CRISPRcruncher: A tool for engineering restriction sites into coding regions

CRISPR/Cas9 genome editing strategies often rely on the placement of an introduced restriction endonuclease (RE) site adjacent to the genomic edit of interest. This allows for rapid initial PCR-based detection of cells and organisms containing the edit of interest and may also be used for subsequent genotyping. Nevertheless, engineering RE sites at optimal locations within coding regions can be difficult due to the many hundreds of potential endonuclease options and the strict requirement to maintain the correct amino acid sequence. Here we report CRISPRcruncher, a computational tool that analyzes an input coding sequence and produces a complete list of all possible changes that could be made that will create new RE sites while preserving the original peptide sequence. Notably, for sequences tested, CRISPRcruncher identified approximately one new RE site per input nucleotide when mining for 4-bp or longer RE motifs and 0.5 new RE sites per input nucleotide when mining for 6-bp or longer motifs. Therefore, CRISPRcruncher represents a powerful new computational tool in the CRISPR arsenal.