Design Considerations for Highly Specific and Efficient Synthetic crRNA Molecules

In order to understand the parameters affecting CRISPR-Cas9 gene editing efficiency, we systematically transfected synthetic CRISPR RNA (crRNA) and trans-activating CRISPR RNA (tracrRNA) reagents targeting components of the proteasome into a reporter cell line in which knockout of proteasome function results in fluorescence of a ubiquitin-EGFP fusion protein that is normally degraded by the proteasome pathway. We evaluated the functionality of > 1100 crRNA sequences in this system; using these data, we developed and trained an algorithm to score crRNAs based on how likely they are to produce functional knockout of targeted genes. We further tested our algorithm by designing synthetic crRNAs to genes unrelated to the proteasome and examined their ability to knock out gene function using additional phenotypic assays. To augment our functionality algorithm, we developed an optimized alignment program to perform rapid, flexible, and complete specificity analysis of crRNAs, including detection of gapped alignments. We have combined this comprehensive specificity check with our functionality algorithm to select and score highly specific and functional crRNAs for any given gene target.