A general onepot-method for nucleic acid detection with CRISPR-Cas12a

Abstract

CRISPR/Cas12a system has been shown promising for nucleic acid diagnostics due to its rapid, portable and accurate features. In combination with isothermal amplification technology, single-copy sensitivity can be achieved. However, cleavage of the amplicons and primers by the cis- and trans-activity of Cas12a hinders the attempts to integrate the amplification and detection steps into a single reaction. Through phosphorothioate modification of primer and design of crRNA that allow for the cutting site locating at the modified site of the primer, we realized onepot detection of SARS-CoV-2 with single-copy sensitivity. We also identified the activated Cas12a has a much higher affinity to C nucleotide-rich reporter than others. By applying such reporters, we significantly reduced the reaction time required for the lateral-flow readout. Furthermore, to improve the specificity of the strip-based assay, we created a novel reporter and, when combined with a customized strip, the unspecific signal could be completely eliminated. This established system termed Targeting DNA by Cas12a-based Eye Sight Testing in Onepot Reaction (TESTOR) was validated using clinical cervical samples for human papillomaviruses (HPVs) detection. Our system represents a general approach to integrating the nucleic acid amplification and detection into a onepot reaction in CRISPR-Cas systems, highlighting its potential as a rapid, portable and accurate detection platform of nucleic acids.

Sensitive quantitative detection of SARS-CoV-2 in clinical samples using digital warm-start CRISPR assay

“…Simultaneously, Cas12a-cRNA complex specifically binds the sites of the amplicons to activate Cas12a’s collateral cleavage activity, thereby indiscriminately cleaving surrounding nontarget ssDNA-FQ reporter to generate increased fluorescence. The ssDNA-FQ is a 5-cytosin nucleotide single-stranded DNA (5′-CCCCC-3′) labeled with FAM (Fluorescein) at 5′ end and Iowa Black FQ quencher at 3′ end, and is used in our assay due to its higher affinity to Cas12a 24 . Fluorescence is quenched via resonance energy transfer in intact ssDNA-FQ reporters, but can be recovered after activated Cas12a cleaves the reporters.…”

CRISPR Technology in Gene-Editing-Based Detection and Treatment of SARS-CoV-2

“…In addition, the treated individual will not suffer repeated disease relapses. Therefore, advances in the CRISPR/Cas system and the success of clinical trials in animal models are critical Table 1 , Guanghui et al, 2020 .…”

Elucidating the Role of CRISPR/Cas in Single-Step Isothermal Nucleic Acid Amplification Testing Assays

“…Leveraging clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated systems (Cas) has in recent years become the focal point of developing novel nucleic acid detection assays. − Indeed, nucleic acid-activated complexes of Cas endonuclease and CRISPR RNA (crRNA) and their subsequent cleavage of single-stranded nucleic acids provide an elegant signal amplification and detection strategy that are compatible with nucleic acid amplification testing (NAAT) methods, as evidenced by a myriad of assays combining various CRISPR/Cas methods (e.g., Cas9, Cas12a, Cas12b, Cas13a, Cas14) and various NAAT methods (e.g., recombinase polymerase amplification (RPA), loop-mediated isothermal amplification (LAMP), polymerase chain reaction (PCR)) reported in the last few years. − Despite many combinations, most of the assays to date keep CRISPR/Cas methods and NAAT methods as separate steps rather than integrating them in a single step, even though single-step assays can be advantageous in requiring less manual operation, simplifying instrumentation requirement, reducing the likelihood of contamination, and accelerating the assay turnaround time. Moreover, although CRISPR/Cas methods and several isothermal NAAT methods have similar reaction temperatures and are therefore compatible for integration, only a handful of such single-step isothermal CRISPR/Cas-assisted NAAT assays have been reported to date. − The paucity thus signals the need for further advance and development.…”