About CRISPinatoR

What is CRISPinatoR?

CRISPinatoR is a web-based sgRNA design platform for the CRISPR/Cas9 system. Our approach focuses on disrupting Exon Splicing Enhancer (ESE) sites in order to induce frame-shift exon skipping that increase the probability of gene-knockouts.

How does it work?

We exploit both ESE mechanisms and exon symmetry to maximizethe probability of gene ablation. In our previous work we have shown that CRISPR/Cas9-induced alterations in ESEs are highly related to exon skipping events.

Now, we define exon symmetry as the match between the starting and ending phase of an exon. If the phases are the same, the exon is symmetric; if the phases are different, the exon is asymmetric. Inducing a skip of an Asymmetric exon will cause the effect of a frame-shift deletion. This can, in turn, generate a Premature Termination Codon (PTC) that induces gene-knockout via nonsense-mediated decay (NMD). Skipping a symmetric exon, on the other hand, will preserve the reading frame and may rescue a shorter version of the protein in question.

Using this information we categorized potential sgRNAs into the following tiers:
  • Tier 1: All guides that affect ESEs in asymmetric exons which have the potential of generating PTCs.
  • Tier 2: sgRNAs that do not affect asymmetric exons and have an off-target score >80.
  • Tier 3: All guides that affect ESEs in symmetric exons which have the potential of producing altered proteins.
  • Removed: sgRNAs with low off-target scores (≤80).

Best Practices

  1. Select multiple sgRNAs for each target gene.
  2. Pick sgRNAs far away from each other.
  3. Target the upstream region of your gene, but avoid the very beginning.
  4. Generally 5 to 65% in the upstream region of the CDS is recommended
    • Targeting too early may induce Alternative Translation Initiation (ATI)
  5. Consider sgRNAs that predict the distance between its PTC to the last Exon-Exon Junction to be >55bp.
    • This increases the likelihood of inducing NMD
Follow our tutorial for more information on how to use CRISPinatoR.

Further Reading

  1. Rubina Tuladhar, Yunku Yeu, John Tyler Piazza, Zhen Tan, Jean Rene Clemenceau, Xiaofeng Wu, Quinn Barrett, Jeremiah Herbert, David Mathews, James Kim, Tae Hyun Hwang, Lawrence Lum Genome buffering mechanisms obfuscate CRISPR/Cas9-directed gene interrogation campaigns in mammalian cells, March 20 2019, bioRxiv 583138; doi: 10.1101/583138