To learn more about Gene Editing watch “Webinar: CRISPR-Cas9 Gene Editing with Synthetic RNA – from start to finish!”
Attendees will learn:
More about the webinar:
The CRISPR-Cas9 system introduces double-strand DNA breaks at a specific locus in the genome by using a complex of the Cas9 nuclease with either a chimeric single guide RNA (sgRNA) or two short RNAs (a CRISPR RNA (crRNA) and a trans-activating RNA (tracrRNA)). The ability of any given sgRNA or crRNA to create a break in the target DNA that causes functional protein disruption can vary based on the guide RNA (gRNA) sequence and position in the targeted gene. Likewise, the overall specificity of RNA-directed DNA cleavage events is not yet completely understood and can hamper its wider application.
While gRNAs targeting one or a few genes can often be chosen ad hoc, performing high throughput genome-scale loss-of-function screens requires gRNAs that have consistently high functional knockout efficiencies. To understand the parameters affecting CRISPR-Cas9 gene editing efficiency, we systematically evaluated over 1100 synthetic crRNAs in a reporter assay that directly measures functional activity of a central cellular process (the ubiquitin-proteasome pathway) and identified characteristics important for functional gene disruption. Using this data, we developed and trained an algorithm to score gRNA sequences 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. Our results demonstrate that high-scoring crRNAs have increased functionality. Further, we developed an optimized alignment program to perform 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.