Abstract
Precision genomic alterations largely rely on homology directed repair (HDR), but targeting without homology using the non-homologous end-joining (NHEJ) pathway has gained attention as a promising alternative. Previous studies demonstrated precise insertions formed by the ligation of donor DNA into a targeted genomic double-strand break in both dividing and non-dividing cells. Here, we demonstrate the use of NHEJ repair to replace genomic segments with donor sequences; we name this method ‘Replace’ editing (Rational end-joining protocol delivering a targeted sequence exchange). Using CRISPR/Cas9, we create two genomic breaks and ligate a donor sequence in-between. This exchange of a genomic for a donor sequence uses neither microhomology nor homology arms. We target four loci in cell lines and show successful exchange of exons in 16–54% of human cells. Using linear amplification methods and deep sequencing, we quantify the diversity of outcomes following Replace editing and profile the ligated interfaces. The ability to replace exons or other genomic sequences in cells not efficiently modified by HDR holds promise for both basic research and medicine.
Highlights
RNA-guided nucleases [1,2,3] have rapidly become foundational tools in facilitating genomic manipulations [4,5]
The canonical non-homologous end-joining (NHEJ) pathway is traditionally viewed as error prone and relegated to disrupting gene function by inducing small insertions and deletions (InDels) during double-strand break (DSB) repair
The highfidelity aspects of NHEJ repair are often underappreciated as mutant InDels are observed, whereas non-mutagenic repair is indistinguishable from the original allele [14]
Summary
RNA-guided nucleases [1,2,3] have rapidly become foundational tools in facilitating genomic manipulations [4,5]. These nucleases target specific genomic loci and form a double-strand break (DSB). Specific genomic changes are made using homology directed repair (HDR) [6,7] with exogenously introduced DNA containing flanking sequences homologous to the targeted locus. Non-mutagenic repair by NHEJ reforms the Cas target site allowing for continued DSB formation. This may result in a final genomic population containing majority InDels despite NHEJ repair being predominately error-free
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