(160x) Amplification-Free Nucleic Acid Detection at Room Temperature Using CRISPR Chain Reaction

Rapid and sensitive detection of nucleic acids is critical for a wide variety of biotechnological and pharmaceutical engineering applications. The ongoing SARS-CoV-2 outbreak has highlighted the need for developing robust diagnostic tests that can accurately identify pathogen nucleic acids in different types of human biological samples. Traditional nucleic acid detection assays based on reverse transcriptase polymerase chain reaction (RT-qPCR) are widely used but are limited by their dependency on expensive reagents, sophisticated equipment, and trained personnel. In recent years, a large number of CRISPR/Cas based diagnostic platforms such as SHERLOCK^[1][2] and DETECTR^[3] have been established. These methods exploit the inherent ability of type V and type VI Cas effectors to produce non-specific collateral cleavage of single-stranded DNA and RNA molecules after target-specific identification and cleavage. While CRISPR-based methods are rapid, cost-effective and can potentially be deployed at point-of-care, they suffer from a low detection sensitivity at room temperature without a target pre-amplification step.

Here, we have developed an amplification-free CRISPR/Cas12 based diagnostic method called CRISPR Chain Reaction (CCR) by combining a primary, on-target, CRISPR/Cas system with a ‘locked’ secondary CRISPR system that consists of an excess of secondary DNA activators and a modified crRNA that is locked for activity. The locked secondary system becomes unlocked and produces an enhanced signal only after the primary CRISPR system finds its target and initiates collateral cleavage. By combining CCR platform with reverse transcriptase, we were able to detect attomolar concentration of a wide variety of RNA targets including HIV-1 and SARS-CoV-2 at room temperature within 60-90 minutes without any target pre-amplification. Our CCR platform is a universal CRISPR amplification system and can be rapidly coupled with any CRISPR-based target detection system to enhance their sensitivity of detection. The CCR also offers the flexibility of combining both Cas12 and Cas13 effectors in a single assay and enables amplification-free detection of both DNA or RNA targets.

References

1. J. Kellner, J.G. Koob, J.S. Gootenberg, O.O. Abudayyeh, F. Zhang, SHERLOCK: nucleic acid detection with CRISPR nucleases. Nat Protoc, 14 (10) (2019), pp. 2986-3012
2. Gootenberg, J. S. et al. Nucleic acid detection with CRISPR-Cas13a/C2c2. Science 356, 438–442 (2017)
3. S. Chen, E. Ma, L.B. Harrington, M. Da Costa, X. Tian, J.M. Palefsky, J.A. Doudna, CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity. Science, 360 (6387) (2018), pp. 436-439