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

1. Broughton JP, Deng X, Yu G, et al. CRISPR-Cas12-based detection of SARS-CoV-2. Nat Biotechnol. Apr 2020;doi:10.1038/s41587-020-0513-4

2. Abudayyeh OO, Gootenberg JS, Kellner MJ, Zhang F. Nucleic Acid Detection of Plant Genes Using CRISPR-Cas13. CRISPR J. Jun 2019;2:165-171. doi:10.1089/crispr.2019.0011

3. Kellner MJ, Koob JG, Gootenberg JS, Abudayyeh OO, Zhang F. SHERLOCK: nucleic acid detection with CRISPR nucleases. Nat Protoc. Oct 2019;14(10):2986-3012. doi:10.1038/s41596-019-0210-2

4. Gootenberg JS, Abudayyeh OO, Kellner MJ, Joung J, Collins JJ, Zhang F. Multiplexed and portable nucleic acid detection platform with Cas13, Cas12a, and Csm6. Science. 04 2018;360(6387):439-444. doi:10.1126/science.aaq0179

5. Myhrvold C, Freije CA, Gootenberg JS, et al. Field-deployable viral diagnostics using CRISPR-Cas13. Science. 04 2018;360(6387):444-448. doi:10.1126/science.aas8836

6. Gootenberg JS, Abudayyeh OO, Lee JW, et al. Nucleic acid detection with CRISPR-Cas13a/C2c2. Science. 04 2017;356(6336):438-442. doi:10.1126/science.aam9321

7. Aquino-Jarquin G. CRISPR-Cas14 is now part of the artillery for gene editing and molecular diagnostic. Nanomedicine. 06 2019;18:428-431. doi:10.1016/j.nano.2019.03.006

8. Chen JS, Ma E, Harrington LB, et al. CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity. Science. 04 2018;360(6387):436-439. doi:10.1126/science.aar6245

9. Nguyen LT, Smith BM, Jain PK. Enhancement of trans-cleavage activity of Cas12a with engineered crRNA enables amplified nucleic acid detection. bioRxiv. 2020:2020.04.13.036079. doi:10.1101/2020.04.13.036079

10. Nguyen LT, Gurijala J, Rananaware SR, Pizzano BLM, Stone BT, Jain PK. CRISPR-ENHANCE: An enhanced nucleic acid detection platform using Cas12a. Methods. Feb 2021;doi:10.1016/j.ymeth.2021.02.001

11. Nguyen LT, Rananaware SR, Pizzano BLM, Stone BT, Jain PK. Engineered CRISPR/Cas12a Enables Rapid SARS-CoV-2 Detection. MedRxiv. 2020;doi:10.1101/2020.12.23.20248725