Comparison of Efficiency and Specificity of CRISPR-Associated (Cas) Nucleases in Plants: An Expanded Toolkit for Precision Genome Engineering. Comparison of Efficiency and Specificity of Cas Nucleases in Plants

Molecular tools adapted from bacterial CRISPR (Clustered Regulatory InterspacedShort Palindromic Repeats) systems for adaptive immunity have become widely usedfor plant genome engineering, both to investigate gene functions and to engineerdesirable traits. A number of different Cas (CRISPR-associated) nucleases are nowused but, as most studies performed to date have engineered different targets using avariety of plant species and molecular tools, it has been difficult to draw conclusionsabout the comparative performance of different nucleases. Due to the time and effortrequired to regenerate engineered plants, efficiency is critical. In addition, there havebeen several reports of mutations at sequences with less than perfect identity to thetarget. While in some plant species it is possible to remove these so-called 'off-targets'by backcrossing to a parental line, the specificity of genome engineering tools isimportant when targeting specific members of closely-related gene families, especiallywhen recent paralogues are co-located in the genome and unlikely to segregate.Specificity is also important for species that take years to reach sexual maturity or thatare clonally propagated. Here, we directly compare the efficiency and specificity of Casnucleases from different bacterial species together with engineered variants of Cas9.We find that the nucleotide content correlates with efficiency and that Cas9 fromStaphylococcus aureus is comparatively most efficient at inducing mutations. We alsodemonstrate that 'high-fidelity' variants of Cas9 can reduce off-target mutations inplants. We present these molecular tools as standardised DNA parts to facilitate theirre-use.