Model Reduces Off-Target Effects in CRISPR

Researchers in Denmark have developed a model that helps refine CRISPR and boost the precision and effectiveness of the genome-editing tool.

Ten years ago, researchers identified a protein in bacteria that can cut DNA, and which uses a so-called guide RNA to recognize where DNA needs to be cut. CRISPR—which makes it possible to remove or insert the exact genes you want in any living organism—has been hailed as a revolution within gene technology ever since.  The technology also makes it possible to cure diseases through the precise correction of errors in genes.

Implementation of the technology, however, requires that the method is effective and precise so that only the desired gene modifications are achieved. A major challenge of CRISPR/Cas9-mediated genome engineering is that not all guide RNAs (gRNAs) cleave DNA efficiently.

To better understand the mechanisms that affect the effectiveness of the CRISPR method, researchers from the University of Copenhagen and Aarhus University used an energy-based model to identify the mechanisms regulating CRISPR-Cas9's activity and specification. The model makes it possible for researchers to design gRNA components that can increase the effectiveness of the method and minimize unintended, or “off-target,” effects.

"Our study shows that, by better understanding the CRISPR/Cas9 protein and its gRNA component, we can more accurately hit and cut the DNA and thereby optimize the effectiveness of gene modification," says Yonglun Luo, co-senior author of the Nature Communications paper. "Unintended off-targets are a major concern when using the CRISPR method to treat diseases, and most of the tools for measuring off-targets have serious limitations and do not include the factors that we have discovered in our study. These discoveries have given us the key to designing CRISPR-gRNA with high effectiveness and precision.”

The computational study uses an energy-based model for CRISPR-gRNA-target binding to systematically analyze the relationship between nucleotides binding and the CRISPR editing activity on a large dataset of 11,062 experimentally validated gRNA efficiencies. The discovery that CRISPR-Cas9 can finetune its binding at the target site by sliding towards overlapping protospacer adjacent motifs (PAMs) to maximize editing efficiency was further validated by measuring SpCas9 cleavage efficiency at 1,024 sites using a new library-based method developed by the team. 

The team behind the study will continue refining the method and the design of the gRNA component to further improve the method's effectiveness and precision. "We’ll also try to find new methods of measuring on-target and off-target areas and developing innovative methods to address the off-target challenges that still limit our ability to use the CRISPR-Cas9 method," says Luo.