On the ever-growing functional versatility of the CRISPR-Cas13 system.

CRISPR-Cas systems evolved in prokaryotes to implement a powerful antiviral immune response as a result of sequence-specific targeting by ribonucleoproteins. One of such systems consists of an RNA-guided RNA endonuclease, known as CRISPR-Cas13. In very recent years, this system is being repurposed in different ways in order to decipher and engineer gene expression programmes. Here, we discuss the functional versatility of the CRISPR-Cas13 system, which includes the ability for RNA silencing, RNA editing, RNA tracking, nucleic acid detection and translation regulation. This functional palette makes the CRISPR-Cas13 system a relevant tool in the broad field of systems and synthetic biology.

Multiplexable and Biocomputational Virus Detection by CRISPR-Cas9-Mediated Strand Displacement.

Recurrent disease outbreaks caused by different viruses, including the novel respiratory virus SARS-CoV-2, are challenging our society at a global scale; so versatile virus detection methods would enable a calculated and faster response. Here, we present a novel nucleic acid detection strategy based on CRISPR-Cas9, whose mode of action relies on strand displacement rather than on collateral catalysis, using the Streptococcus pyogenes Cas9 nuclease. Given a preamplification process, a suitable molecular beacon interacts with the ternary CRISPR complex upon targeting to produce a fluorescent signal. We show that SARS-CoV-2 DNA amplicons generated from patient samples can be detected with CRISPR-Cas9. We also show that CRISPR-Cas9 allows the simultaneous detection of different DNA amplicons with the same nuclease, either to detect different SARS-CoV-2 regions or different respiratory viruses. Furthermore, we demonstrate that engineered DNA logic circuits can process different SARS-CoV-2 signals detected by the CRISPR complexes. Collectively, this CRISPR-Cas9 R-loop usage for the molecular beacon opening (COLUMBO) platform allows a multiplexed detection in a single tube, complements the existing CRISPR-based methods, and displays diagnostic and biocomputing potential.

Programmable regulation of translation by harnessing the CRISPR-Cas13 system.

The ability to control protein expression at both the transcriptional and post-transcriptional levels is instrumental for the cell to integrate multiple molecular signals and then reach high operational sophistication. Although challenging, fully artificial regulations at different levels are required for boosting systems and synthetic biology. Here, we report the development of a novel framework to regulate translation by repurposing the CRISPR-Cas13 immune system, which uses an RNA-guided ribonuclease. By exploiting a cell-free expression system for prototyping gene regulatory structures, our results demonstrate that CRISPR-dCas13a ribonucleoproteins (d means catalytically dead) can be programmed to repress or activate translation initiation. The performance assessment of the engineered systems also revealed guide RNA design principles. Moreover, we show that the system can work in vivo. This development complements the ability to regulate transcription with other CRISPR-Cas systems and offers potential applications.

Diagnostics of Infections Produced by the Plant Viruses TMV, TEV, and PVX with CRISPR-Cas12 and CRISPR-Cas13.

Viral infections in plants threaten food security. Thus, simple and effective methods for virus detection are required to adopt early measures that can prevent virus spread. However, current methods based on the amplification of the viral genome by polymerase chain reaction (PCR) require laboratory conditions. Here, we exploited the CRISPR-Cas12a and CRISPR-Cas13a/d systems to detect three RNA viruses, namely, Tobacco mosaic virus, Tobacco etch virus, and Potato virus X, in Nicotiana benthamiana plants. We applied the CRISPR-Cas12a system to detect viral DNA amplicons generated by PCR or isothermal amplification, and we also performed a multiplexed detection in plants with mixed infections. In addition, we adapted the detection system to bypass the costly RNA purification step and to get a visible readout with lateral flow strips. Finally, we applied the CRISPR-Cas13a/d system to directly detect viral RNA, thereby avoiding the necessity of a preamplification step and obtaining a readout that scales with the viral load. These approaches allow for the performance of viral diagnostics within half an hour of leaf harvest and are hence potentially relevant for field-deployable applications.

CRISPR-Cas12a-Based Detection of SARS-CoV-2 Harboring the E484K Mutation.

The novel respiratory virus SARS-CoV-2 is rapidly evolving across the world with the potential of increasing its transmission and the induced disease. Here, we applied the CRISPR-Cas12a system to detect, without the need of sequencing, SARS-CoV-2 genomes harboring the E484K mutation, first identified in the Beta variant and catalogued as an escape mutation. The E484K mutation creates a canonical protospacer adjacent motif for Cas12a recognition in the resulting DNA amplicon, which was exploited to obtain a differential readout. We analyzed a series of fecal samples from hospitalized patients in Valencia (Spain), finding one infection with SARS-CoV-2 harboring the E484K mutation, which was then confirmed by sequencing. Overall, these results suggest that CRISPR diagnostics can be a useful tool in epidemiology to monitor the spread of escape mutations.