Researchers from Helmholtz Munich and the Technical University of Munich (TUM) report that they have developed a new tool (Cas13d-NCS) that allows CRISPR RNA molecules that are located within the cell to move to the cytoplasm to make it effective at neutralizing RNA viruses. The team says this technique, which was necessary because RNA-targeting tools like CRISPR/Cas13 are powerful but inefficient in the cytoplasm of cells where many RNA viruses replicate, opens doors for precision medicine and proactive viral defense strategies.

The scientists’ study “Engineered, nucleocytoplasmic shuttling Cas13d enables highly efficient cytosolic RNA targeting” appears in Cell Discovery.

Treatment with Cas13d-NCS prevents the spread of SARS-CoV-2 (green). [Wolfgang Wurst, PhD]
Treatment with Cas13d-NCS prevents the spread of SARS-CoV-2 (green). [Wolfgang Wurst, PhD]
“CRISPR/Cas13 systems are programmable tools for manipulating RNAs and are used in a variety of RNA-targeting applications. Within the Cas13 family, Cas13d is the most active subtype in mammalian cells. Recently, Cas13d was harnessed as an antiviral against diverse human RNA viruses,” write the investigators.

“However, Cas13d is barely active in the cytosol of mammalian cells, restricting its activity to the nucleus, which limits applications such as programmable antivirals. Most RNA viruses replicate exclusively in the cytosol, suggesting that current Cas13d-based antivirals rely on uncontrolled nuclear leakage and are therefore limited in their efficiency.

“Here, we show that the nuclear localization of Cas13d crRNAs is the fundamental cause of Cas13d’s nuclear preference. To address this limitation, we engineered nucleocytoplasmic shuttling Cas13d (Cas13d-NCS). Cas13d-NCS transfers nuclear crRNAs to the cytosol, where the protein/crRNA complex binds and degrades complementary target RNAs.

“We screened various designs of shuttling proteins and characterized multiple design parameters of the best-performing system. We show that Cas13d-NCS is superior for degrading mRNAs and a self-replicating RNA derived from the Venezuelan equine encephalitis (VEE) RNA virus.

“Ultimately, we harnessed Cas13d-NCS to completely block the replication of various SARS-CoV-2 strains. Cas13d-NCS, therefore, enables the rational manipulation of the subcellular localization of a CRISPR system.”

Screening and optimization

Through screening and optimization, the researchers developed what they label “a transformative solution: Cas13d-NCS,” a system capable of transferring nuclear CRISPR RNA (crRNAs) into the cytosol to guide the CRISPR-Cas complex to specific target sequences for precise modifications. In the cytosol, the protein/crRNA complex targets complementary RNAs and degrades them with unprecedented precision, according to the researchers.

Cas13d-NCS outperforms its predecessors in degrading mRNA targets and neutralizing self-replicating RNA, including replicating sequences of Venezuelan equine encephalitis (VEE) RNA virus and several variants of SARS-CoV-2, unlocking the full potential of Cas13d as a programmable antiviral-tool, add the scientists, who cite this achievement as representing a significant step towards combating pandemics and strengthening defenses against future outbreaks.

The impact of the study goes beyond traditional antiviral strategies and CRISPR systems and ushers in a new era of precision medicine by enabling the strategic manipulation of subcellular localization of CRISPR-based interventions, maintains Wolfgang Wurst, PhD, director of the Institute of Developmental Genetics at Helmholtz Munich.

“This breakthrough in antiviral development with Cas13d-NCS marks a pivotal moment in our ongoing battle against RNA viruses,” says Wurst, who is coordinator of the study and also chair of developmental genetics at TUM, partner of the German Center for Neurodegenerative Diseases (DZNE) and member of the Munich Cluster for Systems Neurology (SyNergy). “This achievement showcases the power of collaborative innovation and human ingenuity in our quest for a healthier and more resilient world.”

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