Versatile, rapid, and portable sensing of nucleic acids is vital for applications in human health. The RNA-targeting CRISPR-associated enzyme Cas13 has recently been adapted for such purpose. This detection platform, termed SHERLOCK (Specific High Sensitivity Enzymatic Reporter UnLOCKing), can discriminate between inputs that differ by a single nucleotide at very low concentrations and can be lyophilized for portable deployment. However, this technology has several limitations, including the lack of quantitation and reliance on fluorescent detection equipment for readout. Here, we extend the SHERLOCK technology to address these limitations and further develop the utility of this platform. Many applications require detection of more than one target molecule in a single reaction, and we therefore sought to create a multiplex platform that relies on unique cleavage preferences of Cas enzymes. To identify possible candidate enzymes compatible with multiplexing, we biochemically characterized three members of the CRISPR-Cas13a family and fourteen members of the CRISPR-Cas13b family.

We next focused on tuning the output of the SHERLOCK signal to make it more quantitative, sensitive, and robust to broaden the utility of the technology, testing optimizations of the amplification to enable quantitative and able to accept larger input volumes for increased sensitivity.

Another goal of SHERLOCKv2 was engineering a visual readout of activity requiring no additional instrumentation. We designed a lateral-flow readout that was based on the destruction of a FAM-biotin reporter, allowing for detection on commercial lateral flow strips. Abundant reporter accumulates anti-FAM antibody-gold nanoparticle conjugates at the first line on the strip, preventing binding of the antibody-gold conjugates to protein A on the second line; cleavage of reporter would reduce accumulation at the first line and result in signal on the second line. We profiled Cas13 cleavage preferences on homopolymer reporters, and found that most orthologs preferred either uridine, a combination of bases, or adenine. We refined the cleavage sequence preferences by evaluating collateral activity across di-nucleotide motifs, finding a large diversity of di-nucleotide cleavage motif preferences. ). From these di-nucleotide cleavage screens, we found that the activities of LwaCas13a, CcaCas13b, LbaCas13a and PsmCas13b could all be independently measured with the four di-nucleotide reporters AU, UC, AC, and GA, respectively. Additionally, using a random in vitro RNA library motif cleavage screen, we identified numerous RNA 6-mers that allowed for further orthogonality between Cas13 enzymes. When combined with RPA, we detected two DNA targets (the P. aeruginosa acyltransferase gene and the S. aureus thermonuclease gene) down to the attomolar range. These advances in in-sample multiplexing via orthogonal base preferences allow for many targets to be detected at scale and for cheaper cost.

We tested a range of primer concentrations and found that 240nM exhibited the greatest correlation between signal and input, and quantification was sustainable across a large range of sample concentrations down to the attomolar range. By scaling up the pre-amplification RPA step, we found that LwaCas13a could give detection signal for 200, 80, and 8zM input samples and allow for single-molecule volume inputs of 250µL and 540µL.

We tested lateral flow with Cas13 for instrument-free detection of ZIKV or DENV ssRNA, and found that detection was possible in under 90 minutes with sensitivities down to the 2 aM condition. Moreover, we found that we could do rapid genomic DNA extraction from human saliva (<10min) and input this directly into SHERLOCK without purification for rapid genotyping in under 23 minutes by fluorescence and 2 hours by lateral flow. The additional refinements presented here for Cas13-based detection allow for quantitative, visual, more sensitive, and multiplexed readouts, enabling additional applications for nucleic acid detection, especially in settings where portable and instrument-free analysis are necessary. SHERLOCKv2 can be used for multiplexed genotyping to inform pharmacogenomic therapeutic development and application, detecting genetically modified organisms in the field, or determining the presence of co-occurring pathogens. Moreover, the rapid, isothermal readout of SHERLOCKv2, enabled by lateral flow and Csm6, provides an opportunity for detection in settings where power or portable readers are unavailable, even for rare species like circulating DNA. In the future, it might be possible to make solution-based colorimetric readouts and multiplex lateral flow assays containing multiple test strips for different targets. Improved CRISPR-dx nucleic acid tests make it easier to detect the presence of nucleic acids in a range of applications across biotechnology and health and are now field-ready for rapid and portable deployment.

Disclosure No significant relationships.