New type of CRISPR gene scissors discovered

Scientists at the Helmholtz Institute for RNA-based Infection Research (HIRI), in cooperation with Benson Hill and Utah State University, have unexpectedly discovered a nuclease that represents an entirely new type of CRISPR immune defence. Unlike any other previously known nuclease of the CRISPR-Cas immune system, the newly dubbed ‘Cas12a2’ destroys DNA to shut down an infected cell. The team’s findings could lead to new CRISPR technologies for molecular biology diagnostics, and have been published in the journal Nature.

Like humans, bacteria and archaea can be attacked by viruses. Bacterial defences, such as CRISPR-Cas systems, have diverse proteins and functions that help bacteria protect themselves against foreign invaders. The defence is based on a common mechanism: a CRISPR ribonucleic acid (crRNA), serving as a ‘guide RNA’, helps detect regions of a foreign genome, such as the DNA of a virus, for targeted cleavage. The CRISPR-associated (Cas) nuclease directed by a crRNA can cut its target like a pair of scissors: a strategy that humans have since harnessed in many technologies.

“We were exploring CRISPR nucleases that were originally clumped with Cas12a; nucleases that defend bacteria by recognising and cleaving invasive DNA,” said HIRI postdoc Oleg Dmytrenko, the first author of the study. “Once we identified more of them, we realised that they were different enough from Cas12a to warrant a deeper dive. This exploration led us to discover that these nucleases, which we called Cas12a2, do something very different not only from Cas12a but also from any other known CRISPR nuclease.”

The crucial difference lies in the mechanism of their defence action. When Cas12a2 recognises invasive RNA, the nuclease cleaves it but can also damage other RNA and DNA inside the cell, impairing its growth and limiting the spread of the infection.

“In general, such defence strategies that abort the infection have been known in bacteria,” Dmytrenko said. “A few other CRISPR-Cas systems work in this way. However, a CRISPR-based defence mechanism that relies on a single nuclease to recognise the invader and degrade cellular DNA and RNA has not been observed before.”

The protein sequence and architecture of Cas12a2 discriminate this nuclease from Cas12a. Activated by a protospacer-flanking sequence (PFS), Cas12a2 recognises target RNAs that are complementary to its guide RNA. Targeting RNA triggers collateral nucleic acid cleavage that degrades RNA, single-stranded DNA and double-stranded DNA. This activity leads to cell arrest, presumably by damaging DNA and RNA in the cell, which impairs growth. Cas12a2 can be used for molecular diagnostics and direct detection of RNA biomarkers, as demonstrated by proof of principle.

In further structural analysis of the nuclease by a second team that authored a companion paper, Cas12a2 was shown to undergo major structural changes after binding to its RNA target at various stages of the immune response. This, in turn, leads to an exposed cleft in the nuclease that can shred any nucleic acid it encounters — be it RNA, single-stranded DNA or double-stranded DNA. The research also discovered ways to mutate Cas12a2 to alter the nucleic acid that the nuclease degrades after recognising its RNA target, opening up potentially broad technological applications for the future.

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