User:Vusao/Off-target genome editing

GUIDE-Seq
Genome-wide, Unbiased Identification of DSBs Enabled by Sequencing (GUIDE-Seq) is a method that allows for the unbiased in vitro detection of off-target editing events caused by CRISPR/Cas9 as well as other RNA-guided nucleases (RGN) in living cells. Conceived to work in concert next-gen sequencing platforms, the technique relies on the integration of a blunt, double-stranded oligodeoxynucleotide (dsODN) that has been phosphothiorated on two of the phosphate linkages on the 5' end of both strands. The dsODN cassette integrates into any site in the genome that contains a double-stranded break (DSB). This means that along with the target and off-target sites that may exist as a result of the activity of a nuclease, the dsODN cassette will also integrate into any spurious sites in the genome that have a DSB. This makes it critical to have a dsODN only condition that controls for errant and naturally occurring DSBs, and is required to use the GUIDE-seq bioinformatic pipeline.

After integration of the dsODN cassette, genomic DNA (gDNA) is extracted from the cell culture and sheared to 500bp fragments via sonication. The resulting sheared gDNA undergoes end-repair and adapter ligation. From here, DNA specifically containing the dsODN insert is amplified via two rounds of polymerase chain reaction (PCR) that proceeds in a unidirectional manner starting from the primers that are complementary to the dsODN. This allows for reads of the adjacent sequences, both the sense and anti-sense strands, flanking the insert. The final product is a panoply of amplicons, describing the DSB distribution, containing indices for sample differentiation, p5 and p7 Illumina flow-cell adapters, and the sequences flanking the dsODN cassette.

GUIDE-Seq is able to achieve detection of rare DSBs that occur with a 0.1% frequency, however this may be as a result of the limitations of next-gen sequencing platforms. The greater the depth of reads an instrument is able to achieve, the better it can detect rarer events. Additionally, GUIDE-Seq is able to detect sites not predicted by the "in silico" methods which often will predict sites based on sequence similarity and percent mismatch. There have been cases of some RGNs not resulting in any off-target sites as well, suggesting that some RGNs may have no associated off-targets. GUIDE-Seq has been used to show that engineered variants of Cas9 can have reduced off-target effects.

A caveat of GUIDE-Seq is that the off-target sites generated can be cell line specific. This would suggest that it would pertinent for researchers to test multiple cell lines to validate efficacy and accuracy.