scholarly journals MiCas9 increases large size gene knock-in rates and reduces undesirable on-target and off-target indel edits

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Linyuan Ma ◽  
Jinxue Ruan ◽  
Jun Song ◽  
Luan Wen ◽  
Dongshan Yang ◽  
...  
Keyword(s):  
Gene Editing ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Repair Capacity ◽  
Strand Breaks ◽  
Homology Directed Repair ◽  
Insertion And Deletion ◽  
Large Size ◽  
Target Locus ◽  
Non Homologous End Joining

AbstractGene editing nuclease represented by Cas9 efficiently generates DNA double strand breaks at the target locus, followed by repair through either the error-prone non-homologous end joining or the homology directed repair pathways. To improve Cas9’s homology directed repair capacity, here we report the development of miCas9 by fusing a minimal motif consisting of thirty-six amino acids to spCas9. MiCas9 binds RAD51 through this fusion motif and enriches RAD51 at the target locus. In comparison to spCas9, miCas9 enhances double-stranded DNA mediated large size gene knock-in rates, systematically reduces off-target insertion and deletion events, maintains or increases single-stranded oligodeoxynucleotides mediated precise gene editing rates, and effectively reduces on-target insertion and deletion rates in knock-in applications. Furthermore, we demonstrate that this fusion motif can work as a “plug and play” module, compatible and synergistic with other Cas9 variants. MiCas9 and the minimal fusion motif may find broad applications in gene editing research and therapeutics.

scholarly journals Homology Directed Repair by Cas9:Donor Co-localization in Mammalian Cells

2018 ◽  
Author(s):  
Philip J.R. Roche ◽  
Heidi Gytz ◽  
Faiz Hussain ◽  
Christopher J.F. Cameron ◽  
Denis Paquette ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Gene Editing ◽  
Low Frequency ◽  
Cost Effective ◽  
Hek293 Cells ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Strand Breaks ◽  
Homology Directed Repair ◽  
Non Homologous End Joining

AbstractHomology directed repair (HDR) induced by site specific DNA double strand breaks (DSB) with CRISPR/Cas9 is a precision gene editing approach that occurs at low frequency in comparison to indel forming non homologous end joining (NHEJ). In order to obtain high HDR percentages in mammalian cells, we engineered Cas9 protein fused to a high-affinity monoavidin domain to deliver biotinylated donor DNA to a DSB site. In addition, we used the cationic polymer, polyethylenimine, to deliver Cas9 RNP-donor DNA complex into the cell. Combining these strategies improved HDR percentages of up to 90% in three tested loci (CXCR4, EMX1, and TLR) in standard HEK293 cells. Our approach offers a cost effective, simple and broadly applicable gene editing method, thereby expanding the CRISPR/Cas9 genome editing toolbox.SummaryPrecision gene editing occurs at a low percentage in mammalian cells using Cas9. Colocalization of donor with Cas9MAV and PEI delivery raises HDR occurrence.

scholarly journals Replicated chromatin curtails 53BP1 recruitment in BRCA1-proficient and -deficient cells

2020 ◽  
Author(s):  
Jone Michelena ◽  
Stefania Pellegrino ◽  
Vincent Spegg ◽  
Matthias Altmeyer
Keyword(s):  
Single Cell ◽  
Repair Pathway ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Strand Breaks ◽  
Homology Directed Repair ◽  
Dna End Resection ◽  
Non Homologous End Joining ◽  
Cellular Phenotypes ◽  
Template Dna

AbstractDNA double-strand breaks can be repaired by two competing mechanisms, non-homologous end-joining (NHEJ) and homologous recombination (HR). Whether one or the other repair pathway is favored depends on the availability of an undamaged template DNA that allows for homology-directed repair. The tumor suppressor proteins 53BP1 and BRCA1 are considered antagonistic players in this repair pathway choice, as 53BP1 restrains DNA end resection, whereas BRCA1, together with its partner protein BARD1, displaces 53BP1 from damaged replicated chromatin and promotes HR. How cells switch from a 53BP1-dominated to a BRCA1-dominated response as they progress through the cell cycle is incompletely understood. Here we reveal, using high-throughput microscopy and applying single cell normalization to control for increased genome size as cells replicate their DNA, that 53BP1 recruitment to damaged replicated chromatin is inefficient in both BRCA1-proficient and BRCA1-deficient cells, in comparison to 53BP1 accumulation at damaged unreplicated chromatin. These findings substantiate a dual switch model from a 53BP1-dominated response in unreplicated chromatin to a BRCA1-BARD1-dominated response in replicated chromatin, in which replication-coupled dilution of 53BP1’s binding mark H4K20me2 functionally cooperates with BRCA1-BARD1-mediated suppression of 53BP1 binding. More generally, we suggest that appropriate normalization of single cell data, e.g. to DNA content, provides additional layers of information, which can be critical for quantifying and interpreting cellular phenotypes.

scholarly journals Replicated chromatin curtails 53BP1 recruitment in BRCA1-proficient and BRCA1-deficient cells

2021 ◽  
Vol 4 (6) ◽  
pp. e202101023
Author(s):  
Jone Michelena ◽  
Stefania Pellegrino ◽  
Vincent Spegg ◽  
Matthias Altmeyer
Keyword(s):  
Homologous Recombination ◽  
Single Cell ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Strand Breaks ◽  
Homology Directed Repair ◽  
Non Homologous End Joining ◽  
Cellular Phenotypes ◽  
Template Dna ◽  
Cell Data

DNA double-strand breaks can be repaired by non-homologous end-joining or homologous recombination. Which pathway is used depends on the balance between the tumor suppressors 53BP1 and BRCA1 and on the availability of an undamaged template DNA for homology-directed repair. How cells switch from a 53BP1-dominated to a BRCA1-governed homologous recombination response as they progress through the cell cycle is incompletely understood. Here we reveal, using high-throughput microscopy and applying single cell normalization to control for increased genome size as cells replicate their DNA, that 53BP1 recruitment to damaged replicated chromatin is inefficient in both BRCA1-proficient and BRCA1-deficient cells. Our results substantiate a dual switch model from a 53BP1-dominated response in unreplicated chromatin to a BRCA1–BARD1–dominated response in replicated chromatin, in which replication-coupled dilution of 53BP1’s binding mark H4K20me2 functionally cooperates with BRCA1–BARD1–mediated suppression of 53BP1 binding. More generally, we suggest that appropriate normalization of single cell data, for example, to DNA content, provides additional layers of information, which can be critical for quantifying and interpreting cellular phenotypes.

scholarly journals The DNAPKcs long-range C-NHEJ complex is required for blunt DNA end joining when XLF is weakened

2021 ◽  
Author(s):  
Metztli Cisneros-Aguirre ◽  
Felicia Wednesday Lopezcolorado ◽  
Linda Jillianne Tsai ◽  
Ragini Bhargava ◽  
Jeremy M Stark
Keyword(s):  
Long Range ◽  
De Novo ◽  
Dna Double Strand Breaks ◽  
Genome Maintenance ◽  
End Joining ◽  
Strand Breaks ◽  
Homology Directed Repair ◽  
Non Homologous End Joining ◽  
Dna Ends

Canonical non-homologous end joining (C-NHEJ) factors can assemble into a long-range (LR) complex with DNA ends relatively far apart that contains DNAPKcs, XLF, XRCC4, LIG4, and the KU heterodimer and a short-range (SR) complex lacking DNAPKcs that has the ends positioned for ligation. Since the SR complex can form de novo, the role of the LR complex (i.e., DNAPKcs) for chromosomal EJ is unclear. We have examined EJ of chromosomal blunt DNA double-strand breaks (DSBs), and found that DNAPKcs is significantly less important than XLF and XRCC4 for such EJ. However, weakening XLF via disrupting interaction interfaces (e.g., disrupting the XLF homodimer interface) causes a marked requirement for DNAPKcs, its kinase activity, and its ABCDE-cluster autophosphorylation sites for blunt DSB EJ. In contrast, other aspects of genome maintenance are sensitive to DNAPKcs kinase inhibition in a manner that is not further enhanced by XLF loss (i.e., suppression of homology-directed repair and structural variants, and IR-resistance). We suggest that DNAPKcs is required to position a weakened XLF in an LR complex that can transition into a functional SR complex for blunt DSB EJ, but also has distinct functions for other aspects of genome maintenance.

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

2017 ◽  
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.

scholarly journals ATM loss leads to synthetic lethality in BRCA1 BRCT mutant mice associated with exacerbated defects in homology-directed repair

2017 ◽  
Vol 114 (29) ◽  
pp. 7665-7670 ◽  
Author(s):  
Chun-Chin Chen ◽  
Elizabeth M. Kass ◽  
Wei-Feng Yen ◽  
Thomas Ludwig ◽  
Mary Ellen Moynahan ◽  
...  
Keyword(s):  
Synthetic Lethality ◽  
Critical Role ◽  
Nonhomologous End Joining ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Atm Kinase ◽  
Strand Breaks ◽  
Homology Directed Repair ◽  
Dna Damage Signaling ◽  
Mutant Cells

BRCA1 is essential for homology-directed repair (HDR) of DNA double-strand breaks in part through antagonism of the nonhomologous end-joining factor 53BP1. The ATM kinase is involved in various aspects of DNA damage signaling and repair, but how ATM participates in HDR and genetically interacts with BRCA1 in this process is unclear. To investigate this question, we used the Brca1S1598F mouse model carrying a mutation in the BRCA1 C-terminal domain of BRCA1. Whereas ATM loss leads to a mild HDR defect in adult somatic cells, we find that ATM inhibition leads to severely reduced HDR in Brca1S1598F cells. Consistent with a critical role for ATM in HDR in this background, loss of ATM leads to synthetic lethality of Brca1S1598F mice. Whereas both ATM and BRCA1 promote end resection, which can be regulated by 53BP1, 53bp1 deletion does not rescue the HDR defects of Atm mutant cells, in contrast to Brca1 mutant cells. These results demonstrate that ATM has a role in HDR independent of the BRCA1–53BP1 antagonism and that its HDR function can become critical in certain contexts.

scholarly journals Genetic dissection of vertebrate 53BP1: A major role in non-homologous end joining of DNA double strand breaks

DNA Repair ◽  
2006 ◽  
Vol 5 (6) ◽  
pp. 741-749 ◽  
Author(s):  
Kyoko Nakamura ◽  
Wataru Sakai ◽  
Takuo Kawamoto ◽  
Ronan T. Bree ◽  
Noel F. Lowndes ◽  
...  
Keyword(s):  
Double Strand Breaks ◽  
Genetic Dissection ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Strand Breaks ◽  
Non Homologous End Joining

scholarly journals Beyond DNA Repair: DNA-PKcs in Tumor Metastasis, Metabolism and Immunity

Cancers ◽  
2020 ◽  
Vol 12 (11) ◽  
pp. 3389
Author(s):  
Haitang Yang ◽  
Feng Yao ◽  
Thomas M. Marti ◽  
Ralph A. Schmid ◽  
Ren-Wang Peng
Keyword(s):  
Tumor Metastasis ◽  
Immune Escape ◽  
Critical Role ◽  
Large Body ◽  
Tumor Development ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Strand Breaks ◽  
Dependent Protein ◽  
Non Homologous End Joining

The DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is a key component of the DNA-PK complex that has a well-characterized function in the non-homologous end-joining repair of DNA double-strand breaks. Since its identification, a large body of evidence has demonstrated that DNA-PKcs is frequently overexpressed in cancer, plays a critical role in tumor development and progression, and is associated with poor prognosis of cancer patients. Intriguingly, recent studies have suggested novel functions beyond the canonical role of DNA-PKcs, which has transformed the paradigm of DNA-PKcs in tumorigenesis and has reinvigorated the interest to target DNA-PKcs for cancer treatment. In this review, we update recent advances in DNA-PKcs, in particular the emerging roles in tumor metastasis, metabolic dysregulation, and immune escape. We further discuss the possible molecular basis that underpins the pleiotropism of DNA-PKcs in cancer. Finally, we outline the biomarkers that may predict the therapeutic response to DNA-PKcs inhibitor therapy. Understanding the functional repertoire of DNA-PKcs will provide mechanistic insights of DNA-PKcs in malignancy and, more importantly, may revolutionize the design and utility of DNA-PKcs-based precision cancer therapy.

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