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

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.

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Beta Human Papillomavirus 8E6 Attenuates Non-Homologous End Joining by Hindering DNA-PKcs Activity

Cancers ◽

10.3390/cancers12092356 ◽

2020 ◽

Vol 12 (9) ◽

pp. 2356

Author(s):

Changkun Hu ◽

Taylor Bugbee ◽

Monica Gamez ◽

Nicholas A. Wallace

Keyword(s):

Dna Repair ◽

Viral Infections ◽

Dna Lesions ◽

Human Papillomaviruses ◽

Molecular Evidence ◽

Repair Pathway ◽

End Joining ◽

Dsb Repair ◽

Dependent Protein ◽

Non Homologous End Joining

Cutaneous viral infections occur in a background of near continual exposure to environmental genotoxins, like UV radiation in sunlight. Failure to repair damaged DNA is an established driver of tumorigenesis and substantial cellular resources are devoted to repairing DNA lesions. Beta-human papillomaviruses (β-HPVs) attenuate DNA repair signaling. However, their role in human disease is unclear. Some have proposed that β-HPV promotes tumorigenesis, while others suggest that β-HPV protects against skin cancer. Most of the molecular evidence that β-HPV impairs DNA repair has been gained via characterization of the E6 protein from β-HPV 8 (β-HPV 8E6). Moreover, β-HPV 8E6 hinders DNA repair by binding and destabilizing p300, a transcription factor for multiple DNA repair genes. By reducing p300 availability, β-HPV 8E6 attenuates a major double strand DNA break (DSB) repair pathway, homologous recombination. Here, β-HPV 8E6 impairs another DSB repair pathway, non-homologous end joining (NHEJ). Specifically, β-HPV 8E6 acts by attenuating DNA-dependent protein kinase (DNA-PK) activity, a critical NHEJ kinase. This includes DNA-PK activation and the downstream of steps in the pathway associated with DNA-PK activity. Notably, β-HPV 8E6 inhibits NHEJ through p300 dependent and independent means. Together, these data expand the known genome destabilizing capabilities of β-HPV 8E6.

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EP526 Inactivating mutations of theIKgene inhibit endometrial cancer progression through regulating mitosis and non-homologous end joining DNA repair pathway

10.1136/ijgc-2019-esgo.584 ◽

2019 ◽

Author(s):

C Gao ◽

G Jin ◽

Y Wang ◽

ME Forbes ◽

Y Yan ◽

...

Keyword(s):

Dna Repair ◽

Endometrial Cancer ◽

Cancer Progression ◽

Repair Pathway ◽

End Joining ◽

Non Homologous End Joining ◽

Inactivating Mutations

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Faculty Opinions recommendation of Interactome analysis identifies a new paralogue of XRCC4 in non-homologous end joining DNA repair pathway.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.725352916.793503936 ◽

2015 ◽

Author(s):

Susan Lees-Miller

Keyword(s):

Dna Repair ◽

Repair Pathway ◽

End Joining ◽

Non Homologous End Joining

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The (Lack of) DNA Double-Strand Break Repair Pathway Choice During V(D)J Recombination

Frontiers in Genetics ◽

10.3389/fgene.2021.823943 ◽

2022 ◽

Vol 12 ◽

Author(s):

Alice Libri ◽

Timea Marton ◽

Ludovic Deriano

Keyword(s):

Dna Repair ◽

Gene Diversity ◽

Receptor Gene ◽

Strand Break ◽

Repair Pathway ◽

Dna Double Strand Breaks ◽

End Joining ◽

Homology Directed Repair ◽

Pathway Choice ◽

Non Homologous End Joining

DNA double-strand breaks (DSBs) are highly toxic lesions that can be mended via several DNA repair pathways. Multiple factors can influence the choice and the restrictiveness of repair towards a given pathway in order to warrant the maintenance of genome integrity. During V(D)J recombination, RAG-induced DSBs are (almost) exclusively repaired by the non-homologous end-joining (NHEJ) pathway for the benefit of antigen receptor gene diversity. Here, we review the various parameters that constrain repair of RAG-generated DSBs to NHEJ, including the peculiarity of DNA DSB ends generated by the RAG nuclease, the establishment and maintenance of a post-cleavage synaptic complex, and the protection of DNA ends against resection and (micro)homology-directed repair. In this physiological context, we highlight that certain DSBs have limited DNA repair pathway choice options.

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Distinct functions of POT1 proteins contribute to the regulation of telomerase recruitment to telomeres

Nature Communications ◽

10.1038/s41467-021-25799-7 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Peili Gu ◽

Shuting Jia ◽

Taylor Takasugi ◽

Valerie M. Tesmer ◽

Jayakrishnan Nandakumar ◽

...

Keyword(s):

Amino Acids ◽

Dna Damage ◽

Dna Repair ◽

Telomere Length ◽

Repair Pathway ◽

End Joining ◽

C Terminus ◽

Dna Damage Responses ◽

Non Homologous End Joining

AbstractHuman shelterin components POT1 and TPP1 form a stable heterodimer that protects telomere ends from ATR-dependent DNA damage responses and regulates telomerase-dependent telomere extension. Mice possess two functionally distinct POT1 proteins. POT1a represses ATR/CHK1 DNA damage responses and the alternative non-homologous end-joining DNA repair pathway while POT1b regulates C-strand resection and recruits the CTC1-STN1-TEN1 (CST) complex to telomeres to mediate C-strand fill-in synthesis. Whether POT1a and POT1b are involved in regulating the length of the telomeric G-strand is unclear. Here we demonstrate that POT1b, independent of its CST function, enhances recruitment of telomerase to telomeres through three amino acids in its TPP1 interacting C-terminus. POT1b thus coordinates the synthesis of both telomeric G- and C-strands. In contrast, POT1a negatively regulates telomere length by inhibiting telomerase recruitment to telomeres. The identification of unique amino acids between POT1a and POT1b helps us understand mechanistically how human POT1 switches between end protective functions and promoting telomerase recruitment.

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