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UPDATE: New CRISPR Enzyme Variants Could Be Used to Detect Disease

This article has been updated to clarify that a team from Broad Institute and its collaborators originally discovered Cas13a in October 2015.

NEW YORK (GenomeWeb) – A team of University of California, Berkeley researchers led by Jennifer Doudna have published a study describing variants of the Cas13a CRISPR protein that could be used to diagnose disease.

In their study published today in Molecular Cell, Doudna and her colleagues built upon previous research, describing additional homologs of Cas13a. "We have taken our foundational research a step further in finding other homologs of the Cas13a family that have different nucleotide preferences, enabling concurrent detection of different reporters with, say, a red and a green fluorescent signal, allowing a multiplexed enzymatic detection system," first author Alexandra East-Seletsky, a Berkeley graduate student in Doudna's lab, said in a statement.

The latest study follows on the team's previous research that was published in Nature in September showing that Cas13a — then called C2c2 — has two different RNA cleaving functions: one for cutting its target and another for processing its guiding CRISPR RNA. In a statement at the time, Doudna called C2c2 "essentially a self-arming sentinel that attacks all RNAs upon detecting its target. This activity can be harnessed as an auto-amplifying detector that may be useful as a low-cost diagnostic." 

A team from the Broad Institute identified Cas13a in a paper published in late 2015 and noted its targeting capabilities last June. After publishing its Nature paper in September, the Berkeley team continued its work by combing through bacterial genome databases, and found 10 other Cas13a-like proteins. Of those, three differed from the original protein in the way they cut RNA. This led the researchers to conclude that the Cas13a variants can be divided into two distinct functional groups that recognize orthogonal sets of CRISPR RNA (crRNA) and possess different single strand-RNA (ssRNA) cleavage specificities.

"These functional distinctions could not be bioinformatically predicted, suggesting more subtle coevolution of Cas13a enzymes," the authors wrote. "Additionally, we find that Cas13a pre-crRNA processing is not essential for ssRNA cleavage, although it enhances ssRNA targeting for crRNAs encoded internally within the CRISPR array."

Beyond sharing respective sets of crRNAs, the two orthogonal Cas13a subfamilies cleave ssRNAs preferentially at uracil versus adenine. Though the researchers said they don't yet know the evolutionary basis for these substrate preferences, they suggested that ancestral versions of these enzymes may have evolved in response to nucleotide composition of the host and phage transcriptomes, resulting in differing active site molecular architectures.

Further, the researchers found that the kinetics of Cas13a-catalyzed ssRNA cleavage are spread out over seven orders of magnitude, which suggested to them that the enzymes operate in both regulatory and cell-destruction pathways.

"We anticipate that other type VI CRISPR-Cas enzymes, such as Cas13b, may also include subfamilies with divergent nucleotide preferences. Structural studies will be required to elucidate the molecular basis for trans-ssRNA substrate recognition to rationalize these preferences and to potentially engineer alternative cleavage activities," they wrote.

Importantly, the researchers' identification of two distinct subfamilies of Cas13a suggests that the various enzymes could be multiplexed to serve different functions, including disease diagnosis.

"Harnessing orthogonal Cas13a homologs with distinct crRNA specificities within the same application may enable RNA detection or in vivo imaging of distinct RNA species in parallel, expanding the utility of type VI-A systems," the researchers concluded. "Notably, representatives of these enzyme subfamilies have not yet been found to co-exist within a single host genome."