Comprehensive sequence alignments between the human and worm genome identified 7,663 C. elegans coding-genes with human orthologs (2,3), including components of all major signaling pathways and essential cellular machineries. Precise genome editing by CRISPR-Cas9 works efficiently in the C. elegans germline when Cas9 protein is injected along with single guide RNA (sgRNA) and a short linear DNA repair template (4).
To facilitate the use of C. elegans as a model for human genetic disease we launched the WormCoolKit website (http://www.wormcoolkit.com/), which offers bioinformatic tools that assist in finding worm orthologs to human genes and vice versa, selecting CRISPR-Cas9 RNA guides and providing DNA template designs for introducing point mutations in the C. elegans genome.
The WormCoolKit orthologs finder furnishes the user with the most likely C. elegans gene ortholog to their human gene of interest. The tool extracts candidates from the OrthoList2 database and carries several additional filtration steps, aimed to increase the likelihood that these genes are true orthologs to the query.
The amino acid conservation tool is designed to inform whether a specific amino acid in a human protein is conserved in its C. elegans ortholog sequence. If conservation is confirmed, the tool will provide the user with the corresponding site in the worm protein amino acid sequence. If not, the tool will notify whether the amino acid in the worm sequence is similar or not conserved at all. To identify conservation status, the algorithm uses different methods of sequence alignments between the human gene and its ortholog. Therefore, each conserved amino acid is delivered with the number of alignments (out of ~2000) supporting the conservation. The tool also delivers an alignment score for the region surrounding the relevant amino acid, to assess whether the variation lies within a conserved region of the protein.
The WormCoolKit automated CRISPR planner is based on the CRISPR method by Paix A. et al (4,5). The planner is designed to first supply the users with RNA guides suitable for their query (based on IDT’s CRISPR-Cas9 guide RNA design checker) and then, once a guide is chosen, the algorithm defines the relevant parameters such as PAM site, CAS9 double-strand break site and mutation zone. It then goes through all possible options to mutate the strand as needed, including mutations to change the designated amino acid, mutations that prevent re-attachment of the CAS9 complex and insertion or removal of a restriction enzyme site to enable post editing identification of the gene by PCR. Lastly, the tool provides the user with a complete DNA repair template sequence containing the mutations flanked by two homology arms (35nts in length).
The WormCoolKit website was devised to streamline the process of modeling human genetic diseases in C. elegans (figure 1), starting by identifying the most likely worm ortholog of the human gene of interest, checking whether a given amino acid variation in the human gene is in a residue conserved in worms and finally designing the CRISPR strategy to insert the variation into the worm genome. That said, the free tools are generally useful for gene and residue conservation analysis between worms and humans and for providing RNA guides and DNA template designs for CRISPRing any point mutation in a worm gene.