHEJ that results in cisplatin sensitivity in TNBC.

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