Dataset Information

Targeted homology-directed repair in blood stem and progenitor cells with CRISPR nanoformulations.

ABSTRACT: Ex vivo CRISPR gene editing in haematopoietic stem and progenitor cells has opened potential treatment modalities for numerous diseases. The current process uses electroporation, sometimes followed by virus transduction. While this complex manipulation has resulted in high levels of gene editing at some genetic loci, cellular toxicity was observed. We have developed a CRISPR nanoformulation based on colloidal gold nanoparticles with a unique loading design capable of cellular entry without the need for electroporation or viruses. This highly monodispersed nanoformulation avoids lysosomal entrapment and localizes to the nucleus in primary human blood progenitors without toxicity. Nanoformulation-mediated gene editing is efficient and sustained with different CRISPR nucleases at multiple loci of therapeutic interest. The engraftment kinetics of nanoformulation-treated primary cells in humanized mice are better relative to those of non-treated cells, with no differences in differentiation. Here we demonstrate non-toxic delivery of the entire CRISPR payload into primary human blood progenitors.

SUBMITTER: Shahbazi R

PROVIDER: S-EPMC6754292 | biostudies-literature | 2019 Oct

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Targeted homology-directed repair in blood stem and progenitor cells with CRISPR nanoformulations.

Shahbazi Reza R Sghia-Hughes Gabriella G Reid Jack L JL Kubek Sara S Haworth Kevin G KG Humbert Olivier O Kiem Hans-Peter HP Adair Jennifer E JE

Nature materials 20190527 10

Ex vivo CRISPR gene editing in haematopoietic stem and progenitor cells has opened potential treatment modalities for numerous diseases. The current process uses electroporation, sometimes followed by virus transduction. While this complex manipulation has resulted in high levels of gene editing at some genetic loci, cellular toxicity was observed. We have developed a CRISPR nanoformulation based on colloidal gold nanoparticles with a unique loading design capable of cellular entry without the n ...[more]

PMID: 31133730

Similar Datasets

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

Targeted homology-directed repair in blood stem and progenitor cells with CRISPR nanoformulations.

Publications

Targeted homology-directed repair in blood stem and progenitor cells with CRISPR nanoformulations.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets