Straightforward Delivery of Linearized Double-Stranded DNA Encoding sgRNA and Donor DNA for the Generation of Single Nucleotide Variants Based on the CRISPR/Cas9 System

Soyeong Jun, Hyeonseob Lim, Hoon Jang, Wookjae Lee, Jinwoo Ahn, Ji Hyun Lee, Duhee Bang

Research output: Contribution to journalArticle

Abstract

CRISPR/Cas9 for genome editing requires delivery of a guide RNA sequence and donor DNA for targeted homologous recombination. Typically, single-stranded oligodeoxynucleotide, serving as the donor template, and a plasmid encoding guide RNA are delivered as two separate components. However, in the multiplexed generation of single nucleotide variants, this two-component delivery system is limited by difficulty of delivering a matched pair of sgRNA and donor DNA to the target cell. Here, we describe a novel codelivery system called "sgR-DNA" that uses a linearized double-stranded DNA consisting of donor DNA component and a component encoding sgRNA. Our sgR-DNA-based method is simple to implement because it does not require cloning steps. We also report the potential of our delivery system to generate multiplex genomic substitutions in Escherichia coli and human cells.

Original languageEnglish
Pages (from-to)1651-1659
Number of pages9
JournalACS Synthetic Biology
Volume7
Issue number7
DOIs
Publication statusPublished - 2018 Jul 20

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Clustered Regularly Interspaced Short Palindromic Repeats
Nucleotides
DNA
Guide RNA
RNA
Oligodeoxyribonucleotides
Cloning
Homologous Recombination
Escherichia coli
Organism Cloning
Plasmids
Substitution reactions
Genes
Cells

All Science Journal Classification (ASJC) codes

  • Biomedical Engineering
  • Biochemistry, Genetics and Molecular Biology (miscellaneous)

Cite this

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Straightforward Delivery of Linearized Double-Stranded DNA Encoding sgRNA and Donor DNA for the Generation of Single Nucleotide Variants Based on the CRISPR/Cas9 System. / Jun, Soyeong; Lim, Hyeonseob; Jang, Hoon; Lee, Wookjae; Ahn, Jinwoo; Lee, Ji Hyun; Bang, Duhee.

In: ACS Synthetic Biology, Vol. 7, No. 7, 20.07.2018, p. 1651-1659.

Research output: Contribution to journalArticle

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AU - Lee, Ji Hyun

AU - Bang, Duhee

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