Abstract
The Cas9 endonuclease can be targeted to genomic sequences by programming the sequence of an associated single guide RNA (sgRNA). For unknown reasons, the activity of these Cas9–sgRNA combinations varies widely at different genomic loci and in different cell types. Thus, disrupting genes in polyploid cell lines or when using poorly performing sgRNAs can require extensive downstream screening to identify homozygous clones. Here we find that non-homologous single-stranded DNA greatly stimulates Cas9-mediated gene disruption in the absence of homology-directed repair. This stimulation increases the frequency of clones with homozygous gene disruptions and rescues otherwise ineffective sgRNAs. The molecular outcome of enhanced gene disruption depends upon cellular context, stimulating deletion of genomic sequence or insertion of non-homologous DNA at the edited locus in a cell line specific manner. Non-homologous DNA appears to divert cells towards error-prone instead of error-free repair pathways, dramatically increasing the frequency of gene disruption.
Highlights
The Cas[9] endonuclease can be targeted to genomic sequences by programming the sequence of an associated single guide RNA
While investigating parameters to optimize rates of homologydirected repair (HDR) during genome editing experiments, we found that the frequency of error-prone repair outcomes tended to increase when single-stranded HDR donor DNA was present in the editing reaction[9]
We found that the addition of a 127-mer single-stranded DNA oligonucleotide derived from BFP, which lacks homology to the targeted locus and whose sequence is absent in the human genome, markedly increased the appearance of insertions and deletions, as measured by a T7E1 assay (Fig. 1a)
Summary
The Cas[9] endonuclease can be targeted to genomic sequences by programming the sequence of an associated single guide RNA (sgRNA). Cas9–sgRNA combinations vary greatly in apparent cellular activity, from completely inactive to nearly 100% efficient, which can complicate experiments in which functional concerns restrict the genomic location to be targeted[3,4,5,6] This variable activity has been attributed to differences in Cas9’s ability to use sgRNAs of various sequences[7,8], but differences in the activity of a given sgRNA between cell lines and organisms suggests that Cas[9] introduction efficiency and location- or organism-specific modulation of DNA repair outcomes may influence observed sgRNA efficiency. Non-homologous DNA appears to drive cells towards error-prone instead of error-free repair pathways, thereby increasing the frequency of sequence disruption during genome editing
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