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

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.