Understanding the trans-cleavage Activity of CRISPR-Cas Systems for RNA Detection Applications
Creators: Feng, Wei
Publisher: name: University of Alberta Library
Year: 2022

<p>The discovery and characterization of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) systems with trans-cleavage activity led to the rise of CRISPR-based diagnostics. A ribonucleoprotein (RNP), consisting of a CRISPR-associated protein (Cas) and a CRISPR RNA (crRNA) or single guide RNA (sgRNA), recognizes a target nucleic acid sequence. The binding of RNP to the specific nucleic acid activates CRISPR-Cas systems, e.g., CRISPR-Cas12a and CRISPR-Cas13a, which can trans-cleave reporter nucleic acids to generate measurable signals. The performance of such CRISPR-based assays depends on the kinetics of the trans-cleavage activity. The first objective of my thesis is to study and maximize the trans-cleavage activity of CRISPR-Cas13a systems. CRISPR-Cas systems have also been combined with nucleic acid amplification techniques to meet the high sensitivity requirement for molecular detection of pathogens. However, the incompatibility of multiple techniques and reaction conditions poses challenges to the development of highly sensitive and accurate assays with simple operations. The second objective of my thesis is to integrate CRISPR-Cas systems with other nucleic acid amplification techniques for broader applications to RNA detection. <br>
I systematically studied how the length of the RNA target, reagent composition, and reaction conditions affected the trans-cleavage activity of Cas13a. The improved understanding contributed to the development of a sensitive assay for the detection of microRNA at ambient temperature using Cas13a. This broadened the application of Cas13a-based assays to short, small RNA molecules; previous assays were only applicable to longer RNA sequences. My characterization of the effect of short RNA or crRNA on the two most common Cas13a enzymes provided guidance for choosing Cas13a enzymes for different applications. <br>
I observed unusual effects of the reaction temperature on the trans-cleavage activity, and performed an in-depth study on the thermal behavior of two Cas enzymes: LwaCas13a and LbuCas13a. I differentiated the effect of temperature on several reaction steps during the operation of Cas13a. By testing a set of rationally designed target RNA, I was able to precisely assess the temperature preference of trans-cleavage activity upon Cas13a activation. I discovered that upon activation, the preferred temperature for high trans-cleavage activity of Cas13a was around ambient temperature, lower than the temperatures commonly used by others for detection. This feature is useful for simplifying Cas13a-based assays.<br>
Detection of RNA using CRISPR-Cas alone is limited by the slow kinetics of the trans-cleavage process. Integrating CRISPR technology with nucleic acid amplification techniques overcomes the limitation in sensitivity. A strategy to integrate reverse transcription (RT), recombinase polymerase amplification (RPA), and CRISPR-Cas12a nuclease reactions into a single tube was developed, resulting in the sensitive detection of SARS-CoV-2. All reactions were integrated into a single, closed tube, and the assay was achieved under an isothermal condition (40 °C). <br>
The understanding gained from this thesis helps guide the development of CRISPR-based assays. The strategies of integrating CRISPR technology with isothermal amplification techniques can be applied to the detection of various types of RNA molecules.</p>