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TIGER: a tool to predict CRISPR–Cas13 off-target activity

TIGER: a tool to predict CRISPR–Cas13 off-target activity‘ – Written by Charlotte Harrison, Science Writer.

CRISPR technology that targets RNA and uses the Cas13 enzyme is less established than the DNA-targeting CRISPR-Cas9 system, but has important therapeutic potential. A study in Nature Biotechnology describes the first model can that predict both on- and off-target activity of CRISPR–Cas13 guide RNAs. The work will be important for designing highly precise RNA-targeting CRISPR agents. 

Off-target predictions

Previous studies of RNA-targeting CRISPRs focused largely on on-target activity and mismatches; off-target activity such as insertion and deletion mutations are less well researched. 

To find out more about off-target activity, the authors tested ~200,000 Cas13d guide RNAs targeting essential genes in human cells. These RNAs were designed to be perfect match guide RNAs, mismatches between the guide and the target RNA, or insertions and deletions (indels).  

They found that the impact of mismatches and indels on Cas13d activity was position- and context-dependent impact, and mismatches that resulted in G–U wobble pairings were better tolerated than other single-base mismatches.  

The authors then used the large-scale dataset obtained from their cell-based studies to train a deep-learning model that they named TIGER – targeted inhibition of gene expression via gRNA design. TIGER was able to predict both on-target and off-target activity. Moreover, TIGER  outperformed previous models developed for Cas13 on-target guide design. 

Gene tuning

The researchers also demonstrated that TIGER’s off-target predictions could be used to precisely modulate gene expression by enabling gene expression to be partially inhibited in cells with mismatch guides.

“Our deep learning model can tell us not only how to design a guide RNA that knocks down a transcript completely, but can also ‘tune’ it – for instance, having it produce only 70% of the transcript of a specific gene,” said author Andrew Stirn in a press release.

Gene tuning may be useful, for example, in cancers where aberrant gene expression leads to uncontrolled tumour growth.

The authors hope that TIGER will help scientists design CRISPR–Cas13 constructs that avoid undesired off-target CRISPR activity and facilitate new RNA-targeting therapies.

“We believe that the ability to model the effect of nucleotide mismatches not only allows for an enhanced understanding of gRNA on-target specificity and off-target avoidance but also enables precise target knockdown to a defined degree that will be useful for diverse transcriptome engineering applications,” they said.

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