RAPID (RNA/DNA Advanced chimeric, PAM-independent, Integrated Nicking, Diagnostics), a nick-tuned, PAM-duplex-mediated platform for PAM-independent RNA and DNA detection and applies it to detect single-point mutations in ssDNA and RNA substrates, a challenge for traditional Cas12 and Cas13 systems.
Abstract
Abstract CRISPR–Cas nucleases have revolutionized diagnostics and biotechnology by providing programmable specificity. Here, we extend the understanding of Cas12a biology with a screen that, unexpectedly, finds that Cas12a trans-cleavage activity can be modulated by nicks in the protospacer in a position-dependent manner. Wanting to explore the impact of non-conventional trans-cleavage substrates, we subsequently find that non-specific Cas12a cleavage can be significantly reduced with RNA and chimeric (mixed RNA/DNA) reporter sequences. Exploiting these features and building on emerging protospacer adjacent motif (PAM)-independent Cas12a diagnostics that use engineered DNA activators and split-guide architectures, we introduce RAPID (RNA/DNA Advanced chimeric, PAM-independent, Integrated Nicking, Diagnostics), a nick-tuned, PAM-duplex-mediated platform for PAM-independent RNA and DNA detection. By strategically introducing a nick within the spacer region, RAPID expands Cas12a detection to include target RNAs, which can be ligated in situ to create a hybrid protospacer-target with trans-cleavage activity matching conventional Cas12a. We then apply RAPID to detect single-point mutations in ssDNA and RNA substrates, a challenge for traditional Cas12 and Cas13 systems. In combination with RT-LAMP, RAPID is used for PAM-independent RNA detection in clinical samples, achieving sensitivity down to ∼1 aM and 100% concordance with RT-qPCR for samples with Ct ≤ 33.
The potential of terminally engineered PAM-less dsDNA as a structural handle for programming Cas12a activity is highlighted and useful insight is provided for the design of CRISPR-based biosensing strategies.
The CRISPR/Cas12a system has revolutionized molecular diagnostics due to its RNA-guided trans-cleavage activity, enabling programmable and highly accurate nucleic acid detection. However, most Cas12a-based assays are optimized for DNA targets, while direct RNA detection constrained by limited sensitivity, typically at the nanomolar level. Existing strategies to improve the performance of RNA analysis often rely on additional DNA activators or complex auxiliary systems. Here, we report a simple yet effective chemical additive-based strategy that overcomes these limitations. This chemical additives-enhanced CRISPR/Cas12a-based RNA detection (CARD) enables femtomolar-level RNA detection using only a single crRNA, without the need for DNA activators, reverse transcription, or strand-displacement reactions. Notably, this approach can be adapted to single-stranded DNA, enabling ssDNA detection at attomolar levels. Collectively, CARD provides a straightforward, amplification-free, and highly sensitive diagnostic framework that might be readily extended to other CRISPR/Cas systems for ultrasensitive nucleic acid diagnostics.
Jun Chen, Haiyan Zheng, Lucas Guan et al.· Biosensors & bioelectronics· 0 citations
Abstract The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system has become a powerful genome-editing tool that uses RNA–DNA pairing to cleave target DNA with protospacer adjacent motif (PAM) sequences. While its primary function is well-studied, secondary activities remain poorly understood, causing unintended off-target effects. This study reports for the first time that Cas9 specifically cleaves the 5′ overhang of the non-target strand (NTS) in target double-stranded DNA. This specific cleavage requires an additional PAM element at the NTS 5′ region, is mediated by Cas9’s RuvC domain, and is regulated by the HNH domain. It depends on the exact positioning of the NTS 5′ end, but not on the overhang homopolymer sequence or overhang length. Adequate single-guide RNA–DNA complementarity is also essential. This discovery potentially advances our understanding of Cas9’s enzymatic versatility to enhance genome-editing precision and efficacy and offers new nucleic acid detection strategies. Based on this cleavage, we developed a sensitive assay for Severe Acute Respiratory Syndrome Coronavirus 2 pseudovirus down to 2.4 copies μL−1, demonstrated extremely high sensitivity in diagnostic applications.
Gene amplification plays a critical role in evolution and disease and is widely utilized to overexpress valuable gene products in biotechnology. To broaden these applications, we previously developed break-induced replication (BIR)-mediated tandem repeat expansion (BITREx), a method utilizing Cas9 nickase (nCas9) to amplify genetic sequences by driving tandem array expansion through ectopic BIR. Since BITREx efficiency depends on the guide RNA (gRNA) recruiting nCas9 to the array's flanking regions, here we develop a plasmid-based reporter system in budding yeast for the rapid identification of high-performing gRNAs. Furthermore, we introduce BITREx 2.0, a dual-nicking strategy that targets both sides of the gene array. We demonstrate that BITREx 2.0 is effective for both natural and synthetic arrays, enhancing expansion efficiency by up to an order of magnitude compared to the original single-nicking format. These advancements significantly broaden the applicability and efficiency of nCas9-mediated gene amplification across diverse biological and biotechnological contexts.
It is shown that short RNAs can directly occupy the canonical crRNA-binding channel and trigger a catalytically competent trans cleavage state in the absence of PAM recognition or canonical R-loop formation.
I. Iwe, S. Singh, K. Guan et al.· medRxiv· 0 citations
FlexiAsCas12a joins the repertoire of Cas12a PAM variants, enabling access to an increasing number of target sequences by Cas12a nucleases, and expands the range of recognized PAM sequences by Cas12a variants to include NATN, NCCN and GTCN sequences.