(210h) Engineered CRISPR-Enhance System for Clinical Detection of Sars-COV-2 RNA

The Severe Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV-2) has resulted into 2019 novel coronavirus disease (COVID-19) pandemic with confirmed almost 3 million deaths and over 136 million confirmed cases worldwide as of April 12th 2021. There is an urgent need for the development of a rapid, sensitive, specific, inexpensive, easy-to-use, and accessible testing kit for SARS-CoV-2 that can be implemented as a point-of-care diagnostic and a home-based testing kit. Most Class 2 CRISPR/Cas systems, including CRISPR/Cas12 (Type V), CRISPR/Cas14 (Type V), and CRISPR/Cas 13 (Type VI) mediate a nonspecific collateral trans-cleavage of random DNA and RNA after binding or cis-cleavage of their target DNA or RNA. For CRISPR/Cas12a, this multiple-turnover trans-cleavage activity is only initiated once the crRNA/Cas12a complex is bound to its target ssDNA or dsDNA that acts as an activator. This trans-cleavage activity has been widely exploited for nucleic acid detection and has been combined with fluorescence-based, paper-based, and electrochemical-based sensing technologies to develop rapid and sensitive diagnostics. Therefore, Type V and VI CRISPR/Cas systems have recently emerged as diagnostics for detecting nucleic acids including SARS-CoV-2 RNA.^1-11 However, current CRISPR/Cas12a systems are limited to a picomolar detection limit unless the target is either pre-amplified in some manner. The Cas12a-based DETECTR technology from Mammoth Biosciences and Cas13a-based SHERLOCK technology from Sherlock Biosciences have been approved by the FDA under EUA as lab-based diagnostics for detecting SARS-CoV-2 genomic RNA in a fluorescence-based assay.

We recently discovered that crRNAs can tolerate various length extensions with DNA or RNA on both 3’- and 5’-ends, while the phosphorothioate extensions reduced or eliminated the trans-cleavage activity (Nguyen et al., Nature Communications, 2020)⁹. In particular, an engineered crRNA containing a 7-nt DNA extensions on its 3’-end, drastically increased the trans-cleavage activity of CRISPR/Cas12a and increased specificity of detection. By extending the 3’- or 5’-ends of the crRNA with different lengths of DNA, RNA, and phosphorothioate DNA, we discovered a new self-catalytic behavior and an augmented rate of Cas12a-mediated trans-cleavage activity as high as 3.5-fold compared to the wild type crRNA, making it the fastest reported CRISPR/Cas12a in terms of trans-cleavage activity, and termed it as CRISPR-ENHANCE. This in turn reflected an unprecedented improvement in sensitivity and limit of detection of nucleic acid targets down to the femtomolar level for a variety of clinically-relevant nucleic acid targets including prostate cancer antigen 3 (PCA3), HIV, HCV, and SARS-CoV-2 RNA without target pre-amplification within 30 minutes. By combining with a reverse transcription-loop-mediated isothermal amplification (RT-LAMP) step, 100 aM of SARS-CoV-2 RNA could be detected using a lateral flow assay within 60 minutes (Nguyen et al., Methods, 2021)¹⁰.

By further optimization of crRNAs and combining with a target pre-amplification strategy using a reverse transcription-loop-mediated isothermal amplification (RT-LAMP) step, a single molecule detection of SARS-CoV-2 RNA in nasopharyngeal swabs was achieved within 50 minutes. Compared to the wild-type crRNAs, our CRISPR-ENHANCE system demonstrated up to 5-fold higher sensitivity in detecting SARS-CoV-2 RNA. By combining engineered crRNAs with a Cas12a mutant (D156R), a multimodal reporter, and lyophilizing the components, the CRISPR-ENHANCE v2 could be conducted within 33 minutes using both a fluorescence-based assay and a lateral flow assay. The CRISPR-ENHANCE v2 demonstrated high inclusivity and exclusivity and achieved 96.7% sensitivity and 96.7% specificity in clinical samples and was stable for at least 30 days at room temperature (Figure 1, Nguyen et al., MedRxiv, 2021- in revision at Communications Medicine-Nature)¹¹.

References

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