Approaches to Enhance Precise CRISPR/Cas9-Mediated Genome Editing

Modification of the human genome has immense potential for preventing or treating disease. Modern genome editing techniques based on CRISPR/Cas9 show great promise for altering disease-relevant genes. The efficacy of precision editing at CRISPR/Cas9-induced double-strand breaks is dependent on the relative activities of nuclear DNA repair pathways, including the homology-directed repair and error-prone non-homologous end-joining pathways. The competition between multiple DNA repair pathways generates mosaic and/or therapeutically undesirable editing outcomes. Importantly, genetic models have validated key DNA repair pathways as druggable targets for increasing editing efficacy. In this review, we highlight approaches that can be used to achieve the desired genome modification, including the latest progress using small molecule modulators and engineered CRISPR/Cas proteins to enhance precision editing.

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Simultaneous precise editing of multiple genes in human cells

Nucleic Acids Research ◽

10.1093/nar/gkz669 ◽

2019 ◽

Vol 47 (19) ◽

pp. e116-e116 ◽

Cited By ~ 17

Author(s):

Stephan Riesenberg ◽

Manjusha Chintalapati ◽

Dominik Macak ◽

Philipp Kanis ◽

Tomislav Maricic ◽

...

Keyword(s):

Genome Editing ◽

Kinase Inhibitor ◽

Double Strand Breaks ◽

End Joining ◽

Nucleotide Substitutions ◽

Strand Breaks ◽

Homology Directed Repair ◽

A Genome ◽

Dependent Protein ◽

Non Homologous End Joining

Abstract When double-strand breaks are introduced in a genome by CRISPR they are repaired either by non-homologous end joining (NHEJ), which often results in insertions or deletions (indels), or by homology-directed repair (HDR), which allows precise nucleotide substitutions to be introduced if a donor oligonucleotide is provided. Because NHEJ is more efficient than HDR, the frequency with which precise genome editing can be achieved is so low that simultaneous editing of more than one gene has hitherto not been possible. Here, we introduced a mutation in the human PRKDC gene that eliminates the kinase activity of the DNA-dependent protein kinase catalytic subunit (DNA-PKcs). This results in an increase in HDR irrespective of cell type and CRISPR enzyme used, sometimes allowing 87% of chromosomes in a population of cells to be precisely edited. It also allows for precise editing of up to four genes simultaneously (8 chromosomes) in the same cell. Transient inhibition of DNA-PKcs by the kinase inhibitor M3814 is similarly able to enhance precise genome editing.

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Recent Perspectives in Radiation-Mediated DNA Damage and Repair: Role of NHEJ and Alternative Pathways

10.5772/intechopen.96374 ◽

2021 ◽

Author(s):

Ajay Kumar Sharma ◽

Priyanka Shaw ◽

Aman Kalonia ◽

M.H. Yashavarddhan ◽

Pankaj Chaudhary ◽

...

Keyword(s):

Dna Damage ◽

Dna Repair ◽

Double Strand Breaks ◽

Single Strand ◽

End Joining ◽

Strand Breaks ◽

Single Strand Annealing ◽

Repair Pathways ◽

Non Homologous End Joining ◽

Alternative Nhej

Radiation is one of the causative agents for the induction of DNA damage in biological systems. There is various possibility of radiation exposure that might be natural, man-made, intentional, or non-intentional. Published literature indicates that radiation mediated cell death is primarily due to DNA damage that could be a single-strand break, double-strand breaks, base modification, DNA protein cross-links. The double-strand breaks are lethal damage due to the breakage of both strands of DNA. Mammalian cells are equipped with strong DNA repair pathways that cover all types of DNA damage. One of the predominant pathways that operate DNA repair is a non-homologous end-joining pathway (NHEJ) that has various integrated molecules that sense, detect, mediate, and repair the double-strand breaks. Even after a well-coordinated mechanism, there is a strong possibility of mutation due to the flexible nature in joining the DNA strands. There are alternatives to NHEJ pathways that can repair DNA damage. These pathways are alternative NHEJ pathways and single-strand annealing pathways that also displayed a role in DNA repair. These pathways are not studied extensively, and many reports are showing the relevance of these pathways in human diseases. The chapter will very briefly cover the radiation, DNA repair, and Alternative repair pathways in the mammalian system. The chapter will help the readers to understand the basic and applied knowledge of radiation mediated DNA damage and its repair in the context of extensively studied NHEJ pathways and unexplored alternative NHEJ pathways.

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ATM antagonizes NHEJ proteins assembly and DNA-ends synapsis at single-ended DNA double strand breaks

Nucleic Acids Research ◽

10.1093/nar/gkaa723 ◽

2020 ◽

Vol 48 (17) ◽

pp. 9710-9723

Author(s):

Sébastien Britton ◽

Pauline Chanut ◽

Christine Delteil ◽

Nadia Barboule ◽

Philippe Frit ◽

...

Keyword(s):

Dna Repair ◽

Double Strand Breaks ◽

Dna Double Strand Breaks ◽

End Joining ◽

Strand Breaks ◽

Repair Pathways ◽

Non Homologous End Joining ◽

Dna Strand ◽

Dna Ends

Abstract Two DNA repair pathways operate at DNA double strand breaks (DSBs): non-homologous end-joining (NHEJ), that requires two adjacent DNA ends for ligation, and homologous recombination (HR), that resects one DNA strand for invasion of a homologous duplex. Faithful repair of replicative single-ended DSBs (seDSBs) is mediated by HR, due to the lack of a second DNA end for end-joining. ATM stimulates resection at such breaks through multiple mechanisms including CtIP phosphorylation, which also promotes removal of the DNA-ends sensor and NHEJ protein Ku. Here, using a new method for imaging the recruitment of the Ku partner DNA-PKcs at DSBs, we uncover an unanticipated role of ATM in removing DNA-PKcs from seDSBs in human cells. Phosphorylation of DNA-PKcs on the ABCDE cluster is necessary not only for DNA-PKcs clearance but also for the subsequent MRE11/CtIP-dependent release of Ku from these breaks. We propose that at seDSBs, ATM activity is necessary for the release of both Ku and DNA-PKcs components of the NHEJ apparatus, and thereby prevents subsequent aberrant interactions between seDSBs accompanied by DNA-PKcs autophosphorylation and detrimental commitment to Lig4-dependent end-joining.

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Covalent linkage of the DNA repair template to the CRISPR/Cas9 complex enhances homology-directed repair

10.1101/218149 ◽

2017 ◽

Cited By ~ 4

Author(s):

Natasa Savic ◽

Femke Ringnalda ◽

Katja Bargsten ◽

Yizhou Li ◽

Christian Berk ◽

...

Keyword(s):

Dna Repair ◽

Mammalian Cells ◽

Dna Double Strand Breaks ◽

End Joining ◽

Strand Breaks ◽

Homology Directed Repair ◽

Genetic Modifications ◽

Repair Template ◽

Single Base Pair ◽

Non Homologous End Joining

AbstractThe CRISPR/Cas9 targeted nuclease technology allows the insertion of genetic modifications with single base-pair precision. The preference of mammalian cells to repair Cas9-induced DNA double-strand breaks via non-homologous end joining (NHEJ) rather than via homology-directed repair (HDR) however leads to relatively low rates of correctly edited loci. Here we demonstrate that covalently linking the DNA repair template to Cas9 increases the ratio of HDR over NHEJ up to 23-fold, and therefore provides advantages for clinical applications where high-fidelity repair is needed.

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Double strand break (DSB) repair pathways in plants and their application in genome engineering

Genome editing for precision crop breeding - Burleigh Dodds Series in Agricultural Science ◽

10.19103/as.2020.0082.04 ◽

2021 ◽

pp. 27-62

Author(s):

Natalja Beying ◽

◽

Carla Schmidt ◽

Holger Puchta ◽

◽

...

Keyword(s):

Genome Engineering ◽

Plant Cells ◽

Strand Break ◽

End Joining ◽

Dsb Repair ◽

Strand Breaks ◽

Repair Mechanisms ◽

Repair Pathways ◽

Non Homologous End Joining ◽

Underlying Mechanisms

In genome engineering, after targeted induction of double strand breaks (DSBs) researchers take advantage of the organisms’ own repair mechanisms to induce different kinds of sequence changes into the genome. Therefore, understanding of the underlying mechanisms is essential. This chapter will review in detail the two main pathways of DSB repair in plant cells, non-homologous end joining (NHEJ) and homologous recombination (HR) and sum up what we have learned over the last decades about them. We summarize the different models that have been proposed and set these into relation with the molecular outcomes of different classes of DSB repair. Moreover, we describe the factors that have been identified to be involved in these pathways. Applying this knowledge of DSB repair should help us to improve the efficiency of different types of genome engineering in plants.

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The Leukemic Fusion Gene NUP98-HOXD13 Suppresses Non-Homologous End Joining Factors and Impairs DNA Double Strand Break Repair

Blood ◽

10.1182/blood.v120.21.521.521 ◽

2012 ◽

Vol 120 (21) ◽

pp. 521-521

Author(s):

David L. Caudell ◽

Abdul Gafoor A. Puthiyaveetil

Keyword(s):

Dna Repair ◽

B Lymphocytes ◽

Fusion Gene ◽

Double Strand Breaks ◽

End Joining ◽

Strand Breaks ◽

Class Switch ◽

Induction Pattern ◽

Dna Break ◽

Non Homologous End Joining

Abstract Abstract 521 Chromosomal translocations are hallmark features of hematologic malignancies that often require collaborating mutations for malignant transformation. These secondary mutations can occur spontaneously, or rather, be induced by the primary translocation. Mutations can occur as a result of DNA damage and misrepair with DNA double strand breaks being one of the most serious types of cell damage. Double strand breaks are classically repaired by the non-homologous end joining (NHEJ) mechanism and impaired NHEJ has been shown to promote mutagenesis. Transgenic mice expressing the myeloid leukemic fusion gene NUP98-HOXD13 (NHD13) develop Myelodysplastic syndrome and progress to acute leukemia after acquiring secondary mutations. Our studies have shown that B lymphocyte development and class switch recombination are impaired in stimulated B lymphocytes from NHD13 mice. Based on this, we used in vitro class switch recombination (CSR) to delineate the DNA break induction and repair mechanisms in NHD13 B lymphocytes. Naïve B lymphocytes were harvested from wild type (WT) and NHD13 spleens and cultured in the presence of E.coli Lipopolysaccharide (LPS) and IL-4 to induce CSR. The DNA break induction pattern was determined using phosphorylated H2AX labeling combined with confocal microscopy and flow cytometry. Our results showed that NHD13 B lymphocytes had a comparable break induction pattern, but significantly reduced DNA repair. Analysis of the cell cycle pattern of stimulated B cells at 24 hour intervals showed cell cycle arrest at the G2/M phase at 72 hours following stimulation—a hallmark feature of impaired DNA break repair. We analyzed the expression of classical NHEJ and alternative end joining factors including Ku70, Ku80, DNA Protein Kinase catalytic subunit (DNAPKcs), Xrcc4, DNA ligase 4, Ligase 1, and Ligase 3. Our results showed reduced expression of DNAPKcs, Ligase 4 and Xrcc4 in NHD13 B lymphocytes at 72 hours following stimulation, suggesting that cells failed to initiate NHEJ-mediated DNA repair. Our results suggest that a myeloid leukemic gene can impair the DNA repair mechanism and may indirectly promote mutations necessary for malignant transformation. Disclosures: No relevant conflicts of interest to declare.

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Mechanistic and genetic basis of single-strand templated repair at Cas12a-induced DNA breaks in Chlamydomonas reinhardtii

Nature Communications ◽

10.1038/s41467-021-27004-1 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Aron Ferenczi ◽

Yen Peng Chew ◽

Erika Kroll ◽

Charlotte von Koppenfels ◽

Andrew Hudson ◽

...

Keyword(s):

Dna Repair ◽

Chlamydomonas Reinhardtii ◽

Nuclear Dna ◽

Model Organism ◽

Single Strand ◽

Dna Double Strand Breaks ◽

Dna Breaks ◽

End Joining ◽

Non Homologous End Joining ◽

Underlying Mechanisms

AbstractSingle-stranded oligodeoxynucleotides (ssODNs) are widely used as DNA repair templates in CRISPR/Cas precision genome editing. However, the underlying mechanisms of single-strand templated DNA repair (SSTR) are inadequately understood, constraining rational improvements to precision editing. Here we study SSTR at CRISPR/Cas12a-induced DNA double-strand breaks (DSBs) in the eukaryotic model green microalga Chlamydomonas reinhardtii. We demonstrate that ssODNs physically incorporate into the genome during SSTR at Cas12a-induced DSBs. This process is genetically independent of the Rad51-dependent homologous recombination and Fanconi anemia pathways, is strongly antagonized by non-homologous end-joining, and is mediated almost entirely by the alternative end-joining enzyme polymerase θ. These findings suggest differences in SSTR between C. reinhardtii and animals. Our work illustrates the promising potentially of C. reinhardtii as a model organism for studying nuclear DNA repair.

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Programmed genome editing of the omega-1 ribonuclease 1 of the blood fluke,Schistosoma mansoni

10.1101/358424 ◽

2018 ◽

Cited By ~ 2

Author(s):

Wannaporn Ittiprasert ◽

Victoria H. Mann ◽

Shannon E. Karinshak ◽

Avril Coghlan ◽

Gabriel Rinaldi ◽

...

Keyword(s):

Schistosoma Mansoni ◽

Genome Editing ◽

Human Macrophage ◽

Wild Type ◽

End Joining ◽

Chromosomal Breakage ◽

Knock Out ◽

Guide Rna ◽

Homology Directed Repair ◽

Non Homologous End Joining

AbstractCRISPR/Cas9 based genome editing has yet been reported in parasitic or indeed any species of the phylum Platyhelminthes. We tested this approach by targeting omega-1 (ω1) ofSchistosoma mansonias a proof of principle. This secreted ribonuclease is crucial for Th2 priming and granuloma formation, providing informative immuno-pathological readouts for programmed genome editing. Schistosome eggs were either exposed to Cas9 complexed with a synthetic guide RNA (sgRNA) complementary to exon 6 of ω1 by electroporation or transduced with pseudotyped lentivirus encoding Cas9 and the sgRNA. Some eggs were also transduced with a single stranded oligodeoxynucleotide donor transgene that encoded six stop codons, flanked by 50 nt-long 5’-and 3’-microhomology arms matching the predicted Cas9-catalyzed double stranded break (DSB) within ω1. CRISPResso analysis of amplicons spanning the DSB revealed ∼4.5% of the reads were mutated by insertions, deletions and/or substitutions, with an efficiency for homology directed repair of 0.19% insertion of the donor transgene. Transcripts encoding ω1 were reduced >80% and lysates of ω1-edited eggs displayed diminished ribonuclease activity indicative that programmed editing mutated the ω1 gene. Whereas lysates of wild type eggs polarized Th2 cytokine responses including IL-4 and IL-5 in human macrophage/T cell co-cultures, diminished levels of the cytokines followed the exposure to lysates of ω1-mutated schistosome eggs. Following injection of schistosome eggs into the tail vein of mice, the volume of pulmonary granulomas surrounding ω1-mutated eggs was 18-fold smaller than wild type eggs. Programmed genome editing was active in schistosomes, Cas9-catalyzed chromosomal breakage was repaired by homology directed repair and/or non-homologous end joining, and mutation of ω1 impeded the capacity of schistosome eggs both to drive Th2 polarization and to provoke formation of pulmonary circumoval granulomas. Knock-out of ω1 and the impaired immunological phenotype showcase the novel application of programmed gene editing in and functional genomics for schistosomes.

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The Determinant of DNA Repair Pathway Choices in Ionising Radiation-Induced DNA Double-Strand Breaks

BioMed Research International ◽

10.1155/2020/4834965 ◽

2020 ◽

Vol 2020 ◽

pp. 1-12 ◽

Cited By ~ 3

Author(s):

Lei Zhao ◽

Chengyu Bao ◽

Yuxuan Shang ◽

Xinye He ◽

Chiyuan Ma ◽

...

Keyword(s):

Dna Repair ◽

Double Strand Breaks ◽

Ionising Radiation ◽

Repair Pathway ◽

Dna Double Strand Breaks ◽

End Joining ◽

Dsb Repair ◽

Strand Breaks ◽

Dna Dsbs ◽

Repair Pathways

Ionising radiation- (IR-) induced DNA double-strand breaks (DSBs) are considered to be the deleterious DNA lesions that pose a serious threat to genomic stability. The major DNA repair pathways, including classical nonhomologous end joining, homologous recombination, single-strand annealing, and alternative end joining, play critical roles in countering and eliciting IR-induced DSBs to ensure genome integrity. If the IR-induced DNA DSBs are not repaired correctly, the residual or incorrectly repaired DSBs can result in genomic instability that is associated with certain human diseases. Although many efforts have been made in investigating the major mechanisms of IR-induced DNA DSB repair, it is still unclear what determines the choices of IR-induced DNA DSB repair pathways. In this review, we discuss how the mechanisms of IR-induced DSB repair pathway choices can operate in irradiated cells. We first briefly describe the main mechanisms of the major DNA DSB repair pathways and the related key repair proteins. Based on our understanding of the characteristics of IR-induced DNA DSBs and the regulatory mechanisms of DSB repair pathways in irradiated cells and recent advances in this field, We then highlight the main factors and associated challenges to determine the IR-induced DSB repair pathway choices. We conclude that the type and distribution of IR-induced DSBs, chromatin state, DNA-end structure, and DNA-end resection are the main determinants of the choice of the IR-induced DNA DSB repair pathway.

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Impact of chromatin context on Cas9-induced DNA double-strand break repair pathway balance

10.1101/2020.05.05.078436 ◽

2020 ◽

Cited By ~ 1

Author(s):

Ruben Schep ◽

Eva K. Brinkman ◽

Christ Leemans ◽

Xabier Vergara ◽

Ben Morris ◽

...

Keyword(s):

Genome Editing ◽

Strand Break ◽

Double Strand Break ◽

Repair Pathway ◽

End Joining ◽

Dsb Repair ◽

Dna Double Strand Break ◽

Repair Pathways ◽

Non Homologous End Joining ◽

The Impact

AbstractDNA double-strand break (DSB) repair is mediated by multiple pathways, including classical non-homologous end-joining pathway (NHEJ) and several homology-driven repair pathways. This is particularly important for Cas9-mediated genome editing, where the outcome critically depends on the pathway that repairs the break. It is thought that the local chromatin context affects the pathway choice, but the underlying principles are poorly understood. Using a newly developed multiplexed reporter assay in combination with Cas9 cutting, we systematically measured the relative activities of three DSB repair pathways as function of chromatin context in >1,000 genomic locations. This revealed that NHEJ is broadly biased towards euchromatin, while microhomology-mediated end-joining (MMEJ) is more efficient in specific heterochromatin contexts. In H3K27me3-marked heterochromatin, inhibition of the H3K27 methyltransferase EZH2 shifts the balance towards NHEJ. Single-strand templated repair (SSTR), often used for precise CRISPR editing, competes with MMEJ, and this competition is weakly associated with chromatin context. These results provide insight into the impact of chromatin on DSB repair pathway balance, and guidance for the design of Cas9-mediated genome editing experiments.

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