Use of PCR Fragments with Short Homology Arms for Genome Editing

Unmet Need
The RNA-guided DNA endonuclease, Cas9, has emerged as a powerful new tool for genome engineering. Cas9 creates targeted ‘double-strand breaks’ (DSBs) in the genome. Knock-in of specific mutations (precision genome editing) requires ‘homology-directed repair’ (HDR) of the DSB by synthetic donor DNAs containing the desired edits, but HDR has shown considerable variability in efficiency. Guidelines which improve HDR design and integration success would help move forward the field of genome editing.
 
Technology Overview
Hopkins researchers developed a novel method for genome editing in mammalian cells that uses linear donor DNAs to introduce precise edits in the genome. Data demonstrated that PCR fragments containing edits up to 1kb require only 35bp homology arms to initiate repair of Cas9-induced double-strand breaks in human cells and mouse embryos. Furthermore, researchers experimentally determined donor DNA design rules that maximize the recovery of edits without cloning or selection. These findings enable rational design of synthetic donor DNAs for efficient genome editing.
 
Stage of Development
Hopkins researchers have demonstrated that PCR fragments with short homologous arms are efficient donors for genome editing in C. elegans, mouse embryos and human cells.

Publications
Paix A, et al. PNAS 2017;114(50): E10745–E10754.

Category(s):