CRISPR/Cas9 Modified to Increase Its Versatility

A team of molecular biologists has modified the CRISPR/Cas9 gene-editing tool to incorporate an "on switch", which enables the precise manipulation of specific genes in selected cells.

CRISPR/Cas9 is regarded as the cutting edge of molecular biology technology. CRISPRs (clustered regularly interspaced short palindromic repeats) are segments of prokaryotic DNA containing short repetitions of base sequences. Each repetition is followed by short segments of "spacer DNA" from previous exposures to a bacterial virus or plasmid. Since 2013, the CRISPR/Cas9 system has been used in research for gene editing (adding, disrupting, or changing the sequence of specific genes) and gene regulation.

By delivering the Cas9 enzyme and appropriate guide RNAs (sgRNAs) into a cell, the organism's genome can be cut at any desired location. The conventional CRISPR/Cas9 system from Streptococcus pyogenes is composed of two parts: the Cas9 enzyme, which cleaves the DNA molecule and specific RNA guides that shepherd the Cas9 protein to the target gene on a DNA strand. Nonetheless, the CRISPR/Cas9 scaffold is not ideal for fusions or activation by cellular triggers.

To increase the versatility of the CRISPR/Cas9 protein scaffolding, investigators at the University of California, Berkeley (USA) used a circular permutation technique to show that a topological rearrangement of Cas9 would provide an advanced platform for RNA-guided genome modification and protection.

Circular permutation involved cutting the amino-acid string of the Cas9 protein, switching the order of the two segments, and then allowing it to fold into a new three-dimensional configuration. While this process frequently deactivated Cas9, the enzyme remained functional about 10% of the time.

The investigators found that the protein's termini could be positioned adjacent to bound DNA, offering a straightforward mechanism for strategically fusing functional domains. Additionally, circular permutation enabled protease-sensing Cas9s (ProCas9s), a unique class of single-molecule effectors possessing programmable inputs and outputs. ProCas9s could sense a wide range of proteases, and the investigators reported in the January 10, 2019, issue of the journal Cell that ProCas9 could orchestrate a cellular response to pathogen-associated protease activity.

"When we cut the protein and moved the old piece to a new place within the protein, the system became very sensitive to how you linked the two fragments together," said senior author Dr. David Savage, associate professor of molecular and cell biology at the University of California, Berkeley. "We realized that we could use that sensitivity to engineer the protein to have protease recognition sites. We are not stuck with what nature gave us with regards to genome-editing proteins. These proteins can be elaborately optimized and turned into scaffolds not found in nature but possessing the right properties for use in human cells. There are a lot of proteases that regulate signaling pathways in cells, transform normal cells into cancer cells, and are involved in pathogen infection. If we can sense these signals, we can tap into and respond accordingly to these important pathways."

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