Generation of gene-edited rats by delivery of CRISPR/Cas9 protein and donor DNA into intact zygotes using electroporation

Remy, Séverine; Chenouard, Vanessa; Tesson, Laurent; Usal, Claire; Ménoret, Séverine; Brusselle, Lucas; Heslan, Jean‐Marie; Nguyen, Tin; Bellien, Jérémy; Mérot, Jean; Cian, Anne De; Giovannangéli, Carine; Concordet, Jean‐Paul; Anegón, Ignacio

doi:10.1038/s41598-017-16328-y

Cited by 81 publications

(71 citation statements)

References 37 publications

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“…Up to 500 presumptive zygotes can be microinjected one by one into the cytoplasm (2e5 pl of injection mix) in each session. On the other hand, electroporation has recently been described as an alternative approach to deliver small CRISPR reagents into mouse and rat zygotes [30,31] and it is currently used in our laboratory [28,32]. This procedure avoids the technically demanding microinjection technique allowing a high throughput scheme in the laboratory.…”

Section: Embryo Manipulationmentioning

confidence: 99%

CRISPR in livestock: From editing to printing

et al. 2020

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a b s t r a c tPrecise genome editing of large animals applied to livestock and biomedicine is nowadays possible since the CRISPR revolution. This review summarizes the latest advances and the main technical issues that determine the success of this technology. The pathway from editing to printing, from engineering the genome to achieving the desired animals, does not always imply an easy, fast and safe journey. When applied in large animals, CRISPR involves time-and cost-consuming projects, and it is mandatory not only to choose the best approach for genome editing, but also for embryo production, zygote microinjection or electroporation, cryopreservation and embryo transfer. The main technical refinements and most frequent questions to improve this disruptive biotechnology in large animals are presented. In addition, we discuss some CRISPR applications to enhance livestock production in the context of a growing global demand of food, in terms of increasing efficiency, reducing the impact of farming on the environment, enhancing pest control, animal welfare and health. The challenge is no longer technical. Controversies and consensus, opportunities and threats, benefits and risks, ethics and science should be reconsidered to enter into the CRISPR era.

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Section: Embryo Manipulationmentioning

confidence: 99%

CRISPR in livestock: From editing to printing

et al. 2020

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“…The high fidelity of the homologous recombination pathway can be used to create controlled insertions, deletions and substitution of a single nucleotide or large tracks of genomic DNA by HDR. Although reported in other species [17][18][19] , current literature is scarce for CRISPR assisted ssODN-mediated homologous recombination in ruminant species (i.e., sheep, goats and cattle). Recently, Williams et al 5 reported for the first time the effectiveness of this tool to induce a point mutation in sheep, and Eaton et al 20 described the generation of homozygotes lambs by the insertion of a human mutation into the orthologous sheep locus.…”

Section: Discussionmentioning

confidence: 99%

Otoferlin gene editing in sheep via CRISPR-assisted ssODN-mediated Homology Directed Repair

Menchaca

Santos-Neto

Souza-Neves

et al. 2020

Sci Rep

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Different mutations of the OTOF gene, encoding for otoferlin protein expressed in the cochlear inner hair cells, induces a form of deafness that is the major cause of nonsyndromic recessive auditory neuropathy spectrum disorder in humans. We report the generation of the first large animal model of OTOF mutations using the CRISPR system associated with different Cas9 components (mRNA or protein) assisted by single strand oligodeoxynucleotides (ssODN) to induce homology-directed repair (HDR). Zygote microinjection was performed with two sgRNA targeting exon 5 and 6 associated to Cas9 mRNA or protein (RNP) at different concentrations in a mix with an ssODN template targeting HDR in exon 5 containing two STOP sequences. A total of 73 lambs were born, 13 showing indel mutations (17.8%), 8 of which (61.5%) had knock-in mutations by HDR. Higher concentrations of Cas9-RNP induced targeted mutations more effectively, but negatively affected embryo survival and pregnancy rate. This study reports by the first time the generation of OTOF disrupted sheep, which may allow better understanding and development of new therapies for human deafness related to genetic disorders. These results support the use of CRISPR/Cas system assisted by ssODN as an effective tool for gene editing in livestock.

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“…Germline editing with CRISPR-Cas9 has proven remarkably useful for genetically modifying animals (Li et al, 2013;Chapman et al, 2015;Remy et al, 2017). However, germline modifications can produce undesirable developmental phenotypes providing little benefit for studies interrogating gene function in adult animals.…”

Section: Regulable Gene Editing With Inducible Crispr-cas Systemsmentioning

confidence: 99%

Genetically Engineering the Nervous System with CRISPR-Cas

Sandoval

Elahi

Ploski

2020

eNeuro

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The multitude of neuronal subtypes and extensive interconnectivity of the mammalian brain presents a substantial challenge to those seeking to decipher its functions. While the molecular mechanisms of several neuronal functions remain poorly characterized, advances in next-generation sequencing (NGS) and gene-editing technology have begun to close this gap. The clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein (CRISPR-Cas) system has emerged as a powerful genetic tool capable of manipulating the genome of essentially any organism and cell type. This technology has advanced our understanding of complex neurologic diseases by enabling the rapid generation of novel, disease-relevant in vitro and transgenic animal models. In this review, we discuss recent developments in the rapidly accelerating field of CRISPR-mediated genome engineering. We begin with an overview of the canonical function of the CRISPR platform, followed by a functional review of its many adaptations, with an emphasis on its applications for

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Generation of gene-edited rats by delivery of CRISPR/Cas9 protein and donor DNA into intact zygotes using electroporation

Cited by 81 publications

References 37 publications

CRISPR in livestock: From editing to printing

CRISPR in livestock: From editing to printing

Otoferlin gene editing in sheep via CRISPR-assisted ssODN-mediated Homology Directed Repair

Genetically Engineering the Nervous System with CRISPR-Cas

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