Article:
Nucleic Acid Detection with CRISPR-Cas13a/C2c2
Gootenberg JS et al., Science 356, 438-442 (2017)
Rationale:
Microbial clustered regularly interspersed short palindromic repeats (CRISPR) and CRISPR-associated systems (CRISPR-Cas) are evolutionarily diverse systems employed by bacteria and archaea as adaptive immune defenses against invading bacteriophages and plasmids. Engineered CRISPR-Cas systems have revolutionized genome editing but their role in nucleic acid detection and diagnostics has yet to be fully explored.
Methodology:
The authors created a modular system named Specific High-Sensitivity Enzymatic Reporter Unlocking (SHERLOCK) that has three main components:
- Isothermal nucleic acid amplification:
This amplifies nucleic acid sequences of interest without the need for thermal cycling. The lack of thermal cycling is attractive for use in low resource settings as it would not require the electricity or technical expertise needed with PCR. - Cas13a with CRISPR RNA (crRNA) guide sequences:
The crRNA sequences are engineered to detect specific sequences in the pathogen of interest. - Non-specific RNA linked to a fluorescent probe:
Once the pathogen sequence is detected, the Cas13a enzyme it degrades the tagged nucleic acid and causes emits a fluorescent signal.
The authors test the ability of the SHERLOCK system to detect viruses, bacteria, and human genomic DNA polymorphisms.
Results:
In in vitro setting SHERLOCK can detect nucleic acids down to ~2 attomolar (2 x 10-18). The SHERLOCK assay had similar, to slightly better, sensitivity than digital droplet and quantitative PCR with less variation across replicates. It differentiated RNA fragments from Zika and Dengue flaviruses at attomolar sensitivity. The assay performed similarly in Zika detection in urine and serum samples where they could detect 2 x 103 copies/mL sample (3.2 attomolar).
The SHERLOCK system was adapted to a point-of-care paper-based platform and detected Zika virus to a level of 20 attomolar.
SHERLOCK differentiated bacterial DNA amongst clinically-relevant Gram-negative bacteria and discriminated between Klebsiella pneumoniae harboring either carbapenamase or New Delhi beta-lactamase genes.
Furthermore, they found SHERLOCK distinguished between different single nucleotide polymorphisms in human genomic DNA and could detect EGFR and BRAF mutations.
Conclusions:
The use of CRISPR-Cas13a in the SHERLOCK system is a potentially revolutionary tool to detect nucleic acids from viral and bacterial pathogens with high sensitivity and specificity. The ability to design guide crRNA’s to detect single nucleic acid differences allows it to differentiate closely related microbial strains and identify resistance genes. The authors also provide evidence that this technology can be used as a point of care test in low-resource settings. This technology has the potential to transform how we clinically diagnosis infection in pulmonary and critical care medicine. Further work will be needed to compare how SHERLOCK compares to current culture-based and molecular technologies in different clinical settings, patient populations, and sample types.
