Work

Highly efficient genome editing for single-base substitutions using optimized ssODNs with Cas9-RNPs

Year: 2019

Type: article

Abstract: Target-specific genome editing using engineered nucleases has become widespread in various fields. Long gene knock-in and single-base substitutions can be performed by homologous recombination (HR), but the efficiency is usually very low. To improve the efficiency of knock-in with single-stranded oligo DNA nucleotides (ssODNs), we have investigated optimal design of ssODNs in terms of the blocking mutation, orientation, size, and length of homology arms to explore the optimal parameters of ssODN design using reporter systems for the detection of single-base substitutions. We have also investigated the difference in knock-in efficiency among the delivery forms and methods of Cas9 and sgRNA. The knock-in efficiencies for optimized ssODNs were much higher than those for ssODNs with no blocking mutation. We have also demonstrated that Cas9 protein/sgRNA ribonucleoprotein complexes (Cas9-RNPs) can dramatically reduce the re-cutting of the edited sites.

Source: Scientific Reports

DOI: 10.1038/s41598-019-41121-4

Authors: Sachiko Okamoto, Yasunori Amaishi, Izumi Maki, Tatsuji Enoki, Junichi Mineno

Institution: Takara BIO Inc. (Japan)

Language: English

Topic: CRISPR and Genetic Engineering

Subfield: Molecular Biology

Field: Biochemistry, Genetics and Molecular Biology

Domain: Life Sciences

Open Access status: gold

Estimated APC paid: $1,790

OpenAlex ID: W2921058482