Faster and better CRISPR guide RNA design with the Crackling method

Motivation: CRISPR-Cas9 systems have become a leading tool for gene editing. However, the design of the guide RNAs used to target specific regions is not trivial. Design tools need to identify target sequences that will maximise the likelihood of obtaining the desired cut, and minimise the risk of off-target modifications. Achieving this across entire genomes is also computationally challenging. There is a clear need for a tool that can meet both objectives while remaining practical to use on large genomes. Results: Here, we present Crackling, a new method for whole-genome identification of suitable CRISPR targets. We test its performance on 12 genomes, of length 375 to 9965 megabases, and on data from validation studies. The method maximises the efficiency of the guides by combining the results of multiple scoring approaches. On experimental data, the set of guides it selects are better than those produced by existing tools. The method also incorporates a new approach for faster off-target scoring, based on Inverted Signature Slice Lists (ISSL). This approach provides a gain of an order of magnitude in speed, while preserving the same level of accuracy. Overall, this makes Crackling a faster and better method to design guide RNAs at scale. Availability: Crackling is available https://github.com/bmds-lab/Crackling under the Berkeley Software Distribution (BSD) 3-Clause license.