Search

ABSTRACT White spot syndrome virus (WSSV) and Enterocytozoon hepatopenaei (EHP) represent the most economically destructive pathogens in the current shrimp industry. WSSV causes white spot disease (WSD) responsible for rapid shrimp mortality, while EHP stunts growth and therefore reduces overall productivity. Despite the importance of timely disease detection, current diagnostic methods for WSSV and EHP are typically singleplex, and those offering multiplex detection face issues such as complexity, low field compatibility and/or low sensitivity. Here, we introduce an orthogonal, multiplex CRISPR‐Cas assay for concomitant detection of WSSV and EHP. This method combines recombinase polymerase amplification (RPA) for target DNA enrichment with Cas12a and Cas13a enzymes for fluorescent detection. This assay produces distinct fluorescent colours for different diagnostic outcomes, allowing naked eye visualisation without ambiguity. Further validation reveals that the assay detects as few as 20 and 200 copies of target DNA from EHP and WSSV, respectively, while producing no false positives with DNA from other shrimp pathogens. Moreover, the assay excellently agrees with established PCR methods in evaluation of clinical samples. Requiring only 37°C and less than an hour to complete, multiplex CRISPR‐Cas assay presents a promising tool for onsite diagnostics, offering high accuracy while saving time and resources. [ABSTRACT FROM AUTHOR]

Sequence‐specific endonucleases have been key to the study of the mechanisms and control of DNA double‐strand break (DSB) repair and recombination, and the availability of CRISPR‐Cas nucleases over the last decade has driven rapid progress in the understanding and application of targeted recombination in many organisms, including plants. We present here an analysis of recombination at targeted chromosomal 5′ overhang DSB generated by the FnCas12a endonuclease in the plant, Arabidopsis thaliana. The much‐studied Cas9 nuclease cleaves DNA to generate blunt‐ended DSBs, but relatively less is known about the repair of other types of breaks, such as those with 5′‐overhanging ends. Sequencing the repaired breaks clearly shows that the majority of repaired DSB carry small deletions and are thus repaired locally by end‐joining recombination, confirmed by Nanopore sequencing of larger amplicons. Paired DSBs generate deletions at one or both cut‐sites, as well as deletions and reinsertions of the deleted segment between the two cuts, visible as inversions. While differences are seen in the details, overall the deletion patterns are similar between repair at single‐cut and double‐cut events, notwithstanding the fact that only the former involve cohesive DNA overhangs. A strikingly different repair pattern is however observed at breaks flanked by direct repeats. This change in sequence context results in the presence of a major alternative class of repair events, corresponding to highly efficient repair by single‐strand annealing recombination. [ABSTRACT FROM AUTHOR]

SUMMARY: With the advancement of CRISPR technologies, a comprehensive understanding of repair mechanisms following double‐strand break (DSB) formation is important for improving the precision and efficiency of genetic modifications. In plant genetics, two Cas nucleases are widely used, i.e. Cas9 and Cas12a, which differ with respect to PAM sequence composition, position of the DSB relative to the PAM, and DSB‐end configuration (blunt vs. staggered). The latter difference has led to speculations about different options for repair and recombination. Here, we provide detailed repair profiles for LbCas12a in Arabidopsis thaliana, using identical experimental settings previously reported for Cas9‐induced DSBs, thus allowing for a quantitative comparison of both nucleases. For both enzymes, non‐homologous end‐joining (NHEJ) produces 70% of mutations, whereas polymerase theta‐mediated end‐joining (TMEJ) generates 30%, indicating that DSB‐end configuration does not dictate repair pathway choice. Relevant for genome engineering approaches aimed at integrating exogenous DNA, we found that Cas12a similarly stimulates the integration of T‐DNA molecules as does Cas9. Long‐read sequencing of both Cas9 and Cas12a repair outcomes further revealed a previously underappreciated degree of DNA loss upon TMEJ. The most notable disparity between Cas9 and Cas12a repair profiles is caused by how NHEJ acts on DSB ends with short overhangs: non‐symmetric Cas9 cleavage produce 1 bp insertions, which we here show to depend on polymerase Lambda, whereas staggered Cas12a DSBs are not subjected to fill‐in synthesis. We conclude that Cas9 and Cas12a are equally effective for genome engineering purposes, offering flexibility in nuclease choice based on the availability of compatible PAM sequences. [ABSTRACT FROM AUTHOR]

Gene‐editing technology shows great potential in glioblastoma (GBM) therapy. Due to the complexity of GBM pathogenesis, a single gene‐editing‐based therapy is unlikely to be successful; therefore, a multi‐gene knockout strategy is preferred for effective GBM inhibition. Here, a non‐invasive, biodegradable brain‐targeted CRISPR/Cas12a nanocapsule is used that simultaneously targeted dual oncogenes, EGFR and PLK1, for effective GBM therapy. This cargo nanoencapsulation technology enables the CRISPR/Cas12a system to achieve extended blood half‐life, efficient blood‐brain barrier (BBB) penetration, active tumor targeting, and selective release. In U87MG cells, the combinatorial gene editing system resulted in 61% and 33% knockout of EGFR and PLK1, respectively. Following systemic administration, the CRISPR/Cas12a system demonstrated promising brain tumor accumulation that led to extensive EGFR and PLK1 gene editing in both U87MG and patient‐derived GSC xenograft mouse models with negligible off‐target gene editing detected through NGS. Additionally, CRISPR/Cas12a nanocapsules that concurrently targeted the EGFR and PLK1 oncogenes showed superior tumor growth suppression and significantly improved the median survival time relative to nanocapsules containing single oncogene knockouts, signifying the potency of the multi‐oncogene targeting strategy. The findings indicate that utilization of the CRISPR/Cas12a combinatorial gene editing technique presents a practical option for gene therapy in GBM. [ABSTRACT FROM AUTHOR]

SUMMARY: RNA‐guided endonucleases originating from the bacterial CRISPR/Cas system are a versatile tool for targeted gene editing. To determine the functional relevance of a gene of interest, deletion of the entire open reading frame (ORF) by two independent double‐strand breaks (DSBs) is particularly attractive. This strategy greatly benefits from high editing efficiency, which is strongly influenced by the Cas endonuclease version used. We developed two reporter switch‐on assays, for quantitative comparison and optimization of Cas constructs. The assays are based on four components: (i) A reporter gene, the mRNA of which carries a hairpin (HP) loop targeted by (ii) the endoribonuclease Csy4. Cleavage of the mRNA at the HP loop by Csy4 abolishes the translation of the reporter. Csy4 was used as the target for full deletion. (iii) A Cas system targeting sites flanking the Csy4 ORF with a 20‐bp spacer either side to preferentially detect full‐deletion events. Loss of functional Csy4 would lead to reporter gene expression, allowing indirect quantification of Cas‐mediated deletion events. (iv) A reference gene for normalization. We tested these assays on Nicotiana benthamiana leaves and Lotus japonicus calli induced on hypocotyl sections, using Firefly luciferase and mCitrine as reporter genes and Renilla luciferase and hygromycin phosphotransferase II as reference genes, respectively. We observed a >90% correlation between reporter expression and full Csy4 deletion events, demonstrating the validity of these assays. The principle of using the Csy4–HP module as Cas target should be applicable to other editing goals including single DSBs in all organisms. Significance Statement: Cas endonuclease systems have revolutionized genome editing but rapid quantitative assays for the optimization of this process that circumvent the need for DNA extraction from individual plants are rare or lacking. Here, we describe two quantitative reporter switch‐on assays for the direct side‐by‐side comparison of the editing efficiency of different Cas systems, like Cas9 and Cas12a, the principles of which can be applied for optimizing gene editing in any plant species. [ABSTRACT FROM AUTHOR]

Rapid and accurate diagnostic tests are fundamental for improving patient outcomes and combating infectious diseases. The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) Cas12a‐based detection system has emerged as a promising solution for on‐site nucleic acid testing. Nonetheless, the effective design of CRISPR RNA (crRNA) for Cas12a‐based detection remains challenging and time‐consuming. In this study, we propose an enhanced crRNA design system with deep learning for Cas12a‐mediated diagnostics, referred to as EasyDesign. This system employs an optimized convolutional neural network (CNN) prediction model, trained on a comprehensive data set comprising 11,496 experimentally validated Cas12a‐based detection cases, encompassing a wide spectrum of prevalent pathogens, achieving Spearman's ρ = 0.812. We further assessed the model performance in crRNA design for four pathogens not included in the training data: Monkeypox Virus, Enterovirus 71, Coxsackievirus A16, and Listeria monocytogenes. The results demonstrated superior prediction performance compared to the traditional experiment screening. Furthermore, we have developed an interactive web server (https://crispr.zhejianglab.com/) that integrates EasyDesign with recombinase polymerase amplification (RPA) primer design, enhancing user accessibility. Through this web‐based platform, we successfully designed optimal Cas12a crRNAs for six human papillomavirus (HPV) subtypes. Remarkably, all the top five predicted crRNAs for each HPV subtype exhibited robust fluorescent signals in CRISPR assays, thereby suggesting that the platform could effectively facilitate clinical sample testing. In conclusion, EasyDesign offers a rapid and reliable solution for crRNA design in Cas12a‐based detection, which could serve as a valuable tool for clinical diagnostics and research applications. Highlights: Advanced crRNA design system: Developed EasyDesign, utilizing a convolutional neural network (CNN) trained with over 11,000 diagnostic‐target pairs to enable the creation of highly sensitive crRNAs for Cas12a‐based nucleic acid diagnostics.Proven predictive capabilities: EasyDesign demonstrates superior predictive performance for crRNA‐mediated Cas12a detection, validated through its successful application in designing diagnostics for a variety of viruses in clinical settings.User‐friendly web platform: Features an intuitive web platform that streamlines the creation of CRISPR/Cas12a‐based diagnostic tools, thereby enhancing accessibility and usability for both researchers and clinicians. [ABSTRACT FROM AUTHOR]

The RNA‐programmed CRISPR effector protein Cas12a has emerged as a powerful tool for gene editing and molecular diagnostics. However, additional bio‐engineering strategies are required to achieve control over Cas12a activity. Here, we show that Toehold Switch DNA hairpins, presenting a rationally designed locked protospacer adjacent motif (PAM) in the loop, can be used to control Cas12a in response to molecular inputs. Reconfiguring the Toehold Switch DNA from a hairpin to a duplex conformation through a strand displacement reaction provides an effective means to modulate the accessibility of the PAM, thereby controlling the binding and cleavage activities of Cas12a. Through this approach, we showcase the potential to trigger downstream Cas12a activity by leveraging proximity‐based strand displacement reactions in response to target binding. By utilizing the trans‐cleavage activity of Cas12a as a signal transduction method, we demonstrate the versatility of our approach for sensing applications. Our system enables rapid, one‐pot detection of IgG antibodies and small molecules with high sensitivity and specificity even within complex matrices. Besides the bioanalytical applications, the switchable PAM‐engineered Toehold Switches serve as programmable tools capable of regulating Cas12a‐based targeting and DNA processing in response to molecular inputs and hold promise for a wide array of biotechnological applications. [ABSTRACT FROM AUTHOR]

Rapid identification of organisms is essential for many biological and medical disciplines, from understanding basic ecosystem processes, disease diagnosis, to the detection of invasive pests. CRISPR‐based diagnostics offers a novel and rapid alternative to other identification methods and can revolutionize our ability to detect organisms with high accuracy. Here we describe a CRISPR‐based diagnostic developed with the universal cytochrome‐oxidase 1 gene (CO1). The CO1 gene is the most sequenced gene among Animalia, and therefore our approach can be adopted to detect nearly any animal. We tested the approach on three difficult‐to‐identify moth species (Keiferia lycopersicella, Phthorimaea absoluta and Scrobipalpa atriplicella) that are major invasive pests globally. We designed an assay that combines recombinase polymerase amplification (RPA) with CRISPR for signal generation. Our approach has a much higher sensitivity than real‐time PCR assays and achieved 100% accuracy for identification of all three species, with a detection limit of up to 120 fM for P. absoluta and 400 fM for the other two species. Our approach does not require a sophisticated laboratory, reduces the risk of cross‐contamination, and can be completed in less than 1 h. This work serves as a proof of concept that has the potential to revolutionize animal detection and monitoring. [ABSTRACT FROM AUTHOR]

Human mpox is a zoonotic disease, similar to smallpox, caused by the mpox virus, which is further subdivided into Congo Basin and West African clades with different pathogenicity. In this study, a novel diagnostic protocol utilizing clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR‐associated 12a nuclease (CRISPR/Cas12a)‐mediated recombinase polymerase amplification (RPA) was developed to identify mpox in the Congo Basin and West Africa (CRISPR‐RPA). Specific RPA primers targeting D14L and ATI were designed. CRISPR‐RPA assay was performed using various target templates. In the designed CRISPR‐RPA reaction system, the exponentially amplified RPA amplification products with a protospacer adjacent motif (PAM) site can locate the Cas12a/crRNA complex to its target regions, which successfully activates the CRISPR/Cas12a effector and achieves ultrafast trans‐cleavage of a single‐stranded DNA probe. The limit of detection for the CRISPR‐RPA assay was 10 copies per reaction for D14L‐ and ATI‐plasmids. No cross‐reactivity was observed with non‐mpox strains, confirming the high specificity of the CRISPR‐RPA assay for distinguishing between the Congo Basin and West African mpox. The CRISPR‐RPA assay can be completed within 45 min using real‐time fluorescence readout. Moreover, the cleavage results were visualized under UV light or an imaging system, eliminating the need for a specialized apparatus. In summary, the developed CRISPR/RPA assay is a visual, rapid, sensitive, and highly specific detection technique that can be used as an attractive potential identification tool for Congo Basin and West African mpox in resource‐limited laboratories. [ABSTRACT FROM AUTHOR]

SUMMARY: High efficiency site‐directed chromosomal integration of exogenous DNA in plants remains a challenge despite recent advances in genome editing technologies. One approach to mitigate this problem is to increase the effective concentration of the donor DNA at the target site of interest. HUH endonucleases (ENs) coordinate rolling circle replication. In vitro, they can form stable covalent bonds with DNA that carries their recognition motifs. When fused to a CRISPR‐associated endonuclease, HUH ENs may improve integration rates by increasing the local donor concentration through tethering of the donor to the CRISPR nuclease. We tested this hypothesis by using chimeric proteins between LbCas12a as a CRISPR‐associated endonuclease and the HUH EN from Faba Bean Necrotic Yellow Virus in soybean (Glycine max). Two fusion protein configurations were tested to integrate a 70‐nt oligonucleotide donor into a commercially important target site using protoplasts and in planta transformation. Site‐directed integration rates of the donor DNA, when tethered to the fusion protein, reached about 26% in plants and were up to four‐fold higher than in untethered controls. Integrations via canonical homology‐directed repair or non‐homologous end joining were promoted by tethering in a similar fashion. This study is the first demonstration of HUH EN‐associated tethering to improve site‐directed DNA integration in plants. Significance Statement: An exceptionally high rate of site‐directed integration of exogenous DNA into the soybean (Glycine max) genome was achieved using a novel method to increase the local donor DNA concentration around the CRISPR target site of interest. A donor DNA was covalently tethered to a fusion protein between LbCas12a and the HUH endonuclease of Faba Bean Necrotic Yellow Virus, which was delivered into soybean protoplasts and plants as part of a ribonucleoprotein complex. [ABSTRACT FROM AUTHOR]

Summary: The development of CRISPR‐based editors recognizing distinct protospacer‐adjacent motifs (PAMs), or having different spacer length/structure requirements broadens the range of possible genomic applications. We evaluated the natural and engineered variants of Cas12a (FnCas12a and LbCas12a) and Cas9 for their ability to induce mutations in endogenous genes controlling important agronomic traits in wheat. Unlike FnCas12a, LbCas12a‐induced mutations in the wheat genome, even though with a lower rate than that reported for SpCas9. The eight‐fold improvement in the gene editing efficiency was achieved for LbCas12a by using the guides flanked by ribozymes and driven by the RNA polymerase II promoter from switchgrass. The efficiency of multiplexed genome editing (MGE) using LbCas12a was mostly similar to that obtained using the simplex RNA guides and showed substantial increase after subjecting transgenic plants to high‐temperature treatment. We successfully applied LbCas12a‐MGE for generating heritable mutations in a gene controlling grain size and weight in wheat. We showed that the range of editable loci in the wheat genome could be further expanded by using the engineered variants of Cas12a (LbCas12a‐RVR) and Cas9 (Cas9‐NG and xCas9) that recognize the TATV and NG PAMs, respectively, with the Cas9‐NG showing higher editing efficiency on the targets with atypical PAMs compared to xCas9. In conclusion, our study reports a set of validated natural and engineered variants of Cas12a and Cas9 editors for targeting loci in the wheat genome not amenable to modification using the original SpCas9 nuclease. [ABSTRACT FROM AUTHOR]

Accurate, rapid, and multiplexed nucleic acid detection is of great value for applications in biomedicine and agriculture. Here, we demonstrated a one‐step Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) diagnostic method for the digital and multiplexed detection of both single‐stranded (ss) and double‐stranded (ds) DNA on a handheld smartphone device. ssDNA targets exhibited faster reaction kinetics of this single‐step CRISPR–Cas12a assay than dsDNA counterparts. Under optimized conditions, picomolar levels of ssDNA targets can be detected in 96‐well plates by using a benchtop plate reader. The detection sensitivity can be further improved to 5 fM when running the reaction in a microwell‐based digital assay chip. Multiplexed detection of hepatitis B virus and human papillomavirus DNA markers was demonstrated on the smartphone‐based platform. Finally, the one‐step CRISPR–Cas12a assay performed robustly in human serum samples with a recovery rate of ssDNA detection between 96% and 105.6%, suggesting a high potential for clinical diagnostic applications in point‐of‐care settings. [ABSTRACT FROM AUTHOR]

The use of CRISPR‐Cas proteins for the creation of multiplex genome engineering represents an important avenue for crop improvement, and further improvements for creation of knock‐in plant lines via CRISPR‐based technologies may enable the high‐throughput creation of designer alleles. To circumvent limitations of the commonly used CRISPR‐Cas9 system for multiplex genome engineering, we explored the use of Moraxella bovoculi 3 Cas12a (Mb3Cas12a) for multiplex genome editing in Arabidopsis thaliana. We identified optimized cis‐regulatory sequences for driving expression of single‐transcript multiplex crRNA arrays in A. thaliana, resulting in stable germline transmission of Mb3Cas12a‐edited alleles at multiple target sites. By utilizing this system, we demonstrate single‐transcript multiplexed genome engineering using of up to 13 crRNA targets. We further show high target specificity of Mb3Cas12a‐based genome editing via whole‐genome sequencing. Taken together, our method provides a simplified platform for efficient multiplex genome engineering in plant‐based systems. [ABSTRACT FROM AUTHOR]

Cas12a is an RNA‐guided DNA endonuclease of the type V‐A CRISPR‐Cas system that has evolved convergently with the type II Cas9 protein. We previously showed that proline substitutions in the bridge helix (BH) impart target DNA cleavage selectivity in Streptococcus pyogenes (Spy) Cas9. Here, we examined a BH variant of Cas12a from Francisella novicida (FnoCas12aKD2P) to test mechanistic conservation. Our results show that for RNA‐guided DNA cleavage (cis‐activity), FnoCas12aKD2P accumulates nicked products while cleaving supercoiled DNA substrates with mismatches, with certain mismatch positions being more detrimental for linearization. FnoCas12aKD2P also possess reduced trans‐single‐stranded DNA cleavage activity. These results implicate the BH in substrate selectivity in both cis‐ and trans‐cleavages and show its conserved role in target discrimination among Cas nucleases. [ABSTRACT FROM AUTHOR]

Rapid pathogen detection is critical for prompt treatment, interrupting transmission routes, and decreasing morbidity and mortality. The V‐type CRISPR system had been used for rapid pathogen detection. However, whether single‐stranded DNA in CRISPR system can cause false positives remains undetermined. Herein, we show that high molar concentration of Cas12a effector tolerated more mismatches on ssDNA and activated its trans‐cleavage activity at six base matches. Reducing Cas12a and crRNA molar concentration increased the minimal base‐match number required for Cas12a ssDNA activation to 11, which reducing nonspecific activation. We then established a Cas12a‐based M tuberculosis detection system with a primer having an 8 bp overlap with crRNA. This system did not exhibit primer‐induced false positives, and minimum detection copy reached 1 copy/uL (inputting 1‐μL sample) in standard strains. The Cas12a‐based M tuberculosis detection system showed 80.0% sensitivity and 100.0% specificity in verification using clinical specimens, compared with Xpert MTB/RIF, which showed 72.0% sensitivity and 90.9% specificity. All these results prove that appropriate concentration of cas12a effector can effectively perform nucleic acid detection. [ABSTRACT FROM AUTHOR]

Keywords: CRISPR-Cas; Cas12a; Chimeric crRNA EN CRISPR-Cas Cas12a Chimeric crRNA 87 90 4 01/29/21 20210101 NES 210101 RNA residues in the guide region of crRNA for Cas12a can be partially replaced by DNA. (d) DNA cleavage reaction rate (kobs) (right) of Cas12a guided by chimeric crRNAs containing various numbers of DNA residues (left). gl Recently, the effect of DNA sugar backbone in guide RNAs of Cas9 has been investigated. In contrast, the chimeric crRNAs containing nine or more DNA residues (9 DNA, 10 DNA, 11 DNA, and 14 DNA) showed substantially lowered rate constants (0.36-0.09 min SP -1 sp ). (c) Melting temperatures (Tm) of 21-mer RNA/DNA duplexes with various numbers of DNA replacement in the RNA strand. gl We next examined whether the cleavage activity could be dependent on the thermal stability of the crRNA/target-DNA duplex. [Extracted from the article]

Summary: Recently, CRISPR‐Cas12a (Cpf1) from Prevotella and Francisella was engineered to modify plant genomes. In this report, we employed CRISPR‐LbCas12a (LbCpf1), which is derived from Lachnospiraceae bacterium ND2006, to edit a citrus genome for the first time. First, LbCas12a was used to modify the CsPDS gene successfully in Duncan grapefruit via Xcc‐facilitated agroinfiltration. Next, LbCas12a driven by either the 35S or Yao promoter was used to edit the PthA4 effector binding elements in the promoter (EBEPthA4‐CsLOBP) of CsLOB1. A single crRNA was selected to target a conserved region of both Type I and Type II CsLOBPs, since the protospacer adjacent motif of LbCas12a (TTTV) allows crRNA to act on the conserved region of these two types of CsLOBP. CsLOB1 is the canker susceptibility gene, and it is induced by the corresponding pathogenicity factor PthA4 in Xanthomonas citri by binding to EBEPthA4‐CsLOBP. A total of seven 35S‐LbCas12a‐transformed Duncan plants were generated, and they were designated as #D35s1 to #D35s7, and ten Yao‐LbCas12a‐transformed Duncan plants were created and designated as #Dyao1 to #Dyao10. LbCas12a‐directed EBEPthA4‐CsLOBP modifications were observed in three 35S‐LbCas12a‐transformed Duncan plants (#D35s1, #D35s4 and #D35s7). However, no LbCas12a‐mediated indels were observed in the Yao‐LbCas12a‐transformed plants. Notably, transgenic line #D35s4, which contains the highest mutation rate, alleviates XccΔpthA4:dCsLOB1.4 infection. Finally, no potential off‐targets were observed. Therefore, CRISPR‐LbCas12a can readily be used as a powerful tool for citrus genome editing. [ABSTRACT FROM AUTHOR]

The accelerating pace of scientific discovery is being driven by the development of transformative technologies that are expanding traditional scientific boundaries. In ecology, the concept that ecosystem structure can be monitored through the stew of genetic material released into the environment by local organisms is now a highly active area of research. At the same time that the use of environmental DNA (eDNA) has gained popularity in ecology, CRISPR genome editing technology has been revolutionizing organismal biology and the biomedical sciences. In a From the Cover article in this issue of Molecular Ecology Resources, Williams et al. (2019) report about the fusion of CRISPR technology with molecular ecology to improve the in situ processing capabilities of eDNA applications. Piloting this new CRISPR technology on aquatic systems, the authors describe tools to accurately identify the presence of Atlantic salmon (Salmo salar) from eDNA, using conditions that are highly amenable to implementation in the field. This overcomes a major barrier restricting the use of eDNA, opening a door to expanded use of the technology in ecological research. [ABSTRACT FROM AUTHOR]