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8 recordsLinked to original sources
PubMed2026

Repeat region engineering of Cas13a crRNA enables conformational gating-based autocatalytic CRISPR biosensing.

CrRNA engineering has emerged as a pivotal strategy for extending CRISPR-Cas13a biosensing. However, structural modulation of the direct repeat (DR) region remains exceptionally challenging due to its intricate architecture and the high energetic barrier of the Cas13a-crRNA interface, which is conventionally viewed as a rigid and immutable scaffold. Here, we demonstrate that the DR region is instead a programmable topological element with unexpected structural plasticity. By systematically engineering the DR through sequence insertion and structural splitting, we identified multiple DR variants that retain robust catalytic activity. Crucially, this topological reconfiguration enables Cas13a activity to be precisely gated by unmodified nucleic acid blockers, a level of regulation unattainable with the wild-type crRNA. Building on this flexible modulation, we developed Dre-CRISPR, a DR-engineered platform that couples target-triggered DR restoration to a self-reinforcing autocatalytic loop. This self-amplifying system provides a 2 × 106-fold sensitivity enhancement over nonamplified systems. Furthermore, the Dre-CRISPR platform extends the diagnostic scope of Cas13a to a broader spectrum of analytes, ranging from microRNAs to enzymatic activities and heavy metal ions. Our findings redefine the crRNA scaffold as a versatile signaling node and provide a generalizable framework for developing high-sensitivity, self-amplifying CRISPR biosensors through topology-driven guide RNA engineering.

CRISPR-Associated Proteins↗
PubMed2026

Engineering CRISPR for Point-of-Care Tests.

CRISPR-based molecular diagnostics have emerged as powerful and programmable platforms that enable sensitive and specific detection for disease management and epidemiological surveillance. Advances in CRISPR engineering and assay design are driving the emergence of next-generation detection platforms that are highly sensitive, rapid, and amenable to field deployment. These engineering breakthroughs have the potential to reshape point-of-care tests (POCT) and transform how emerging and persistent health threats are monitored in decentralized and resource-limited settings. Herein, we systematically review the recent advancements in CRISPR engineering strategies aimed at improving detection sensitivity and specificity, eliminating the dependence on preamplification, and enabling robust POC deployment. The discussed strategies encompass both the rational engineering of CRISPR ribonucleoproteins (RNPs) and the optimization of downstream signaling modules for molecular diagnostic applications. We further highlight key challenges and future perspectives that may inspire impactful research directions and accelerate the advancement of CRISPR engineering strategies toward robust, field-deployable POCT platforms.

CRISPR-Cas Systems↗
PubMed2026

Visual Detection and Stratification of Pathogenic mtDNA SNV Heteroplasmy by Balancing FnCas12a Signal Output and Allelic Discrimination.

Assessment of pathogenic mitochondrial DNA (mtDNA) single-nucleotide variant (SNV) heteroplasmy is important for molecular diagnostics, yet rapid visual profiling remains analytically challenging because an assay must combine single-nucleotide allelic discrimination, mutant-fraction-associated readout, and suitable target access. Herein, we report VISTA (visual identification and stratification of targeted mtDNA alleles), a broad-PAM FnCas12a assay that rebalances trans-cleavage signal output and mutant-wild-type discrimination for visual mtDNA SNV heteroplasmy analysis. VISTA uses unmodified FnCas12a with relaxed TTN PAM recognition and integrates crRNA spacer-length engineering with PEG8000/acBSA reaction tuning to improve the practical signal-discrimination balance without nuclease engineering. At the m.3243A>G model locus, spacer truncation enhanced mutant-wild-type discrimination, while molecular-dynamics simulations identified spacer-dependent differences between matched and mismatched complexes at the crRNA-DNA interface. The optimized assay resolved defined synthetic m.3243A>G heteroplasmy gradients by fluorescence imaging and was further adapted to lateral-flow detection. In locus-specific analyses of a deidentified collection of 74 peripheral-blood samples, fluorescence and lateral-flow readouts achieved ROC AUC values above 0.9 for mutant-allele classification after target-region amplification. Fluorescence supported heteroplasmy-associated profiling, whereas lateral flow provided a visual, semiquantitative readout for relative ranking based on the T/C ratio rather than absolute heteroplasmy measurement. VISTA therefore provides an accessible dual-readout analytical strategy for visual detection and heteroplasmy-associated profiling by tuning the FnCas12a signal output and allelic discrimination.

DNA, Mitochondrial↗
PubMed2026

DNA-guided CRISPR-Cas12 for cellular RNA targeting.

Here, we present ΨDNA, a DNA-based guide that enables RNA targeting by Cas12 nucleases, overcoming the traditional reliance on RNA-guided systems. We engineer ΨDNA to mimic a CRISPR RNA (crRNA) scaffold in reverse orientation, allowing AsCas12a and Cas12i1 to recognize RNA and trigger strong single-stranded DNA trans-cleavage for sensitive detection of diverse RNA species, including 100% accurate hepatitis C virus RNA detection in clinical samples. ΨDNA also achieves 70-95% multiplex knockdown of endogenous intracellular RNA transcripts through ribosome stalling across multiple human cell lines. Mechanistic studies reveal that activity depends on a stem loop that stabilizes a catalytically competent Cas12-ΨDNA-RNA complex. Lastly, codelivery of crRNA and ΨDNA enables simultaneous DNA editing and RNA knockdown with a single effector and modular fusions of different enzymes to AsCas12a extend ΨDNA to RNase H-mediated RNA degradation and METTL3-based epitranscriptomic editing. Together, ΨDNA guides constitute an adaptable toolkit that extends Cas12 systems beyond genome editing and diagnostics to enable precise, programmable control of cellular transcriptomes and their epitranscriptomic marks.

Journal Article↗
PubMed2024

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. ΨDNAs are effective in sensing short and long RNAs and demonstrated 100% accuracy for detecting HCV RNA in clinical samples. We discovered that ΨDNAs can guide certain Cas12 enzymes for RNA targeting in cells, enhancing mRNA degradation via ribosome stalling and enabling multiplex knockdown of multiple RNA transcripts. This study establishes ΨDNA as a robust alternative to RNA guides, augmenting the potential of CRISPR-Cas12 for diagnostic applications and targeted RNA modulation in cellular environments.

Journal Article↗
PubMed2025

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. ΨDNAs are effective in sensing short and long RNAs and demonstrated 100% accuracy for detecting HCV RNA in clinical samples. We discovered that ΨDNAs can guide certain Cas12 enzymes for RNA targeting in cells, enhancing mRNA degradation via ribosome stalling and enabling multiplex knockdown of multiple RNA transcripts. This study establishes ΨDNA as a robust alternative to RNA guides, augmenting CRISPR-Cas12's potential for diagnostic applications and for targeted RNA modulation in cellular environments.

Journal Article↗
PubMed2026

Synergistic engineering of Casδ nuclease for robust genome editing.

Casδ is a recently identified evolutionary transitional CRISPR system characterized by its compact size (~900 amino acids), broad temperature tolerance, and guidance by a short crRNA without the requirement of a tracrRNA. However, the low editing efficiency of Casδ in eukaryotic cells limits its application. Here, we have developed a hierarchical engineering strategy to improve the genome editing activity of Casδ-1, with optimization focused on enhancing its interactions with the crRNA, the protospacer adjacent motif (PAM) duplex, the single-stranded DNA substrate, and the RNA-DNA heteroduplex. Through this strategy, we successfully generated an activity-enhanced Casδ-1 variant, designated enCasδ, which harbors 9 amino acid substitutions that synergistically augment its editing efficiency. In human cell lines, enCasδ showed 1.3- to 29.3-fold higher editing activity than the wild-type Casδ-1 across ten tested genomic loci, with an average editing efficiency of 54.6%. In addition, enCasδ also mediated robust genome editing in maize; its editing efficiency increased by an average of 5.3-fold relative to Casδ-1, and reached up to an average of 80% at the TS4 and PSY1 loci in stable transgenic lines. The overall editing performance of enCasδ was comparable to that of Streptococcus pyogenes Cas9 (SpCas9) and other Cas12 nucleases. Collectively, enCasδ represents a highly optimized Casδ-1 variant that broadens the applicability of the Casδ CRISPR system and facilitates robust genome editing in both animal cells and plants.

Gene Editing↗
PubMed2025

Engineering and comparison of cas12a-based genome editing systems in plants.

While Cas9 and Cas12a are both RNA-guided endonucleases used for genome editing, only Cas12a is able to process pre-crRNA via its additional ribonuclease activity. This feature reduces the complexity of Cas12a versus Cas9-based genome editing systems thus providing an attractive alternative for generating site-specific mutations in plants. Here we aimed to improve the efficiency of the cas12a-based generation of two double-strand breaks flanking the open reading frame of a target gene, leading to its full deletion. To this end, we compared the relative impact of different components on cas12a-based gene deletion efficiency in three different eudicotyledons, Arabidopsis thaliana, Lotus japonicus, and Nicotiana benthamiana. We detected the highest cas12a-based editing efficiency with a combination of suitable promoters for crRNA and cas12a expression, a tandem terminator to control cas12a expression, a re-coded cas12a, adapted to the codon usage of Arabidopsis and engineered to carry introns, and encoding a Cas12a flanked by a nuclear localization signal at both ends. Our work revealed the high potential for improving cas12a-based genome editing systems for plant genetic research.

Gene Editing↗