Designing highly functional and specific guide RNAs for knockout and achieving precise knockin using the homology-directed repair pathway

The CRISPR-Cas9 system requires a Cas9 nuclease and two short RNAs, a crRNA and tracrRNA, to introduce double-strand breaks for functional protein disruption (knockout) or for insertion or replacement of genetic content, such as a selection marker (knockin). Our approach to better understand the parameters affecting CRISPR-Cas9 editing efficiency for functional gene knockout utilized synthetic crRNAs and tracrRNA, which can be chemically synthesized rapidly without the need for cloning and sequencing. We systematically transfected > 1100 synthetic crRNA:tracrRNAs into a GFP-reporter cell line to develop an algorithm to predict functional gene knockout. To minimize potential off-targets, we developed a rigorous specificity tool that is able to detect mismatches as well as gapped alignments that are typically missed using most existing specificity tools. Together, we are able to design highly functional and specific guide RNAs. In addition, we have expanded the use of synthetic crRNA:tracrRNA by demonstrating its utility in knockin applications using short single-stranded DNA as donor templates for small insertions, such as SNPs, or DNA vectors as donor templates for large insertions, such as a GFP reporter. Finally, we provide guidelines for design of these donor templates for optimal knockin efficiency.