CRISPR/Cas9 has rapidly become the wonder tool of cell biology, giving researchers the ability to slice and dice genomes with effortless precision. Except that is not always the case. To gauge what affects the accuracy and effectiveness of CRISPR/Cas9, ASCB member Thoru Pederson and colleagues in his lab at the University of Massachusetts Medical School, Worcester, and at the University of Central Florida, took a closer look at its dual components, the non-specific endonuclease Cas9, which cleaves DNA strands and the synthetic guide RNA (gRNA) of ~20 nucleotides that is the targeting device. The ability of researchers to customize the synthetic guide RNA to home in on so many different targets of choice is what gives CRPISPR/Cas9 its reputation as the Swiss Army knife of genomics.
Pederson et al report that a single mismatch between the guide RNA and the targeted sequence can severely limit the effectiveness of the whole procedure. In some cases, a single mismatched nucleotide shortened the time on target for the CRISPR/Cas9 complex from three hours to two minutes. The researchers show that the longer the time of target “residence,” the more effective CRISPR will be in delivering the desired genome edits.