Dataset Information

A Consensus Model of Homology-Directed Repair Initiated by CRISPR/Cas Activity.

ABSTRACT: The mechanism of action of ssODN-directed gene editing has been a topic of discussion within the field of CRISPR gene editing since its inception. Multiple comparable, but distinct, pathways have been discovered for DNA repair both with and without a repair template oligonucleotide. We have previously described the ExACT pathway for oligo-driven DNA repair, which consisted of a two-step DNA synthesis-driven repair catalyzed by the simultaneous binding of the repair oligonucleotide (ssODN) upstream and downstream of the double-strand break. In order to better elucidate the mechanism of ExACT-based repair, we have challenged the assumptions of the pathway with those outlines in other similar non-ssODN-based DNA repair mechanisms. This more comprehensive iteration of the ExACT pathway better described the many different ways where DNA repair can occur in the presence of a repair oligonucleotide after CRISPR cleavage, as well as how these previously distinct pathways can overlap and lead to even more unique repair outcomes.

SUBMITTER: Bloh K

PROVIDER: S-EPMC8067812 | biostudies-literature | 2021 Apr

REPOSITORIES: biostudies-literature

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A Consensus Model of Homology-Directed Repair Initiated by CRISPR/Cas Activity.

Bloh Kevin K Rivera-Torres Natalia N

International journal of molecular sciences 20210407 8

The mechanism of action of ssODN-directed gene editing has been a topic of discussion within the field of CRISPR gene editing since its inception. Multiple comparable, but distinct, pathways have been discovered for DNA repair both with and without a repair template oligonucleotide. We have previously described the ExACT pathway for oligo-driven DNA repair, which consisted of a two-step DNA synthesis-driven repair catalyzed by the simultaneous binding of the repair oligonucleotide (ssODN) upstre ...[more]

PMID: 33917142

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Project description:BackgroundMicroalgae are considered as a sustainable feedstock for the production of biofuels and other value-added compounds. In particular, Nannochloropsis spp. stand out from other microalgal species due to their capabilities to accumulate both triacylglycerol (TAG) and polyunsaturated fatty acids (PUFAs). However, the commercialization of microalgae-derived products is primarily hindered by the high production costs compared to less sustainable alternatives. Efficient genome editing techniques leading to effective metabolic engineering could result in strains with enhanced productivities of interesting metabolites and thereby reduce the production costs. Competent CRISPR-based genome editing techniques have been reported in several microalgal species, and only very recently in Nannochloropsis spp. (2017). All the reported CRISPR-Cas-based systems in Nannochloropsis spp. rely on plasmid-borne constitutive expression of Cas9 and a specific guide, combined with repair of double-stranded breaks (DSB) by non-homologous end joining (NHEJ) for the target gene knockout.ResultsIn this study, we report for the first time an alternative approach for CRISPR-Cas-mediated genome editing in Nannochloropsis sp.; the Cas ribonucleoproteins (RNP) and an editing template were directly delivered into microalgal cells via electroporation, making Cas expression dispensable and homology-directed repair (HDR) possible with high efficiency. Apart from widely used SpCas9, Cas12a variants from three different bacterium were used for this approach. We observed that FnCas12a from Francisella novicida generated HDR-based targeted mutants with highest efficiency (up to 93% mutants among transformants) while AsCas12a from Acidaminococcus sp. resulted in the lowest efficiency. We initially show that the native homologous recombination (HR) system in N. oceanica IMET1 is not efficient for easy isolation of targeted mutants by HR. Cas9/sgRNA RNP delivery greatly enhanced HR at the target site, generating around 70% of positive mutant lines.ConclusionWe show that the delivery of Cas RNP by electroporation can be an alternative approach to the presently reported plasmid-based Cas9 method for generating mutants of N. oceanica. The co-delivery of Cas-RNPs along with a dsDNA repair template efficiently enhanced HR at the target site, resulting in a remarkable higher percentage of positive mutant lines. Therefore, this approach can be used for efficient generation of targeted mutants in Nannochloropsis sp. In addition, we here report the activity of several Cas12a homologs in N. oceanica IMET1, identifying FnCas12a as the best performer for high efficiency targeted genome editing.

Dataset Information

A Consensus Model of Homology-Directed Repair Initiated by CRISPR/Cas Activity.

Publications

A Consensus Model of Homology-Directed Repair Initiated by CRISPR/Cas Activity.

Similar Datasets

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