Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR), is a prokaryotic adaptive immune system against viruses and plasmids from most of the archaea and bacteria. Recently, isothermal assays with CRISPR, such as Specific High-Sensitivity Enzymatic Reporter UnLOCKing (SHERLOCK), have been proved to enable rapid and efficient nucleic acid detection which is promising for the development of the point-of-care testing systems. The elegant trans cleavage reporter unlocking mechanism built on the programmable crRNA guided CRISPR nucleases ensure superior specificity and sensitivity. However, the typical CRISPR-based approaches separate the amplification and detection into a two-step process which not only induces potential contamination but also increases the complexity using digi...[ Read more ]

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR), is a prokaryotic adaptive immune system against viruses and plasmids from most of the archaea and bacteria. Recently, isothermal assays with CRISPR, such as Specific High-Sensitivity Enzymatic Reporter UnLOCKing (SHERLOCK), have been proved to enable rapid and efficient nucleic acid detection which is promising for the development of the point-of-care testing systems. The elegant trans cleavage reporter unlocking mechanism built on the programmable crRNA guided CRISPR nucleases ensure superior specificity and sensitivity. However, the typical CRISPR-based approaches separate the amplification and detection into a two-step process which not only induces potential contamination but also increases the complexity using digital quantification. This thesis addresses the challenges in developing all-in-one CRISPR assays that are compatible with isothermal recombinase polymerase amplification and rolling circle amplification respectively, and implementation of the digital detection of these assays for absolute quantification in combination with microfluidic technology.

In the first project, we first developed all-in-one SHERLOCK with enhanced molecular mobility so as to improve the reaction robustness. We further applied this one-pot SHERLOCK assay using droplet microfluidics for digital quantification. The Microfluidics-Enabled Digital Isothermal Cas13a Assay (MEDICA), a digital format of one-pot SHERLOCK, leverages microfluidic technology to allow the separation of the magnesium initiator from the target-included master mix, allowing MEDICA to completely eliminated undesired premature amplification, as well as the compartmentation of the assay into picoliter reactions for absolute quantification by direct counting of the droplet numbers. MEDICA demonstrated single-molecule detection ability, more rapid and stable qualitative (10 min) and quantitative (25 min) results. We also demonstrated that MEDICA successfully identified and quantified all viral HPV 16 and HPV 18 in clinical specimens, demonstrating good accordance with qPCR results.

In the second project, we developed a digital rolling circle amplification (RCA)-based assay with Cas12a for microRNA detection. We designed a barcode strategy that encodes PAM and detection sequences with a padlock probe. After ligating the target, the circled template was labeled with RCA-amplified Cas12a activation information. The RCA method uses a typical two-step process that involves ligation and amplification. Considering the same reaction temperature of DNA polymerase and Cas12a, we fused these two steps together. Combined with droplet digital detection, the sensitivity achieved 1 fM compared to the picomolar level sensitivity in bulk reaction.

In addition, we investigated the nicking influence of LbCas12a on the targeting strand. We discovered that the dsDNA indicated more robust against the nicking influence while the ssDNA was easily affected by the nicking site. Furthermore, the middle of the protospacer is not the most sensitive part since the collateral activity can be triggered by a nick. These discoveries guide the future molecular circuit design based on nicking influence.

The thesis presents novel nucleic acid detection methods using CRISPR in combination with isothermal amplification. We have shown the Cas13a and Cas 12a based assays can be applied using droplet microfluidics for digital quantification of viral DNA and micro RNA. Due to the merits of isothermal, sensitive and specific collateral detection of the CRISPR system, we expect that the CRISPR bases approach holds great potential for system integration and miniaturization for point-of-care testing.