Design considerations for specific & efficient crRNA molecules for CRISPR-Cas9 genome editing
The discovery that the machinery of the CRISPR-Cas9 bacterial immune system can be re-purposed to easily create deletions, insertions, and replacements in the mammalian genome has revolutionized the field of genome engineering. While gene disruption using crRNAs to target one or a few genes can often be chosen in an ad hoc manner, performing high throughput loss-of-function screens requires gRNAs that have consistently high functional knockout efficiencies. In order to understand the parameters affecting CRISPR-Cas9 gene editing efficiency, we systematically transfected synthetic tracrRNA and crRNAs 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 to identify parameters that are important for DNA cleavage and subsequent functional gene disruption. Using this 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, as well as their cleavage efficiency using next-generation sequencing analysis. 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. Recent work has demonstrated gene editing by crRNAs containing bulges of up to 4 nucleotides, but existing design tools are unable to detect putative off-targets based on gapped alignments. We have combined this comprehensive specificity check with our functionality algorithm to select and score highly specific and functional gRNAs for any given gene target.
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