scholarly journals Cas9-induced large deletions and small indels are controlled in a convergent fashion

2020 ◽  
Author(s):  
Michael Kosicki ◽  
Felicity Allen ◽  
Allan Bradley
Keyword(s):  
Embryonic Stem ◽  
Basic Research ◽  
Large Deletion ◽  
End Joining ◽  
Small Indels ◽  
Repair Processes ◽  
Large Deletions ◽  
Non Homologous End Joining ◽  
Deletion Frequency ◽  
In Cells

Repair of Cas9-induced double-stranded breaks results primarily in formation of small indels, but can also cause potentially harmful large deletions. While mechanisms leading to the creation of small indels are relatively well understood, very little is known about the origins of large deletions. Using a novel library of clonal mouse embryonic stem cells bona fide deficient for 32 DNA repair genes, we have shown that large deletion frequency increases in cells impaired for non-homologous end joining and decreases in cells deficient for the central resection gene Nbn and the microhomology-mediated end joining gene Polq. Across deficient clones, increase in large deletion frequency was closely correlated with the increase in the extent of microhomology and the size of small indels, implying a continuity of repair processes across different genomic scales. Furthermore, by targeting diverse genomic sites, we identified examples of repair processes that were highly locus-specific, discovering a novel role for exonuclease Trex1. Finally, we present evidence that indel sizes increase with the overall efficiency of Cas9 mutagenesis. These findings may have impact on both basic research and clinical use of CRISPR-Cas9, in particular in conjunction with repair pathway modulation.

Characterisation of large F9 deletions in seven unrelated patients with severe haemophilia B

2014 ◽  
Vol 112 (09) ◽  
pp. 459-465 ◽  
Author(s):  
Qiulan Ding ◽  
Guoling You ◽  
Jing Dai ◽  
Xiaodong Xi ◽  
Hongli Wang ◽  
...  
Keyword(s):  
Large Deletion ◽  
The Other ◽  
Severe Haemophilia ◽  
End Joining ◽  
Haemophilia B ◽  
Large Deletions ◽  
Non Homologous End Joining ◽  
Break Induced Replication ◽  
Long Interspersed Nuclear Element ◽  
Female Carriers

SummaryLarge deletions in the F9 gene are detected in approximately 5% of patients with severe haemophilia B, but only a few deletion breakpoints have been characterised precisely until now. In this study we identified a total of seven large F9 deletions in the index patients and nine female carriers by the AccuCopy technique. We also successfully characterised the exact breakpoints for each large deletion including four deletions encompassing the entire F9 gene by the genome walking method combined with primer walking strategy. The extents of deletion regions ranged from 11.1 to 884 kb. Microhomologies ranged from 2 to 6 bp were identified in the breakpoint junctions of six deletions. The other deletion occurred between two highly homologous sequences of the same long interspersed nuclear element 1 (LINE/L1). Non-homologous end joining (NHEJ) and microhomology-mediated break-induced replication (MMBIR) may be the main causative mechanisms for the six large deletions with microhomologies. Non-allelic homologous recombination (NAHR) may mediate the deletion occurred between the two tandem LINEs in the other large deletion. Repetitive elements and non-B DNA forming motifs identified in the junction regions may contribute to DNA breakage leading to large deletions.

scholarly journals Sequence and Structure Characteristics of 22 Deletion Breakpoints in Intron 44 of the DMD Gene Based on Long-Read Sequencing

2021 ◽  
Vol 12 ◽  
Author(s):  
Chang Geng ◽  
Yuanren Tong ◽  
Siwen Zhang ◽  
Chao Ling ◽  
Xin Wu ◽  
...  
Keyword(s):  
Becker Muscular Dystrophy ◽  
Repetitive Elements ◽  
Loop Formation ◽  
End Joining ◽  
Origin Firing ◽  
Large Deletions ◽  
Long Read ◽  
Non Homologous End Joining ◽  
Dmd Gene ◽  
Palindromic Sequences

Purpose: Exon deletions make up to 80% of mutations in the DMD gene, which cause Duchenne and Becker muscular dystrophy. Exon 45-55 regions were reported as deletion hotspots and intron 44 harbored more than 25% of deletion start points. We aimed to investigate the fine structures of breakpoints in intron 44 to find potential mechanisms of large deletions in intron 44.Methods: Twenty-two dystrophinopathy patients whose deletion started in intron 44 were sequenced using long-read sequencing of a DMD gene capture panel. Sequence homology, palindromic sequences, and polypyrimidine sequences were searched at the breakpoint junctions. RepeatMasker was used to analyze repetitive elements and Mfold was applied to predict secondary DNA structure.Results: With a designed DMD capture panel, 22 samples achieved 2.25 gigabases and 1.28 million reads on average. Average depth was 308× and 99.98% bases were covered at least 1×. The deletion breakpoints in intron 44 were scattered and no breakpoints clustered in any region less than 500 bp. A total of 72.7% of breakpoints located in distal 100 kb of intron 44 and more repetitive elements were found in this region. Microhomologies of 0–1 bp were found in 36.4% (8/22) of patients, which corresponded with non-homologous end-joining. Microhomologies of 2–20 bp were found in 59.1% (13/22) of patients, which corresponded with microhomology-mediated end-joining. Moreover, a 7 bp insertion was found in one patient, which might be evidence of aberrant replication origin firing. Palindromic sequences, polypyrimidine sequences, and small hairpin loops were found near several breakpoint junctions. No evidence of large hairpin loop formation in deletion root sequences was observed.Conclusion: This study was the first to explore possible mechanisms underlying exon deletions starting from intron 44 of the DMD gene based on long-read sequencing. Diverse mechanisms might be associated with deletions in the DMD gene.

scholarly journals Effects of mitoTALENs-Directed Double-Strand Breaks on Plant Mitochondrial Genomes

Genes ◽  
2021 ◽  
Vol 12 (2) ◽  
pp. 153
Author(s):  
Shin-ichi Arimura
Keyword(s):  
Double Strand Breaks ◽  
Mitochondrial Genomes ◽  
Transcription Activator ◽  
End Joining ◽  
Strand Breaks ◽  
Large Deletions ◽  
Naturally Occurring ◽  
Copy Numbers ◽  
Nuclear Genomes ◽  
Non Homologous End Joining

Mitochondrial genomes in flowering plants differ from those in animals and yeasts in several ways, including having large and variable sizes, circular, linear and branched structures, long repeat sequences that participate in homologous recombinations, and variable genes orders, even within a species. Understanding these differences has been hampered by a lack of genetic methods for transforming plant mitochondrial genomes. We recently succeeded in disrupting targeted genes in mitochondrial genomes by mitochondria-targeted transcription activator-like effector nucleases (mitoTALENs) in rice, rapeseed, and Arabidopsis. Double-strand breaks created by mitoTALENs were repaired not by non-homologous end-joining (NHEJ) but by homologous recombination (HR) between repeats near and far from the target sites, resulting in new genomic structures with large deletions and different configurations. On the other hand, in mammals, TALENs-induced DSBs cause small insertions or deletions in nuclear genomes and degradation of mitochondrial genomes. These results suggest that the mitochondrial and nuclear genomes of plants and mammals have distinct mechanisms for responding to naturally occurring DSBs. The different responses appear to be well suited to differences in size and copy numbers of each genome.

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 oca2 targeting using CRISPR/Cas9 in the Malawi cichlid Astatotilapia calliptera

2022 ◽  
Author(s):  
Bethan Clark ◽  
Joel Elkin ◽  
Aleksandra Marconi ◽  
George F Turner ◽  
Alan M Smith ◽  
...  
Keyword(s):  
Lake Malawi ◽  
Large Deletion ◽  
Colour Pattern ◽  
Coding Region ◽  
Genetic Loci ◽  
End Joining ◽  
Knock Out ◽  
Homology Directed Repair ◽  
Non Homologous End Joining

Identifying genetic loci underlying trait variation provides insights into the mechanisms of diversification, but demonstrating causality and characterising the role of genetic loci requires testing candidate gene function, often in non-model species. Here we establish CRISPR/Cas9 editing in Astatotilapia calliptera, a generalist cichlid of the remarkably diverse Lake Malawi radiation. By targeting the gene oca2 required for melanin synthesis in other vertebrate species, we show efficient editing and germline transmission. Gene edits include indels in the coding region, likely a result of non-homologous end joining, and a large deletion in the 3′ UTR due to homology-directed repair. We find that oca2 knock-out A. calliptera lack melanin, which may be useful for developmental imaging in embryos and studying colour pattern formation in adults. As A. calliptera resembles the presumed generalist ancestor of the Lake Malawi cichlids radiation, establishing genome editing in this species will facilitate investigating speciation, adaptation and trait diversification in this textbook radiation.

scholarly journals A Homology Independent Sequence Replacement Strategy in Human Cells Using a CRISPR Nuclease

2020 ◽  
Author(s):  
Eric Danner ◽  
Mikhail Lebedin ◽  
Kathrin de la Rosa ◽  
Ralf Kühn
Keyword(s):  
Basic Research ◽  
Strand Break ◽  
End Joining ◽  
Linear Amplification ◽  
Promising Alternative ◽  
Independent Sequence ◽  
Replacement Strategy ◽  
Homology Directed Repair ◽  
Dividing Cells ◽  
Non Homologous End Joining

AbstractPrecision genomic alterations largely rely on Homology Directed Repair (HDR), but targeting without homology using the Non-Homologous End Joining (NHEJ) pathway has gained attention as a promising alternative. Previous studies demonstrated precise insertions formed by the ligation of donor DNA into a targeted genomic double strand break in both dividing and non-dividing cells. Here we extend this idea and use NHEJ repair to replace genomic segments with donor sequences; we name this method ‘Replace’ editing (Rational end-joining protocol delivering a targeted sequence exchange). Using CRISPR/Cas9 we create two genomic breaks and ligate a donor sequence in-between. This exchange of a genomic for a donor sequence uses neither microhomology nor homology arms. We target four loci and show successful exchange of exons in 16% to 54% of cells. Using linear amplification methods and deep sequencing pipelines we quantify the diversity of outcomes following Replace editing and profile mutations formed at the ligated interfaces. The ability to replace exons or other genomic sequences in cells not efficiently modified by HDR holds promise for both basic research and medicine.

scholarly journals Efficient biallelic knock-in in mouse embryonic stem cells by in vivo-linearization of donor and transient inhibition of DNA Polymerase θ/DNA-PK

2021 ◽  
Author(s):  
Daisuke Arai ◽  
Yoichi Nakao
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Dna Polymerase ◽  
Embryonic Stem ◽  
Mouse Embryonic Stem Cells ◽  
End Joining ◽  
Homology Directed Repair ◽  
Low Efficiency ◽  
Non Homologous End Joining

CRISPR/Cas9-mediated homology-directed repair (HDR) is used for error-free targeted knock-in of foreign donor DNA. However, the low efficiency of HDR-mediated knock-in hinders establishment of knock-in clones. Double-strand breaks (DSBs) induced by CRISPR/Cas9 are preferentially repaired by non-homologous end joining (NHEJ) or microhomology-mediated end joining (MMEJ) before HDR can occur, thereby preventing HDR-mediated knock-in. NHEJ/MMEJ also cause random integrations, which give rise to false-positive knock-in events, or silently disrupt the genome. In this study, we optimized an HDR-mediated knock-in method for mouse embryonic stem cells (mESCs). We succeeded in improving efficiency of HDR-mediated knock-in of a plasmid donor while almost completely suppressing NHEJ/MMEJ-based integration by combining in vivo-linearization of the donor plasmid, transient knockdown of DNA Polymerase θ, and chemical inhibition of DNA-dependent protein kinase (DNA-PK) by M3814. This method also dramatically improved the efficiency of biallelic knock-in; at the Rosa26a locus, 95% of HDR-mediated knock-in clones were biallelic. We designate this method BiPoD (Biallelic knock-in assisted by Pol θ and DNA-PK inhibition). BiPoD achieved simultaneous efficient biallelic knock-in into two loci. BiPoD, therefore, enables rapid and easy establishment of biallelic knock-in mESC lines.

scholarly journals Necessities in the Processing of DNA Double Strand Breaks and Their Effects on Genomic Instability and Cancer

Cancers ◽  
2019 ◽  
Vol 11 (11) ◽  
pp. 1671 ◽  
Author(s):  
George Iliakis ◽  
Emil Mladenov ◽  
Veronika Mladenova
Keyword(s):  
High Speed ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Strand Breaks ◽  
Large Deletions ◽  
Single Strand Annealing ◽  
Modes Of Processing ◽  
Non Homologous End Joining ◽  
Strand Annealing

Double strand breaks (DSBs) are induced in the DNA following exposure of cells to ionizing radiation (IR) and are highly consequential for genome integrity, requiring highly specialized modes of processing. Erroneous processing of DSBs is a cause of cell death or its transformation to a cancer cell. Four mechanistically distinct pathways have evolved in cells of higher eukaryotes to process DSBs, providing thus multiple options for the damaged cells. The homologous recombination repair (HRR) dependent subway of gene conversion (GC) removes IR-induced DSBs from the genome in an error-free manner. Classical non-homologous end joining (c-NHEJ) removes DSBs with very high speed but is unable to restore the sequence at the generated junction and can catalyze the formation of translocations. Alternative end-joining (alt-EJ) operates on similar principles as c-NHEJ but is slower and more error-prone regarding both sequence preservation and translocation formation. Finally, single strand annealing (SSA) is associated with large deletions and may also form translocations. Thus, the four pathways available for the processing of DSBs are not alternative options producing equivalent outcomes. We discuss the rationale for the evolution of pathways with such divergent properties and fidelities and outline the logic and necessities that govern their engagement. We reason that cells are not free to choose one specific pathway for the processing of a DSB but rather that they engage a pathway by applying the logic of highest fidelity selection, adapted to necessities imposed by the character of the DSB being processed. We introduce DSB clusters as a particularly consequential form of chromatin breakage and review findings suggesting that this form of damage underpins the increased efficacy of high linear energy transfer (LET) radiation modalities. The concepts developed have implications for the protection of humans from radon-induced cancer, as well as the treatment of cancer with radiations of high LET.

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