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CRISPR-based diagnostics use the CRISPR-Cas system trans -cleavage activity to identify specific target sequences. When activated, this activity cleaves surrounding reporter molecules, producing a detectable signal. This technique has great specificity, sensitivity, and rapid detection, making it an important molecular diagnostic tool for medical and infectious disease applications. Despite its potential, the present CRISPR/Cas system has challenges with its single-stranded DNA reporters, characterized by low stability and limited sensitivity, restricting effective application in complex biological settings. In this work, we investigate the trans -cleavage activity of CRISPR/Cas12a on substrates utilizing fluorescent polystyrene microspheres to detect tetracycline. This innovative discovery led to the development of microsphere probes addressing the stability and sensitivity issues associated with CRISPR/Cas biosensing. By attaching the ssDNA reporter to polystyrene microspheres, we discovered that the Cas12a system exhibits robust and sensitive trans -cleavage activity. Further work revealed that the trans -cleavage activity of Cas12a on the microsphere surface is significantly dependent on the concentration of the ssDNA reporters. Building on these intriguing discoveries, we developed microsphere-based fluorescent probes for CRISPR/Cas aptasensors, which showed stability and sensitivity in tetracycline biosensing. We demonstrated a highly sensitive detection of tetracycline with a detection limit of 0.1 μM. Finally, the practical use of a microsphere-based CRISPR/Cas aptasensor in spiked food samples was proven successful. These findings highlighted the remarkable potential of microsphere-based CRISPR/Cas aptasensors for biological research and medical diagnosis.

Cas12a is a widely used programmable nuclease for genome editing across a variety of organisms, but its application is limited by its PAM recognition restriction. To alleviate these PAM constraints, protein engineering efforts have been applied to expand the PAM recognition range. In this study, we designed and constructed 990 synthetic hybrid Cas12a chimeras through domain shuffling and screened an efficient hybrid Cas12a (ehCas12a) that could recognize a broad range PAM of 5'-TYYN-3' (Y is T or C and N is A, T, C, or G). Furthermore, we constructed an ehCas12a variant, ehCas12a RRVR (T167R/N572R/K578V/N582R), with expanded PAM preference to 5'-TNYN, TWRV-3' (W is A or T, R is A or G, and V is A, C, or G), which can efficiently recognize -2* A/G PAMs that are barely recognized by Cas12a-type proteins and their mutants. Finally, we demonstrated that the DNase-inactivated ehCas12a RRVR base editor (dehCas12a RRVR-BE) was capable of targeting noncanonical PAMs in vivo and disease-related loci for potential therapeutic applications. Overall, our findings highlight the modular design and reconfiguration of Cas proteins for enhanced functionality.

Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas12a, which exhibits excellent target DNA-activated trans -cleavage activity under the guidance of a programmable CRISPR RNA (crRNA), has shown great promise in next-generation biosensing technology. However, current CRISPR-Cas12a-based biosensors usually improve sensitivity by the initial nucleic acid amplification, while the distinct programmability and predictability of the crRNA-guided target binding process has not been fully exploited. Herein, we, for the first time, propose a modular and sensitive CRISPR-Cas12a fluorometric aptasensor by integrating an enzyme-free and robust crRNA-mediated catalytic nucleic acid network, namely, Cas12a-CMCAN, in which crRNA acts as an initiator to actuate cascade toehold-mediated strand displacement reactions (TM-SDRs). As a proof of concept, adenosine triphosphate (ATP) was selected as a model target. Owing to the multiturnover of CRISPR-Cas12a trans -cleavage and the inherent recycling amplification network, this method achieved a limit of detection value of 0.16 μM (20-fold lower than direct Cas12a-based ATP detection) with a linear range from 0.30 to 175 μM. In addition, Cas12a-CMCAN can be successfully employed to detect ATP levels in diluted human serum samples. Considering the simplicity, sensitivity, and easy to tune many targets by changing aptamer sequences, the Cas12a-CMCAN sensing method is expected to offer a heuristic idea for the development of CRISPR-Cas12a-based biosensors and unlock its potential for general and convenient molecule diagnostics.

Bacteroides is the most abundant genus in the human gut microbiome and has been increasingly used as model organisms for studying the function and ecology of the gut microbiome. However, genome editing tools for such commensal gut microbes are still lacking. Here we developed a versatile, highly efficient CRISPR/Cas-based genome editing tool that allows markerless gene deletion and insertion in human gut Bacteroides species. We constructed multiple CRISPR/Cas systems in all-in-one Bacteroides - E. coli shuttle plasmids and systematically evaluated the genome editing efficiency in Bacteroides thetaiotaomicron , including the mode of Cas protein expression (constitutive, inducible), different Cas proteins (FnCas12a, SpRY, SpCas9), and sgRNAs. Using the anhydrotetracycline (aTc)-inducible CRISPR/FnCas12a system, we successfully deleted large genomic fragments up to 50 kb to study the function of metabolic gene clusters. Furthermore, we demonstrated that CRISPR/FnCas12a can be broadly applied to engineer multiple human gut Bacteroides species, including Bacteroides fragilis , Bacteroides ovatus , Bacteroides uniformis , and Bacteroides vulgatus . We envision that CRISPR/Cas-based genome editing tools for Bacteroides will greatly facilitate mechanistic studies of the gut commensal and the development of engineered live biotherapeutics.

CRISPR-Cas12a is a powerful platform for DNA-based diagnostics. The detection scheme relies on unselective shredding of a fluorescent ssDNA reporter upon target DNA recognition. To extend the reporter library beyond ssDNAs, we discovered a fluorescent reporter type using a dsDNA template. In this design, the fluorescence of the dsDNA reporter is quenched via contact-quenching mechanism. Upon detection, the quenched fluorescence recovers with the activation Cas12a complex. Here, we compared the probing performance of two dsDNA reporters with two ssDNA reporters. The rate of the Cas12a trans-cleavage reaction was studied using one of the dsDNA reporters under different settings. The detection of different sizes of dsDNA or ssDNA targets was studied systematically under three different temperatures. Lower thresholds for ssDNA and dsDNA target size were identified. The mismatch tolerance and target specificity were examined for both ssDNA and dsDNA targets, separately. The probing performance of the dsDNA reporter was evaluated in a random DNA pool with and without target strands. We report that dsDNA can serve as a tunable fluorescence reporter template expanding the toolbox for Cas12a-based diagnostics.

Transcriptional perturbation using inactivated CRISPR-nucleases (dCas) is a common method in eukaryotic organisms. While rare examples of dCas9-based tools for prokaryotes have been described, multiplexing approaches are limited due to the used effector nuclease. For the first time, a dCas12a derived tool for the targeted activation and repression of genes was developed. Therefore, a previously described SoxS activator domain was linked to dCas12a to enable the programmable activation of gene expression. A proof of principle of transcriptional regulation was demonstrated on the basis of fluorescence reporter assays using the alternative host organism Paenibacillus polymyxa as well as Escherichia coli . Single target and multiplex CRISPR interference targeting the exopolysaccharide biosynthesis of P. polymyxa was shown to emulate polymer compositions of gene knockouts. The simultaneous expression of 11 gRNAs targeting multiple lactate dehydrogenases and a butanediol dehydrogenase resulted in decreased lactate formation, as well as an increased butanediol production in microaerobic fermentation processes. Even though Cas12a is more restricted in terms of its genomic target sequences compared to Cas9, its ability to efficiently process its own guide RNAs in vivo makes it a promising tool to orchestrate sophisticated genetic reprogramming of bacterial cells or to screen for engineering targets in the genome. The developed tool will accelerate metabolic engineering efforts in the alternative host organism P. polymyxa and might be also applied for other bacterial cell factories.

In cyanobacteria, metabolic engineering using synthetic biology tools is limited to build a biosolar cell factory that converts CO 2 to value-added chemicals, as repression of essential genes has not been achieved. In this study, we developed a dCas12a-mediated CRISPR interference system (CRISPRi-dCas12a) in cyanobacteria that effectively blocked the transcriptional initiation by means of a CRISPR-RNA (crRNA) and 19-nt direct repeat, resulting in 53-94% gene repression. The repression of multiple genes in a single crRNA array was also successfully achieved without a loss in repression strength. In addition, as a demonstration of the dCas12a-mediated CRISPRi for metabolic engineering, photosynthetic squalene production was improved by repressing the essential genes of either acnB encoding for aconitase or cpcB 2 encoding for phycocyanin β-subunit in Synechococcus elongatus PCC 7942. The ability to regulate gene repression will promote the construction of biosolar cell factories to produce value-added chemicals.

Accessing the full biosynthetic potential encoded in the genomes of fungi is limited by the low expression of most biosynthetic gene clusters (BGCs) under common laboratory culture conditions. CRISPR-mediated transcriptional activation (CRISPRa) of fungal BGCs could accelerate genomics-driven bioactive secondary metabolite discovery. In this work, we established the first CRISPRa system for filamentous fungi. First, we constructed a CRISPR/dLbCas12a-VPR-based system and demonstrated the activation of a fluorescent reporter in Aspergillus nidulans . Then, we targeted the native nonribosomal peptide synthetase-like (NRPS-like) gene micA in both chromosomal and episomal contexts, achieving increased production of the compound microperfuranone. Finally, multigene CRISPRa led to the discovery of the mic cluster product as dehydromicroperfuranone. Additionally, we demonstrated the utility of the variant dLbCas12a D156R -VPR for CRISPRa at room temperature culture conditions. Different aspects that influence the efficiency of CRISPRa in fungi were investigated, providing a framework for the further development of fungal artificial transcription factors based on CRISPR/Cas.

Because DNA has the merit of high capacity and complexity, DNA steganography, which conceals DNA-encoded messages, is very promising in information storage. The classical DNA steganography method hides DNA with a "secret message" in a mount of junk DNA, and the message can be extracted by polymerase chain reaction (PCR) using specific primers (key), followed by DNA sequencing and sequence decoding. As leakage of the primer information may result in message insecurity, new methods are needed to better secure the DNA information. Here, we develop a pre-key by either mixing specific primers (real key) with nonspecific primers (fake key) or linking a real key with 3'-end redundant sequences. Then, the single-stranded DNA (ssDNA) trans cleavage activity of CRISPR/Cas12a is employed to cut a fake key or remove the 3'-end redundant sequences, generating a real key for further information extraction. Therefore, with the Cas12a-assisted DNA steganography method, both storage and transfer of DNA-encoding data can be better protected.