Abstract
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 ~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.
Highlights
The foundations of contemporary gene editing were laid with two key observations from a plasmid-based study of DNA homologous recombination-dependent DNA repair pathways
To measure the ability of the CRISPR/Cas[9] system to create double-stranded break (DSB) in the CFTR gene, cystic fibrosis tracheal epithelial (CFTE) cells were co-transfected with plasmids encoding Cas[9] and either guide RNA (gRNA)-in[10] or gRNA-ex[11]
Seventy-two hours post-transfection genomic DNA was analysed by deep sequencing to determine the frequency and size of deletions caused by non-homologous end joining (NHEJ) repair of Cas9/ gRNA-induced DSBs
Summary
The foundations of contemporary gene editing were laid with two key observations from a plasmid-based study of DNA homologous recombination-dependent DNA repair pathways. By using donor plasmids with up to eight single nucleotide changes, each creating a novel restriction site without changing the coding sequence of the NeoR gene across a 745 bp region, they were able to map the extent of repair tract length; analysis of 80 recombinant clones revealed that the majority (75%) of repair tracts were 12 bp or shorter[4] They reported a small number of very long tracts in the same study (up to 511 bp), this seminal analysis of repair tracts, along with the original observation from plasmid studies that a DSB close to the target site enhances recombination frequency, have become widely cited in gene editing studies as the rationale for creating DSBs at or close to the target sequence to be modified when using a donor sequence for homology-directed repair (HDR) with programmable and/or RNA-guided endonucleases[5,6,7,8,9]. Use of a selection-free system allowed us to detect and characterise template-independent non-homologous end joining (NHEJ) events that occur both independently, and in combination with HDR events
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