Peer-Reviewed Journal Details
Mandatory Fields
Hollywood, JA,Lee, CM,Scallan, MF,Harrison, PT
2016
August
Scientific Reports
Analysis of gene repair tracts from Cas9/gRNA double-stranded breaks in the human CFTR gene
Validated
Optional Fields
PLURIPOTENT STEM-CELLS ZINC-FINGER NUCLEASES CYSTIC-FIBROSIS HOMOLOGOUS RECOMBINATION SACCHAROMYCES-CEREVISIAE MAMMALIAN-CELLS GENOME DNA CAS9 RNA
6
To maximise the efficiency of template-dependent gene editing, most studies describe programmable and/or RNA-guided endonucleases that make a double-stranded break at, or close to, the target sequence to be modified. The rationale for this design strategy is that most gene repair tracts will be very short. Here, we describe a CRISPR Cas9/gRNA selection-free strategy which uses deep sequencing to characterise repair tracts from a donor plasmid containing seven nucleotide differences across a 216 bp target region in the human CFTR gene. We found that 90% of the template-dependent repair tracts were >100 bp in length with equal numbers of uni-directional and bi-directional repair tracts. The occurrence of long repair tracts suggests that a single gRNA could be used with variants of the same template to create or correct specific mutations within a 200 bp range, the size of similar to 80% of human exons. The selection-free strategy used here also allowed detection of non-homologous end joining events in many of the homology-directed repair tracts. This indicates a need to modify the donor, possibly by silent changes in the PAM sequence, to prevent creation of a second double-stranded break in an allele that has already been correctly edited by homology-directed repair.
10.1038/srep32230
Grant Details