DNA-guided CRISPR/Cas12 for RNA targeting.

CRISPR-Cas nucleases are transforming genome editing, RNA editing, and diagnostics but have been limited to RNA-guided systems. We present ΨDNA, a DNA-based guide for Cas12 enzymes, engineered for specific and efficient RNA targeting. ΨDNA mimics a crRNA but with a reverse orientation, enabling stable Cas12-RNA assembly and activating trans-cleavage without RNA components.

Exploring the Thermostability of CRISPR-Cas12b using Molecular Dynamics Simulations.

CRISPR (clustered regularly interspaced short palindromic repeat)-based diagnostics are at the forefront of rapid detection platforms of infectious diseases. The integration of reverse transcription-loop-mediated isothermal amplification (RT-LAMP) with CRISPR-Cas protein systems has led to the creation of advanced one-pot assays. The sensitivity of these assays has been bolstered by the utilization of a thermophilic Cas12 protein, BrCas12b, and its engineered variant, which exhibits enhanced thermal stability and allows for broader operation temperatures of the assay.

Harnessing noncanonical crRNAs to improve functionality of Cas12a orthologs.

There is a broad diversity among Cas12a endonucleases that possess nucleic acid detection and gene-editing capabilities, but few are studied extensively. Here, we present an exhaustive investigation of 23 Cas12a orthologs, with a focus on their cis- and trans-cleavage activities in combination with noncanonical crRNAs. Through biochemical assays, we observe that some noncanonical crRNA:Cas12a effector complexes outperform their corresponding wild-type crRNA:Cas12a.

CRISPR-based diagnostics detects invasive insect pests.

Rapid identification of organisms is essential for many biological and medical disciplines, from understanding basic ecosystem processes, disease diagnosis, to the detection of invasive pests. CRISPR-based diagnostics offers a novel and rapid alternative to other identification methods and can revolutionize our ability to detect organisms with high accuracy. Here we describe a CRISPR-based diagnostic developed with the universal cytochrome-oxidase 1 gene (CO1).

Programmable RNA detection with CRISPR-Cas12a.

Cas12a, a CRISPR-associated protein complex, has an inherent ability to cleave DNA substrates and is utilized in diagnostic tools to identify DNA molecules. We demonstrate that multiple orthologs of Cas12a activate trans-cleavage in the presence of split activators. Specifically, the PAM-distal region of the crRNA recognizes RNA targets provided that the PAM-proximal seed region has a DNA target.

CRISPR-based diagnostics detects invasive insect pests.

Rapid identification of organisms is essential across many biological and medical disciplines, from understanding basic ecosystem processes and how organisms respond to environmental change, to disease diagnosis and detection of invasive pests. CRISPR-based diagnostics offers a novel and rapid alternative to other identification methods and can revolutionize our ability to detect organisms with high accuracy. Here we describe a CRISPR-based diagnostic developed with the universal cytochrome-oxidase 1 gene (CO1).

Engineering highly thermostable Cas12b via de novo structural analyses for one-pot detection of nucleic acids.

CRISPR-Cas-based diagnostics have the potential to elevate nucleic acid detection. CRISPR-Cas systems can be combined with a pre-amplification step in a one-pot reaction to simplify the workflow and reduce carryover contamination. Here, we report an engineered Cas12b with improved thermostability that falls within the optimal temperature range (60°C-65°C) of reverse transcription-loop-mediated isothermal amplification (RT-LAMP).

Programmable RNA detection with CRISPR-Cas12a.

CRISPR is a prominent bioengineering tool and the type V CRISPR-associated protein complex, Cas12a, is widely used in diagnostic platforms due to its innate ability to cleave DNA substrates. Here we demonstrate that Cas12a can also be programmed to directly detect RNA substrates without the need for reverse transcription or strand displacement. We discovered that while the PAM-proximal "seed" region of the crRNA exclusively recognizes DNA for initiating trans-cleavage, the PAM-distal region or 3'-end of the crRNA can tolerate both RNA and DNA substrates.

Programmable RNA detection with CRISPR-Cas12a.

CRISPR is a prominent bioengineering tool and the type V CRISPR-associated protein complex, Cas12a, is widely used in diagnostic platforms due to its innate ability to cleave DNA substrates. Here we demonstrate that Cas12a can also be programmed to directly detect RNA substrates without the need for reverse transcription or strand displacement. We discovered that while the PAM-proximal "seed" region of the crRNA exclusively recognizes DNA for initiating cleavage, the PAM-distal region or 3'-end of the crRNA can tolerate both RNA and DNA substrates.

Rapid SARS-CoV-2 diagnosis using disposable strips and a metal-oxide-semiconductor field-effect transistor platform.

The SARS-CoV-2 pandemic has had a significant impact worldwide. Currently, the most common detection methods for the virus are polymerase chain reaction (PCR) and lateral flow tests. PCR takes more than an hour to obtain the results and lateral flow tests have difficulty with detecting the virus at low concentrations.

Clinical validation of engineered CRISPR/Cas12a for rapid SARS-CoV-2 detection.

Background: The coronavirus disease (COVID-19) caused by SARS-CoV-2 has swept through the globe at an unprecedented rate. CRISPR-based detection technologies have emerged as a rapid and affordable platform that can shape the future of diagnostics.

Methods: We developed ENHANCEv2 that is composed of a chimeric guide RNA, a modified LbCas12a enzyme, and a dual reporter construct to improve the previously reported ENHANCE system.

CRISPR-ENHANCE: An enhanced nucleic acid detection platform using Cas12a.

Rapid detection of nucleic acids is essential for clinical diagnosis of a wide range of infectious and non-infectious diseases. CRISPR-based diagnostic platforms are well-established for rapid and specific detection of nucleic acids but suffer from a low detection sensitivity without a target pre-amplification step. Our recently developed detection system, called CRISPR-ENHANCE, employs engineered crRNAs and optimized conditions to achieve a significantly higher sensitivity and enable femtomolar levels of nucleic acid detection even without target pre-amplification.