Publications

Rapid Whole-Genome Identification of High Quality CRISPR Guide RNAs with the Crackling Method

Jake Bradford, Timothy Chappell, Dimitri Perrin

The CRISPR Journal, 2022

The design of CRISPR-Cas9 guide RNAs is not trivial and is a computationally demanding task. Design tools need to identify target sequences that will maximize the likelihood of obtaining the desired cut, while minimizing off-target risk. There is a need for a tool that can meet both objectives while remaining practical to use on large genomes. In this study, we present Crackling, a new method that is more suitable for meeting these objectives. We test its performance on 12 genomes and on data from validation studies. Crackling maximizes guide efficiency by combining multiple scoring approaches. On experimental data, the guides it selects are better than those selected by others. It also incorporates Inverted Signature Slice Lists (ISSL) for faster off-target scoring. ISSL provides a gain of an order of magnitude in speed compared with other popular tools, such as Cas-OFFinder, Crisflash, and FlashFry, while preserving the same level of accuracy. Overall, this makes Crackling a faster and better method to design guide RNAs at scale. Crackling is available at https://github.com/bmds-lab/Crackling under the Berkeley Software Distribution (BSD) 3-Clause license.

Reduced thermal tolerance in a coral carrying CRISPR-induced mutations in the gene for a heat-shock transcription factor

Phillip Cleves, Amanda Tinoco, Jake Bradford, Dimitri Perrin, Line Bay, John Pringle

Proceedings of the National Academy of Sciences (PNAS), 2020

Coral reefs are biodiversity hotspots of great ecological, economic, and aesthetic importance. Their global decline due to climate change and other stressors has increased the urgency of understanding the molecular bases of corals’ responses to stress. Analyses of coral genomes and gene-expression patterns have identified many genes that may be important in stress resistance, but rigorous testing of their function will require the analysis of appropriate mutants. Here, we used CRISPR technology to show that mutational loss of a putative regulator of gene expression in response to heat stress indeed produced a loss of heat tolerance. Such use of CRISPR to generate mutations in corals should illuminate many aspects of coral biology and, thus, help to guide conservation efforts.

Improving CRISPR guide design with consensus approaches

Jake Bradford, Dimitri Perrin

BMC Genomics, 2019

Background CRISPR-based systems are playing an important role in modern genome engineering. A large number of computational methods have been developed to assist in the identification of suitable guides. However, there is only limited overlap between the guides that each tool identifies. This can motivate further development, but also raises the question of whether it is possible to combine existing tools to improve guide design. Results We considered nine leading guide design tools, and their output when tested using two sets of guides for which experimental validation data is available. We found that consensus approaches were able to outperform individual tools. The best performance (with a precision of up to 0.912) was obtained when combining four of the tools and accepting all guides selected by at least three of them. Conclusions These results can be used to improve CRISPR-based studies, but also to guide further tool development. However, they only provide a short-term solution as the time and computational resources required to run four tools may be impractical in certain applications.

A benchmark of computational CRISPR-Cas9 guide design methods

Jake Bradford, Dimitri Perrin

PLOS Computational Biology, 2019

The popularity of CRISPR-based gene editing has resulted in an abundance of tools to design CRISPR-Cas9 guides. This is also driven by the fact that designing highly specific and efficient guides is a crucial, but not trivial, task in using CRISPR for gene editing. Here, we thoroughly analyse the performance of 18 design tools. They are evaluated based on runtime performance, compute requirements, and guides generated. To achieve this, we implemented a method for auditing system resources while a given tool executes, and tested each tool on datasets of increasing size, derived from the mouse genome. We found that only five tools had a computational performance that would allow them to analyse an entire genome in a reasonable time, and without exhausting computing resources. There was wide variation in the guides identified, with some tools reporting every possible guide while others filtered for predicted efficiency. Some tools also failed to exclude guides that would target multiple positions in the genome. We also considered two collections with over a thousand guides each, for which experimental data is available. There is a lot of variation in performance between the datasets, but the relative order of the tools is partially conserved. Importantly, the most striking result is a lack of consensus between the tools. Our results show that CRISPR-Cas9 guide design tools need further work in order to achieve rapid whole-genome analysis and that improvements in guide design will likely require combining multiple approaches.