Low-Background CRISPR/Cas12a Sensors for Versatile Live-Cell Biosensing

Abstract

The trans-cleavage activity of CRISPR/Cas12a has been widely used in biosensing. However, many CRISPR/Cas12a-based biosensors, especially those that work in “on–off–on” mode, usually suffer from high background and thus impossible intracellular application. Herein, this problem is efficiently overcome by elaborately designing the activator strand (AS) of CRISPR/Cas12a using the “RESET” effect found by our group. The activation ability of the as-designed AS to CRISPR/Cas12a can be easily inhibited, thus assuring a low background for subsequent biosensing applications, which not only benefits the detection sensitivity improvement of CRISPR/Cas12a-based biosensors but also promotes their applications in live cells as well as makes it possible to design high-performance biosensors with greatly improved flexibility, thus achieving the analysis of a wide range of targets. As examples, by using different strategies such as strand displacement, strand cleavage, and aptamer–substrate interaction to reactivate the inhibited enzyme activity, several CRISPR/Cas12a-based biosensing systems are developed for the sensitive and specific detection of different targets, including nucleic acid (miR-21), biological small molecules (ATP), and enzymes (hOGG1), giving the detection limits of 0.96 pM, 8.6 μM, and 8.3 × 10–5 U/mL, respectively. Thanks to the low background, these biosensors are demonstrated to work well for the accurate imaging analysis of different biomolecules in live cells. Moreover, we also demonstrate that these sensing systems can be easily combined with lateral flow assay (LFA), thus holding great potential in point-of-care testing, especially in poorly equipped or nonlaboratory environments.

Allosteric Activator-Regulated CRISPR/Cas12a System Enables Biosensing and Imaging of Intracellular Endogenous and Exogenous Targets

“…The resulting high background makes sensitive detection of the target impossible. This result suggests that the activation ability of an AS with long complementary sequence to crRNA cannot be efficiently deprived by the means of competitive hybridization, which is consistent with our previous finding . In contrast, when the complementary sequence of AS was reduced to 16 bases, negligible CRISPR/Cas12a activation ability was observed whether it was embedded in the hairpin (hAS 16 and AS 16 , Figure B).…”

“…This result suggests that the activation ability of an AS with long complementary sequence to crRNA cannot be efficiently deprived by the means of competitive hybridization, which is consistent with our previous finding. 20 In contrast, when the complementary sequence of AS was reduced to 16 bases, negligible CRISPR/Cas12a activation ability was observed whether it was embedded in the hairpin (hAS 16 and AS 16 , Figure 1B). However, with the addition of target DNA (tDNA), the fluorescence of the sensing system containing hAS16 was greatly enhanced, suggesting that hAS 16 and tDNA could together activate CRISPR/Cas12a, which can be interpreted by our reported "RESET" effect.…”

“…Typically, the design of “on–off–on” mode sensors based on the CRISPR/Cas12 system mainly focuses on programming the activator strand (AS) to convert to the trans -cleavage activity of Cas12a into target information. , Initially, an external auxiliary probe is usually designed to put the activator strand in a “blocked” state, thereby inhibiting the trans -cleavage activity of the CRISPR/Cas12a system. However, this design strategy usually has higher requirements on the length and dosage of auxiliary probes to ensure the lowest background, as well as a high signal-to-noise ratio.…”

Photoactivatable CRISPR/Cas12a Sensors for Biomarkers Imaging and Point-of-Care Diagnostics

“…As a member of the CRISPR family, the CRISPR/Cas12a system is found to possess high trans -cleavage activity (collateral effect) and indiscriminate cleavage of ambient single-stranded DNA (ssDNA) when specific guide CRISPR RNA (crRNA) is activated by a matching target DNA. , Due to its ability to cleave nontargeted ssDNA, the activity of Cas12a can be converted into the enhancement of the fluorescence signal caused by the cleavage of FQ-Repoter. − These findings show the potential of the CRISPR/Cas12a system as an excellent actuator for developing novel biosensors, not only converting biometric events into detectable signals but also dramatically increasing the reporting signals. Till now, many CRISPR/Cas12a-based diagnostic methods have been established for the highly sensitive and specific detection of nucleic acid and non-nucleic acid analytes, such as small molecules, protein biomarkers , and so on.…”

Ligase detection reaction amplification-activated CRISPR-Cas12a for single-molecule counting of FEN1 in breast cancer tissues

“…S1, ESI†). 3 Recently, CRISPR-Cas12a has been broadly applied in the field of genome editing, transcription regulation, molecular diagnostics, and cancer therapy. 4 CRISPR-Cas12a has been used for one-step detection of DNAs and RNAs, 5 but the application of CRISPR-Cas12a for protein detection is still a challenge due to the low abundance of proteins in organisms.…”