An engineered Cas12a enables higher-order combinatorial functional genomic screens using CRISPR interference.

Synthetic DNA guides (crDNA) reprogram Cas12a nucleases for RNA targeting.

Single-cell CRISPR (Perturb-seq) screens have primarily relied on Cas9 whereas Cas12a, despite its unique effectiveness for multiplex guide expression, remains underexplored. This may be due to Cas12a...

The anti-CRISPR protein AcrVA2 specifically interrupts Cas12a biogenesis by triggering co-translational mRNA degradation.

An engineered Cas12a enables higher-order combinatorial functional genomic screens using CRISPR interference.

A review discusses the current state of CRISPR-mediated genetic manipulation in human cells, animals, and plants and considers its future potential.

CRISPR-Cas12a, an RNA-based DNA targeting system, is widely used for genome editing and biomarker detection. To mitigate the off-target DNA cleavage of Cas12a, we previously developed a Francisella novicida Cas12a variant (FnoCas12aKD2P) by introducing double proline substitutions (K969P/D970P) in a conserved arginine-rich helix called the bridge helix (BH). In this work, we use a combinatorial approach to understand the molecular mechanisms of BH-mediated activation of Cas12a for DNA cleavage. We report five structures of FnoCas12aKD2P that are at different states of conformational activation. Comparison of the variant and wild-type (FnoCas12aWT) structures, along with activity assays and computational simulations, establishes the loop-to-helical transition and bending of the BH as an allosteric trigger for RNA-DNA hybrid propagation. These changes track with the previously reported coupled remodeling of BH and helix 1 of RuvC motif-II as well as the REC lobe movements needed to accom

The CRISPR/Cas12a system has emerged as a highly useful tool for genomic editing due to its ability to process multiple guide RNAs (gRNAs) from a single promoter. In this study, we explored the potent...

CRISPR nuclease-mediated gene knock-out is limited by suboptimal sgRNAs, inaccessible target sites, and silent mutations. Here, we present a Cas12a-based system that targets each gene with four sgRNAs...

Synthetic DNA guides (crDNA) reprogram Cas12a nucleases for RNA targeting.

The anti-CRISPR protein AcrVA2 specifically interrupts Cas12a biogenesis by triggering co-translational mRNA degradation.

The detection of food contamination in a swift and sensitive manner is essential for safeguarding public health. Clustered regularly interspaced short palindromic repeats (CRISPR)-based assays for nucleic acid detection are renowned for their high specificity and convenient, related studies have focused on refining the Cas protein and optimizing the CRISPR (cr)RNAs design within CRISPR-based assays for enhancing the sensitivity of nucleic acid detection. Our research offers innovative insights into enhancing the fluorescence signal output intensity from a physical standpoint, thereby presenting a practical and cost-effective strategy to lower the detection thresholds in CRISPR-based assays. By a layer of microsphere arrays was spread onto the bottom of the microfluidic chip to enhance the fluorescence signal of the sample via self-assembly of the microspheres. Recombinase polymerase amplification (RPA) was used to amplify target sequences, followed by crRNA binding to activate Cas enzyme, cleaving fluorescein amidite (FAM)-labeled reporters and emitting a fluorescent signal. The method successfully identified SARS-CoV-2 positive samples (10 clinical samples and 8 environmental contamination samples) and distinguished them from negative samples. Meanwhile, it successfully detected 4 food contamination Shigella samples and 5 clinical Shigella samples. In this study, the developed method exhibited a detection limit (LoD) of 75 fM for SARS-CoV-2 (POCT with USB camera: 50 fM) and 100 fM for Shigella (POCT with USB camera: 75 fM). It also demonstrated promising sensitivity (100%) and specificity (100%) in a small-sample validation. Combined portable and automated detection was achieved using a smartphone to receive and process the fluorescent signals obtained from the samples. The detection platform developed in this study is not only applicable for the detection of pathogens in cold-chain food products, but also extends to pathogen detection in community hospitals and resource-limited areas, providing an efficient solution for rapid pathogen screening in different settings. Moreover, different nucleic acid samples can be detected by changing the RPA primer and CRISPR crRNA. This method provides a paradigm for studying enhanced fluorescence signaling and holds significant potential to advance the commercialization and practical use of CRISPR fluorescence sensors.

Biolistic delivery of intron-optimized LbCas12a and its variants into shoot apical meristems results in efficient multiplexed genome editing in common whea