Design considerations for highly specific and efficient synthetic crRNA molecules

Optimal guide RNAs balance functionality and specificity

Functional gene knockout using CRISPR-Cas9 has become an important tool for understanding a gene’s role in a biological system, and has the potential to advance basic and applied research. Despite how effective this tool is at targeting and editing specific genes, not all gene cleavage events due to guide RNAs result in functional knockout of the target protein.

In this poster, the parameters affecting CRISPR-Cas9 gene editing efficiency are explored; 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 to develop and train 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 a rigorous 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.

Takeaways from the poster

  • crRNAs vary in specificity and in efficiency for creating functional gene knockouts
  • We used a high-throughput fluorescence assay to develop and train an algorithm to score crRNAs for likelihood of producing functional gene knockouts
  • We developed an optimized alignment program to perform rapid and complete specificity analysis of crRNAs
  • We demonstrate that targets with gaps in either the RNA or DNA strand can be cleaved and are therefore important to identify during specificity checking

View the poster now! »

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