In the CRISPR system, a short RNA guide sequence and a variant of the Cas9 protein are introduced into cells. The RNA will bind to an identical sequence in the genome, as long as it is followed by the PAM sequence (usually GG), and mark it for subsequent cutting or modification by the Cas9 protein. However, it is wellknown to most biologists from primers and siRNAs that nucleotide binding does not require a 100% identical sequence and single base pair mismatches are often tolerated. So unsurprisingly, CRISPR modifications were soon found at “off-target” positions in the genome, at chromosomal locations that were not intended but nevertheless cut in cells.

Off-target effects have evolved from a minor scientific detail to a topic picked up by television news. This is because off-targets could potentially be fatal for the whole CRISPR technique. If CRISPR enzymes cleave random locations in the genome, this could render the technique useless for most applications. The first studies (Hsu et al. 2013) using systematic mutations of a selected target sequence found that some mutations of the target sequence are tolerated and that the nucleotides closer to the CRISPR recognition PAM site are less tolerant, similar to what was known from siRNA binding. Then, a series of studies from different labs using high-throughput sequencing of cell cultures found many more off-targets than expected, up to thousands for a single guide (Tsai