CRISPETa Software Developed to Efficiently Delete Noncoding DNA Regions

Cas9 proteins (scissors) are guided to their target sites by single guide RNAs (sgRNAs, orange ribbons). The target region in between is removed. CRISPETa software enables researcher to design such deletion experiments quickly and conveniently. [Pulido-Quetglas et al./CC BY]

Scientists at the Computational Biology of RNA Processing laboratory of the Centre for Genomic Regulation (CRG) in Barcelona, Spain recently created a tool based on CRISPR/Cas9 called DECKO, which can be used to delete any desired piece of noncoding DNA. CRISPR/Cas9 is composed of two components: a molecular barcode, called single guide RNA (sgRNA), which is designed by researchers to recognize one precise location in the genome, and the Cas9 protein, which binds to a structured loop in the sgRNA.

The unique advantage of DECKO is that it uses two individual sgRNAs, acting like two molecular scissors that snip out a piece of DNA, according to Rory Johnson, Ph.D., former staff scientist at the CRG and now group leader at the National Center of Competence in Research RNA & Disease and department of clinical research at the University of Bern.

While working on DECKO, Dr. Johnson and colleagues in the CRG lab of Roderic Guigo Serra, Ph.D., realized that no software was available for designing the pairs of sgRNAs that are required, meaning that designing deletion experiments was time consuming. To overcome this, a master's degree student, Carlos Pulido-Quetglas, designed a software pipeline called CRISPETa for designing CRISPR deletion experiments. The user tells CRISPETa what region they wish to delete and the software returns a set of optimized pairs of sgRNAs that can directly be used by experimental researchers.

One of the key features is that it can create designs at high scales, with future screening experiments in mind. Importantly, CRISPETa is designed for use by nonexperts and is available in a user-friendly website, making CRISPR deletion available to the widest possible number of scientific and biomedical researchers, notes the CRG scientific team.

In the CRISPETa study, (“Scalable Design of Paired CRISPR Guide RNAs for Genomic Deletion”), published in PLOS Computational Biology, the researchers also introduced a new version of DECKO that, reportedly, is cheaper and faster than the previous one. The researchers showed that CRISPETa designs efficiently delete their desired targets in human cells. Most importantly, in those regions that give rise to RNA molecules, the researchers showed that the RNA molecules also carry the deletion.

CRISPETa users may, for example, delete a suspected functional region of noncoding DNA and test the outcome on cellular or molecular activity, explains Pulido, adding that this software will also be potentially valuable for groups aiming to utilize CRISPR deletion for therapeutic purposes by for example, deleting a region of noncoding DNA that is suspected to cause a disease state. 

“Ultimately, we expect that CRISPR deletion and other genome engineering tools to lead to a revolution in our ability to understand the genomic basis of disease, particularly in the 99% of DNA that does not encode proteins. Apart from being used as a basic research tool, CRISPR may even be used in the future as a powerful therapeutic to reverse disease-causing mutations,”  says Dr. Johnson.