Framework-Hotspot Enhanced Trans Cleavage of CRISPR-Cas12a for Clinical Samples Detection

The trans-cleavage property of CRISPR-Cas12a system makes it an excellent tool for disease diagnosis. Nevertheless, most methods based on CRISPR-Cas system still require pre-amplification of the target to achieve the desired detection sensitivity. Here we generate Framework-Hotspot reporters (FHRs) with different local densities to investigate their effect on trans-cleavage activity of Cas12a. We find that the cleavage efficiency increases and the cleavage rate accelerates with increasing reporter density. We further construct a modular sensing platform with CRISPR-Cas12a-based target recognition and FHR-based signal transduction. Encouragingly, this modular platform enables sensitive (100 fM) and rapid (<15 min) detection of pathogen nucleic acids without pre-amplification, as well as detection of tumor protein markers in clinical samples. The design provides a facile strategy for enhanced trans cleavage of Cas12a, which accelerates and broadens its applications in biosensing.     

Highly efficient and safe genome editing by CRISPR-Cas12a using CRISPR RNA with a ribosyl-2′-O-methylated uridinylate-rich 3′-overhang in mouse zygotes

The CRISPR-Cas12a system has been developed to harness highly specific genome editing in eukaryotic cells. Given the relatively small sizes of Cas12a genes, the system has been suggested to be most applicable to gene therapy using AAV vector delivery. Previously, we reported that a U-rich crRNA enabled highly efficient genome editing by the CRISPR-Cas12a system in eukaryotic cells. In this study, we introduced methoxyl modifications at C2 in riboses in the U-rich 3′-overhang of crRNA. When mixed with Cas12a effector proteins, the ribosyl-2′-O-methylated (2-OM) U-rich crRNA enabled improvement of dsDNA digestibility. Moreover, the chemically modified U-rich crRNA achieved very safe and highly specific genome editing in murine zygotes. The engineered CRISPR-Cas12a system is expected to facilitate the generation of various animal models. Moreover, the engineered crRNA was evaluated to further improve a CRISPR genome editing toolset.Subject terms: Gene targeting, Genetic engineering     

MnO2 nanosheets as a carrier and accelerator for improved live-cell biosensing application of CRISPR/Cas12a

Besides gene-editing, the CRISPR/Cas12a system has also been widely used in in vitro biosensing, but its applications in live-cell biosensing are rare. One reason is lacking appropriate carriers to synchronously deliver all components of the CRISPR/Cas12a system into living cells. Herein, we demonstrate that MnO₂ nanosheets are an excellent carrier of CRISPR/Cas12a due to the two important roles played by them. Through a simple mixing operation, all components of the CRISPR/Cas12a system can be loaded on MnO₂ nanosheets and thus synchronously delivered into cells. Intracellular glutathione (GSH)-induced decomposition of MnO₂ nanosheets not only results in the rapid release of the CRISPR/Cas12a system in cells but also provides Mn²⁺ as an accelerator to promote CRISPR/Cas12a-based biosensing of intracellular targets. Due to the merits of highly efficient delivery, rapid intracellular release, and the accelerated signal output reaction, MnO₂ nanosheets work better than commercial liposome carriers in live-cell biosensing analysis of survivin messenger RNA (mRNA), producing much brighter fluorescence images in a shorter time. The use of MnO₂ nanosheets might provide a good carrier for different CRISPR/Cas systems and achieve the rapid and sensitive live-cell biosensing analysis of different intracellular targets, thus paving a promising way to promote the applications of CRISPR/Cas systems in living cells.

Herein, we demonstrate that MnO₂ nanosheets are an excellent carrier of CRISPR/Cas12a due to the two important roles played by them.     

CRISPR-Cas System for RNA Detection and Imaging

The system of clustered regularly interspaced short palindromic repeats(CRISPR)and CRISPR-associated endonucleases(Cas)have been widely used in gene editing,disease treatment,molecular diagnosis and chromosome imaging.On account of the programmable target recognition of CRISPR-Cas system and the specific targeting function toward RNA of type Ⅵ class Ⅱ Cas proteins,CRISPR-Cas system has been deployed as RNA recognition and detection tools,exhibiting promising application potentials in the field of RNA detection and imaging.In this review,we summarize the latest research progresses as well as development prospects of CRISPR-Cas system in RNA diagnosis and live cell RNA imaging.     

PAM-less conditional DNA substrates leverage trans-cleavage of CRISPR-Cas12a for versatile live-cell biosensing

The CRISPR-Cas system has been repurposed as a powerful live-cell imaging tool, but its utility is limited to genomic loci and mRNA imaging in living cells. Here, we demonstrated the potential of the CRISPR-Cas system as a generalizable live-cell biosensing tool by extending its applicability to monitor diverse intracellular biomolecules. In this work, we engineered a CRISPR-Cas12a system with a generalized stimulus-responsive switch mechanism based on PAM-less conditional DNA substrates (pcDNAs). The pcDNAs with stimulus-responsiveness toward a trigger were constructed from the DNA substrates featuring no requirement of a protospacer-adjacent motif (PAM) and a bubble structure. With further leveraging the trans-cleavage activity of CRISPR-Cas12a for signal reporting, we established a versatile CRISPR-based live-cell biosensing system. This system enabled the sensitive sensing of various intracellular biomolecules, such as telomerase, ATP, and microRNA-21, making it a helpful tool for basic biochemical research and disease diagnostics.

This work developed the PAM-less conditional DNA substrates that leverage the trans-cleavage effect of CRISPR-Cas12a to sense various biomolecules in living cells.     

An Update of Nucleic Acids Aptamers Theranostic Integration with CRISPR/Cas Technology

The clustered regularly interspaced short palindromic repeat (CRISPR)/Cas system is best known for its role in genomic editing. It has also demonstrated great potential in nucleic acid biosensing. However, the specificity limitation in CRISPR/Cas has created a hurdle for its advancement. More recently, nucleic acid aptamers known for their high affinity and specificity properties for their targets have been integrated into CRISPR/Cas systems. This review article gives a brief overview of the aptamer and CRISPR/Cas technology and provides an updated summary and discussion on how the two distinctive nucleic acid technologies are being integrated into modern diagnostic and therapeutic applications     

2′-O-Methyl modified guide RNA promotes the single nucleotide polymorphism (SNP) discrimination ability of CRISPR–Cas12a systems

The CRISPR–Cas12a system has been widely applied to genome editing and molecular diagnostics. However, off-target cleavages and false-positive results remain as major concerns in Cas12a practical applications. Herein, we propose a strategy by utilizing the 2′-O-methyl (2′-OMe) modified guide RNA (gRNA) to promote the Cas12a''s specificity. Gibbs free energy analysis demonstrates that the 2′-OMe modifications at the 3′-end of gRNA effectively suppress the Cas12a''s overall non-specific affinity while maintaining high on-target affinity. For general application illustrations, HBV genotyping and SARS-CoV-2 D614G mutant biosensing platforms are developed to validate the enhanced Cas12a''s specificity. Our results indicate that the 2′-OMe modified gRNAs could discriminate single-base mutations with at least two-fold enhanced specificity compared to unmodified gRNAs. Furthermore, we investigate the enhancing mechanisms of the 2′-OMe modified Cas12a systems by molecular docking simulations and the results suggest that the 2′-OMe modifications at the 3′-end of gRNA reduce the Cas12a''s binding activity to off-target DNA. This work offers a versatile and universal gRNA design strategy for highly specific Cas12a system development.

This study illustrates that 2′-O-methyl modified gRNAs improve the specificity of the CRISPR–Cas12a system (mg-CRISPR) via suppressing the Cas12a''s affinity to off-target DNA and provides an efficient strategy for high-specificity gRNA design.     

Programmable Live-Cell CRISPR Imaging with Toehold-Switch-Mediated Strand Displacement

The widespread application of CRISPR-Cas9 has transformed genome engineering. Nevertheless, the precision to control the targeting activity of Cas9 requires further improvement. We report a toehold-switch-based approach to engineer the conformation of single guide RNA (sgRNA) for programmable activation of Cas9. This activation circuit is responsive to multiple inputs and can regulate the conformation of the sgRNA through toehold-switch-mediated strand displacement. We demonstrate the orthogonal suppression and activation of Cas9 with orthogonal DNA inputs. Combination of toehold switches leads to a variety of intracellular Cas9 activation programs with simultaneous and orthogonal responses, through which multiple genome loci are displayed in different colors in a controllable manner. This approach provides a new route for programing CRISPR in living cells for genome imaging and engineering.     

Towards CRISPR powered electrochemical sensing for smart diagnostics

Even though global health has been steadily improved, the global disease burden associated with communicable and non-communicable diseases extensively increased healthcare expenditure. The present COVID-19 pandemic scenario has again ascertained the importance of clinical diagnostics as a basis to make life-saving decisions. In this context, there is a need for developing next-generation integrated smart real-time responsive biosensors with high selectivity and sensitivity. The emergence of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas biosensing systems has shown remarkable potential for developing next-generation biosensors. CRISPR/Cas integrated electrochemical biosensors (E-CRISPR) stands out with excellent properties. In this opinionated review, we illustrate the rapidly evolving applications for E-CRISPR-integrated detection systems towards biosensing and the future scope associated with E-CRISPR based diagnostics.     

A Conformational Restriction Strategy for the Control of CRISPR/Cas Gene Editing with Photoactivatable Guide RNAs**

The CRISPR/Cas system is one of the most powerful tools for gene editing. However, approaches for precise control of genome editing and regulatory events are still desirable. Here, we report the spatiotemporal and efficient control of CRISPR/Cas9- and Cas12a-mediated editing with conformationally restricted guide RNAs (gRNAs). This approach relied on only two or three pre-installed photo-labile substituents followed by an intramolecular cyclization, representing a robust synthetic method in comparison to the heavily modified linear gRNAs that often require extensive screening and time-consuming optimization. This tactic could direct the precise cleavage of the genes encoding green fluorescent protein (GFP) and the vascular endothelial growth factor A (VEGFA) protein within a predefined cutting region without notable editing leakage in live cells. We also achieved light-mediated myostatin (MSTN) gene editing in embryos, wherein a new bow-knot-type gRNA was constructed with excellent OFF/ON switch efficiency. Overall, our work provides a significant new strategy in CRISPR/Cas editing with modified circular gRNAs to precisely manipulate where and when genes are edited.     

CRISPR-Cas13a mediated nanosystem for attomolar detection of canine parvovirus type 2

The CPV-2 DNA extracted from dog's intestine was amplified using RPA and transcribed into an RNA copy. The RNA copy was incubated with Cas13a and crRNA, upon activation collateral cleavage activity of Cas13a cleaves all the target RNA and nonspecific RNA in the vicinity producing fluorescent signals within 30 min.     

DNA Origami Post-Processing by CRISPR-Cas12a

Customizable nanostructures built through the DNA-origami technique hold tremendous promise in nanomaterial fabrication and biotechnology. Despite the cutting-edge tools for DNA-origami design and preparation, it remains challenging to separate structural components of an architecture built from—thus held together by—a continuous scaffold strand, which in turn limits the modularity and function of the DNA-origami devices. To address this challenge, here we present an enzymatic method to clean up and reconfigure DNA-origami structures. We target single-stranded (ss) regions of DNA-origami structures and remove them with CRISPR-Cas12a, a hyper-active ssDNA endonuclease without sequence specificity. We demonstrate the utility of this facile, selective post-processing method on DNA structures with various geometrical and mechanical properties, realizing intricate structures and structural transformations that were previously difficult to engineer. Given the biocompatibility of Cas12a-like enzymes, this versatile tool may be programmed in the future to operate functional nanodevices in cells.     

Optical Control of a CRISPR/Cas9 System for Gene Editing by Using Photolabile crRNA

Currently CRISPR/Cas9 is a widely used efficient tool for gene editing. Precise control over the CRISPR/Cas9 system with high temporal and spatial resolution is essential for studying gene regulation and editing. Here, we synthesized a novel light-controlled crRNA by coupling vitamin E and a photolabile linker at the 5′ terminus to inactivate the CRISPR/Cas9 system. The vitamin E modification did not affect ribonucleoprotein (RNP) formation of Cas9/crRNA/tracrRNA complexes but did inhibit the association of RNP with the target DNA. Upon light irradiation, vitamin E-caged crRNA was successfully activated to achieve light-induced genome editing of vascular endothelial cell-growth factor A (VEGFA) in human cells through a T7E1 assay and Sanger sequencing as well as gene knockdown of EGFP expression in EGFP stably expressing cells. This new caging strategy for crRNA could provide new methods for spatiotemporal photoregulation of CRISPR/Cas9-mediated gene editing.     

A Photolabile Semiconducting Polymer Nanotransducer for Near‐Infrared Regulation of CRISPR/Cas9 Gene Editing

Noninvasive regulation of CRISPR/Cas9 gene editing is conducive to understanding of gene function and development of gene therapy; however, it remains challenging. Herein, a photolabile semiconducting polymer nanotransducer (pSPN) is synthesized to act as the gene vector to deliver CRISPR/Cas9 plasmids into cells and also as the photoregulator to remotely activate gene editing. pSPN comprises a ¹O₂‐generating backbone grafted with polyethylenimine brushes through ¹O₂‐cleavable linkers. NIR photoirradiation spontaneously triggers the cleavage of gene vectors from pSPN, resulting in the release of CRISPR/Cas9 plasmids and subsequently initiating gene editing. This system affords 15‐ and 1.8‐fold enhancement in repaired gene expression relative to the nonirradiated controls in living cells and mice, respectively. As this approach does not require any specific modifications on biomolecular components, pSPN represents the first generic nanotransducer for in vivo regulation of CRISPR/Cas9 gene editing.     

A DNA Origami-Based Gene Editing System for Efficient Gene Therapy in Vivo

DNA nanostructures have played an important role in the development of novel drug delivery systems. Herein, we report a DNA origami-based CRISPR/Cas9 gene editing system for efficient gene therapy in vivo. In our design, a PAM-rich region precisely organized on the surface of DNA origami can easily recruit and load sgRNA/Cas9 complex by PAM-guided assembly and pre-designed DNA/RNA hybridization. After loading the sgRNA/Cas9 complex, the DNA origami can be further rolled up by the locking strands with a disulfide bond. With the incorporation of DNA aptamer and influenza hemagglutinin (HA) peptide, the cargo-loaded DNA origami can realize the targeted delivery and effective endosomal escape. After reduction by GSH, the opened DNA origami can release the sgRNA/Cas9 complex by RNase H cleavage to achieve a pronounced gene editing of a tumor-associated gene for gene therapy in vivo. This rationally developed DNA origami-based gene editing system presents a new avenue for the development of gene therapy.     

Construction of Smart Stimuli-Responsive DNA Nanostructures for Biomedical Applications

DNA nanostructures have recently attracted increasing interest in biological and biomedical applications by virtue of their unique properties, such as structural programmability, multi-functionality, stimuli-responsive behaviors, and excellent biocompatibility. In particular, the intelligent responsiveness of smart DNA nanostructures to specific stimuli has facilitated their extensive development in the field of high-performance biosensing and controllable drug delivery. This minireview begins with different self-assembly strategies for the construction of various DNA nanostructures, followed by the introduction of a variety of stimuli-responsive functional DNA nanostructures for assembling metastable soft materials and for facilitating amplified biosensing. The recent achievements of smart DNA nanostructures for controllable drug delivery are highlighted. Finally, the current challenges and possible developments of this promising research are discussed in the fields of intelligent nanomedicine.     

Thermo‐triggered Release of CRISPR‐Cas9 System by Lipid‐Encapsulated Gold Nanoparticles for Tumor Therapy

CRISPR/Cas9 system is a powerful toolbox for gene editing. However, the low delivery efficiency is still a big hurdle impeding its applications. Herein, we report a strategy to deliver Cas9‐sgPlk‐1 plasmids (CP) by a multifunctional vehicle for tumor therapy. We condensed CPs on TAT peptide‐modified Au nanoparticles (AuNPs/CP, ACP) via electrostatic interactions, and coated lipids (DOTAP, DOPE, cholesterol, PEG2000‐DSPE) on the ACP to form lipid‐encapsulated, AuNPs‐condensed CP (LACP). LACP can enter tumor cells and release CP into the cytosol by laser‐triggered thermo‐effects of the AuNPs; the CP can enter nuclei by TAT guidance, enabling effective knock‐outs of target gene (Plk‐1) of tumor (melanoma) and inhibition of the tumor both in vitro and in vivo. This AuNPs‐condensed, lipid‐encapsulated, and laser‐controlled delivery system provides a versatile method for high efficiency CRISPR/Cas9 delivery and targeted gene editing for treatment of a wide spectrum of diseases.     

光控CRISPR技术的研究进展

成簇规则间隔短回文重复序列和成簇规则间隔短回文重复序列相关(CRISPR-Cas)系统提供了用于可编程基因组编辑的多功能工具. CRISPR与光遗传学及光化学生物学技术的结合产生了很多新的成果. 光激活的CRISPR-Cas系统能够在空间和时间上更好地调控RNA引导的核酸酶的活性. 近年来, 科学家结合CRISPR和多种光学技术, 开发出了一系列光激活的CRISPR工具. 这些工具让研究人员能够在空间、 时间和基因组坐标上进行高分辨率的生命活动研究. 本文概述了CRISPR系统、 基因编辑技术、 光遗传学和光化学生物学的研究进展, 并对光诱导的CRISPR技术的发展进行了展望.