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The novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a worldwide epidemic of the lethal respiratory coronavirus disease (COVID-19), necessitating urgent development of specific and effective therapeutic tools. Among several therapeutic targets of coronaviruses, the spike protein is of great significance due to its key role in host invasion. Here, we report a potential anti-SARS-CoV-2 strategy based on the CRISPR-Cas13a system. Methods: A comprehensive set of bioinformatics methods, including sequence alignment, structural comparison, and molecular docking, was utilized to identify a SARS-CoV-2-spike(S)-specific segment. A tiling crRNA library targeting this specific RNA segment was designed, and optimal crRNA candidates were selected using in-silico methods. The efficiencies of the crRNA candidates were tested in human HepG2 and AT2 cells. Results: The most effective crRNA sequence inducing a robust cleavage effect on S and a potent collateral cleavage effect were identified. Conclusions: This study provides a rapid design pipeline for a CRISPR-Cas13a-based antiviral tool against SARS-CoV-2. Moreover, it offers a novel approach for anti-virus study even if the precise structures of viral proteins are indeterminate.

Cas13a has already been successfully applied to virus detection. However, as a new gene interference tool, its potential in cancer treatment was not fully explored until now. This study constructed a new Cas13a expression vector, decoy minimal promoter-Cas13a-U6-guide RNA (DMP-Cas13a-U6-gRNA [DCUg]), by controlling the Cas13a and gRNA expression with a nuclear factor κB (NF-κB)-specific promoter and U6 promoter, respectively. DCUg could specifically and effectively knock down the expression of reporter genes in the 293T and HepG2 cells. DCUg could also similarly knock down the expression of endogenous oncogenes (TERT, EZH2, and RelA) at both mRNA and protein levels in a human hepatoma cell HepG2, which led to significant apoptosis and growth inhibition. In contrast, the same transfection did not affect the target gene expression, cell apoptosis, and growth of a human normal liver cell HL7702. Finally, DCUg targeting these oncogenes was packaged into adeno-associated virus (AAV) and treated four cells (HepG2, HL7702, WEHI-3, and Hepa1-6) and tumor-bearing mice. As results, the recombinant AAV significantly inhibited the growth of three cancer cells (HepG2, Hepa1-6, and WEHI-3) in vitro and the xenografted Hepa1-6 and WEHI-3 tumors in mice. This study therefore developed a new tool for the CRISPR-Cas13a-based cancer gene therapy., Graphical Abstract, This study constructed a new Cas13a expression vector, DMP-Cas13a-U6-gRNA, by controlling the Cas13a and gRNA expression with a NF-κB-specific promoter and U6 promoter, respectively. This vector provides a new tool for the CRISPR-Cas13a-based cancer gene therapy, which can specifically knock down oncogenes in cancer cells in vitro and in vivo.

First identified as a new circovirus in Hunan Province in China in 2019, porcine circovirus (PCV4) is now widely detected in other Chinese provinces and South Korea. In recent years, the virus has threatened pig health and operations in the pig industry. Hence, early PCV4 detection and regular surveillance are required to control the spread of infection and prevent collateral damage to the industry. Due to PCV4 being difficult to isolate in vitro, molecular detection methods, such as conventional PCR and real-time PCR, and serological assays are currently the main methods used for the detection of PCV4 infection. However, they are time-consuming, labor-intensive, and complex and require professional personnel. To facilitate rapid pen-side PCV4 diagnoses, we used clustered regularly interspaced short palindromic repeats (CRISPR) and Cas13a technology to develop a quick testing kit. Five recombinase-aided amplification (RPA) primer sets were designed based on the conserved PCV4-Cap gene nucleotide region, which were used to determine several key lateral flow strip (LFD) characteristics (sensitivity, specificity, and accuracy). The results showed that the RPA-Cas13a-LFD reaction could detect PCV4 within 1.5 h in genomic DNA harboring a minimum of a single copy. Furthermore, the assay showed good specificity and absence of cross-reactivity with PCV2, PCV3, or other porcine viruses. When we tested 15 clinical samples, a high accuracy was also recorded. Therefore, we successfully developed a detection assay that was simple, fast, accurate, and suitable for on-site PCV4 testing.

The rapid and simple detection of Helicobacter pylori (H. pylori) is essential for its clinical eradication. Although various methods for detecting H. pylori have been well established, such as endoscopy in combination with histology or culture, rapid urease test (RUT) and molecular tests using clinical specimens, it is of great importance to develop an ultrasensitive and accurate nucleic acid detection platform and apply it to identify H. pylori. To meet these demands, a novel method based on PCR and CRISPR-Cas13a, called PCR-Cas13a, was developed and validated using the DNA of 84 clinical strains and 71 clinical specimens. PCR primers for the pre-amplification of conservative sequence and CRISPR RNA (crRNA) for the detection of specific sequence were designed according to the principle. The designed primers and crRNA were specific to H. pylori, and the assay showed a high degree of specificity compared with other common pathogens. Our detection system can screen H. pylori with a limit of 2.2 copies/μL within 30 mins after PCR amplification. Using a coincidence analysis with traditional methods, our method exhibited 100% accuracy for the detection of H. pylori. Furthermore, its diagnostic performance was compared, in parallel with a q-PCR. The PCR-Cas13a demonstrates 98% sensitivity and 100% specificity. Moreover, our approach had a lower limit of detection (LOD) than q-PCR. Herein, we present a diagnostic system for the highly sensitive screening of H. pylori and distinguish it from other pathogens. All the results demonstrated that this PCR-based CRISPR assay has wide application prospects for the detection of H. pylori and other slow-growth pathogens.

Salmonella spp. is one of the most common foodborne disease-causing pathogens that can cause severe diseases in very low infectious doses. Rapid and sensitive detecting Salmonella spp. is advantageous to the control of its spread. In this study, a conserved short fragment of the Salmonella invA gene was selected and used to design primers and specific crRNA (CRISPR RNA) for establishing a one-tube and two-step reaction system for Salmonella spp. detection, by combining recombinase polymerase amplification (RPA) with CRISPR-Cas13a (Clustered Regularly Interspaced Short Palindromic Repeats associated protein 13a) cleavage. The established one-tube RPA-Cas13a method can complete the detection within 20 min and the two-step RPA-Cas13a method detection time within 45 min. The designed primers were highly specific to Salmonella spp. and had no cross-reaction with the other nine diarrheal bacteria. The one-tube RPA-Cas13a could detect the Salmonella genome with the limit of 102 copies, which was the same as real-time polymerase chain reaction (PCR), but less sensitive than two-step RPA-Cas13a (100 copies). The detection results of one-tube or two-step RPA-Cas13a and real-time PCR were highly consistent in clinical samples. One-tube RPA-Cas13a developed in this study provides a simple, rapid, and specific detection method for Salmonella spp. While two-step assay was more sensitive and suitable for samples at low abundance.

Supervisor:塚原 俊文, 先端科学技術研究科, 博士

As one of the top 10 plant viruses, the severity of losses to crop productivity caused by the tomato spotted wilt virus (TSWV) has resulted in an urgent need to develop a more sensitive and rapid method of detection. In this study, we developed a CRISPR/Cas13a-based detection system to diagnose TSWV in tomato and western flower thrips (Frankliniella occidentalis). The detection system relies on recombinase polymerase amplification and Cas13a-mediated collateral cleavage activity. Positive results can be distinguished after 20 min by a significantly enhanced fluorescence signal. We tested the sensitivity of CRISPR/Cas13a-based detection system and found that the detection system that we developed has limits of detection that reaches 2.26 × 102 copies/μl and a 10-fold increase compared with the sensitivity of using RT-PCR to detect the virus. Furthermore, the CRISPR/Cas13a-based detection system has a high selectivity for the TSWV without interference from other viruses. The CRISPR/Cas13a-based detection system was utilized to detect the TSWV in samples of tomato leaves and the transmission vector F. occidentalis that were fully consistent with the results when RT-PCR was used to detect the virus., Graphical Abstract Schematic of CRISPR/Cas13-based virus detection.

Salmonella spp. is one of the most common foodborne disease-causing pathogens that can cause severe diseases in very low infectious doses. Rapid and sensitive detecting Salmonella spp. is advantageous to the control of its spread. In this study, a conserved short fragment of the Salmonella invA gene was selected and used to design primers and specific crRNA (CRISPR RNA) for establishing a one-tube and two-step reaction system for Salmonella spp. detection, by combining recombinase polymerase amplification (RPA) with CRISPR-Cas13a (Clustered Regularly Interspaced Short Palindromic Repeats associated protein 13a) cleavage. The established one-tube RPA-Cas13a method can complete the detection within 20 min and the two-step RPA-Cas13a method detection time within 45 min. The designed primers were highly specific to Salmonella spp. and had no cross-reaction with the other nine diarrheal bacteria. The one-tube RPA-Cas13a could detect the Salmonella genome with the limit of 102 copies, which was the same as real-time polymerase chain reaction (PCR), but less sensitive than two-step RPA-Cas13a (100 copies). The detection results of one-tube or two-step RPA-Cas13a and real-time PCR were highly consistent in clinical samples. One-tube RPA-Cas13a developed in this study provides a simple, rapid, and specific detection method for Salmonella spp. While two-step assay was more sensitive and suitable for samples at low abundance.

Bovine Viral Diarrhea Virus (BVDV) is the main pathogen of bovine viral diarrhea disease (BVD), which leads to enormous economic losses in the cattle industry. A sensitive and specific detection for BVDV is advantageous to the control of BVDV. Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems have been used for detecting virus RNA. In this study, the expression and purification of LwCas13a protein was optimized and the RNase activity of LwCas13a in vitro was verified. CRISPR-LwCas13a system could detect BVDV virus and BVDV RNA with high specificity and simplicity. The detection limit of the LwCas13a system was 103 pM, and there were no cross-reactions with HEK293T and MDBK. In summary, a sensitive, specific, and simple nucleic acid detection method based on CRISPR-Cas13a was developed for BVDV. This method provides a new detection strategy for early diagnosis of BVDV.

The continuing pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, which causes coronavirus disease 2019 (COVID-19), has spread globally and its reliable diagnosis is one of the foremost priorities for protecting public health. Herein a rapid (, Graphical abstract Image 1

A major bottleneck in scaling-up COVID-19 testing is the need for sophisticated instruments and well-trained healthcare professionals, which are already overwhelmed due to the pandemic. Moreover, the high-sensitive SARS-CoV-2 diagnostics are contingent on an RNA extraction step, which, in turn, is restricted by constraints in the supply chain. Here, we present CASSPIT (Cas13AssistedSaliva-based &SmartphoneIntegratedTesting), which will allow direct use of saliva samples without the need for an extra RNA extraction step for SARS-CoV-2 detection. CASSPIT utilizes CRISPR-Cas13a based SARS-CoV-2 RNA detection, and lateral-flow assay (LFA) readout of the test results. The sample preparation workflow includes an optimized chemical treatment and heat inactivation method, which, when applied to COVID-19 clinical samples, showed a 97% positive agreement with the RNA extraction method. With CASSPIT, LFA based visual limit of detection (LoD) for a given SARS-CoV-2 RNA spiked into the saliva samples was ~200 copies; image analysis-based quantification further improved the analytical sensitivity to ~100 copies. Upon validation of clinical sensitivity on RNA extraction-free saliva samples (n = 76), a 98% agreement between the lateral-flow readout and RT-qPCR data was found (Ct

Cas13a, an effector of type VI CRISPR-Cas systems, is an RNA guided RNase with multiplexing and therapeutic potential. This study employs the Leptotrichia shahii (Lsh) Cas13a and a repeat-based CRISPR RNA (crRNA) to track and eliminate toxic RNA aggregates in myotonic dystrophy type 1 (DM1) – a neuromuscular disease caused by CTG expansion in the DMPK gene. We demonstrate that LshCas13a cleaves CUG repeat RNA in biochemical assays and reduces toxic RNA load in patient-derived myoblasts. As a result, LshCas13a reverses the characteristic adult-to-embryonic missplicing events in several key genes that contribute to DM1 phenotype. The deactivated LshCas13a can further be repurposed to track RNA-rich organelles within cells. Our data highlights the reprogrammability of LshCas13a and the possible use of Cas13a to target expanded repeat sequences in microsatellite expansion diseases.

RNAi technology has significant potential as a future therapeutic and could theoretically be used to knock down disease-specific RNAs. However, due to frequent off-target effects, low efficiency, and limited accessibility of nuclear transcripts, the clinical application of the technology remains challenging. In this study, we first assessed the stability of Cas13a mRNA and guide RNA. Next, we titrated Cas13a and guide RNA vectors to achieve effective knockdown of firefly luciferase (FLuc) RNA, used as a target transcript. The interference specificity of Cas13a on guide RNA design was next explored. Subsequently, we targeted the EML4-ALK v1 transcript in H3122 lung cancer cells. As determined by FLuc assay, Cas13a exhibited activity only toward the orientation of the crRNA&ndash, guide RNA complex residing at the 5&prime, of the crRNA. The activity of Cas13a was maximal for guide RNAs 24&ndash, 30 bp in length, with relatively low mismatch tolerance. After knockdown of the EML4-ALK transcript, cell viability was decreased up to 50%. Cas13a could effectively knock down FLuc luminescence (70&ndash, 76%), mCherry fluorescence (72%), and EML4-ALK at the protein (&gt, 80%) and transcript levels (26%). Thus, Cas13a has strong potential for use in RNA regulation and therapeutics, and could contribute to the development of personalized medicine.

Enhancers are cis-acting elements that can promote the expression of target genes and respond to estrogen to induce the transcription of eRNAs, which are closely associated with cancer development. Further study on eRNAs may lead to a better understanding of the significance of transcriptional regulation and the progression of malignant tumors. SMAD7 enhancer RNA (SMAD7e) is an estrogen-responsive eRNA. However, the relationship between SMAD7e and bladder cancer remains unclear. SMAD7e was significantly upregulated in bladder cancer tissues and estrogen-stimulated cells. Knockdown of SMAD7e by CRISPR-Cas13a suppressed cell proliferation and migration, and induced cell apoptosis and inhibited cell invasion. Estrogen caused overexpression of SMAD7e and played a facilitating role in bladder cancer cells. Furthermore, knockdown of SMAD7e by CRISPR-Cas13a prevented the cancer-promoting effects of estrogen on bladder cancer both in vitro and in vivo. The present study suggested the crucial role of SMAD7e in bladder cancer. Estrogen might promote the development of bladder cancer by inducing SMAD7e production. These findings may provide a potential target for CRISPR-mediated gene therapy for bladder cancer in the future.

In the CRISPR-Cas systems, Cas13a is an RNA-guided RNA nuclease specifically targeting single strand RNA. We developed a Cas13a mediated CRISPR interference tool to target mRNA for gene silencing in mosquitoes. A Cas13a expressing plasmid was delivered to mosquitoes by intrathoracic injection, and Cas13a transcripts were detectable at least 10 days post-delivery. The target specific crRNA was synthesized in vitro using T7 RNA polymerase. The Cas13a plasmid and target crRNA can be delivered by intrathoracic injection together, or the Cas13a construct can be provided first, and then target crRNA can be given later when appropriate. The machinery was tested in two mosquito species. In Anopheles gambiae, vitellogenin gene was silenced by Cas13a/Vg-crRNA, which was accompanied by a significant reduction in egg production. In Aedes aegypti, the α- and δ-subunits of COPI genes were silenced by Cas13a/crRNA, which resulted in mortality and fragile midguts, reproducing a phenotype reported previously. Co-silencing genes simultaneously is achievable when a cocktail of target crRNAs is given. No detectable collateral cleavages of non-target transcripts were observed in the study. In addition to dsRNA or siRNA mediated RNA interference, the programmable CRISPR interference method offers an alternative to knock down genes in mosquitoes.

In article number https://doi.org/10.1002/advs.201901299, Chunsheng Kang and co‐workers demonstrate the collateral effect of the CRISPR‐Cas13a system in glioma cells. EGFRvIII is a unique EGFR mutant subtype in glioma, and the CRISPR‐Cas13a system induces death in EGFRvIII‐overexpressing glioma cells. Furthermore, CRISPR‐Cas13a inhibits the formation of glioma intracranial tumors in mice. The results indicate the powerful tumor eliminating potential of this system.

Graphical abstract 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., Canine parvovirus type 2 (CPV-2) infection is the most lethal disease of dogs with higher mortality in puppies worldwide. In today’s world, dogs are an integral part of our communities as well as dogs breeding and rearing has become a lucrative business. Therefore, a fast, accurate, portable, and cost-effective CPV-2 detection method with the ability for on-site detection is highly desired. In this study, we for the first time proposed a nanosystem for CPV-2 DNA detection with RNA-guided RNA endonuclease Cas13a, which upon activation results in collateral RNA degradation. We expressed LwCas13a in prokaryotic expression system and purified it through nickel column. Activity of Cas13a was verified by RNA-bound fluorescent group while using a quenched fluorescent probe as signals. Further Cas13a was combined with Recombinase polymerase amplification (RPA) and T7 transcription to establish molecular detection system termed specific high-sensitivity enzymatic reporter un-locking (SHERLOCK) for sensitive detection of CPV-2 DNA. This nanosystem can detect 100 amol/L CPV-2 DNA within 30 min. The proposed nanosystem exhibited high specificity when tested for CPV-2 and other dog viruses. This CRISPR-Cas13a mediated sensitive detection approach can be of formidable advantage during CPV-2 outbreaks because it is time-efficient, less laborious and does not involve the use of sophisticated instruments.

Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas13a, previously known as CRISPR-C2c2, is the most recently identified RNA-guided RNA-targeting CRISPR-Cas system and has the unique characteristics of both targeted and collateral single-stranded RNA (ssRNA) cleavage activities, which was first identified in Leptotrichia shahii CRISPR-Cas13a. Here, the complete whole genome sequences of 11 Leptotrichia strains were determined and compared with 18 publicly available Leptotrichia genomes in regard to the composition, occurrence and diversity of the CRISPR-Cas13a and other CRISPR-Cas systems. Various types of CRISPR-Cas systems, I-B, II-C, III-A, III-D, III-like and VI-A, were unevenly distributed among the Leptotrichia genomes, accounting for 10 (34.4%), 1 (2.6%), 6 (15.4%), 6 (15.4%), 3 (7.7%) and 11 (37.9%) of the 29 strains, respectively, while 8 (20.5%) strains had no CRISPR-Cas system. The Cas13a effectors were highly divergent with amino acid sequence similarities ranging from 61% to 90% to that of L. shahii and their collateral ssRNA cleavage activities, resulting in cytotoxicity to host cell, were found to be maintained. CRISPR-Cas spacers represent a sequential achievement of former intruder encounters, and the retained spacers reflect the evolutionary phylogeny or relatedness of strains. Analysis of spacer contents and numbers among Leptotrichia species showed considerable diversity with only 2 (0.5%) of 400 spacers through CRIPSR-Cas I-VI were shared by two strains. The organisation and distribution of CRISPR-Cas systems (type I-VI) encoded by all registered Leptotrichia species revealed that effector or spacer sequences of the CRISPR-Cas systems were very divergent, and the prevalence of types I, III and VI was almost equal. There was only one strain carrying type II, while none carried type IV or V. These results provide new insights into the characteristics and divergences of CRISPR-Cas systems among Leptotrichia species.

Enhancers are transcriptional regulatory elements that increase target gene expression. It has reported that enhancers could universally transcribe into enhancer RNAs (eRNAs) with stimulation. Increasing evidence showed eRNAs participated in various disease processes including malignant tumors. P2RY2 enhancer RNA (P2RY2e) is an estrogen-responsive eRNA and involved in the development of breast cancer. However, the relationship between P2RY2e and bladder cancer (BCa) is unclear. In the study, we discovered that P2RY2e was upregulated in BCa tissues and estrogen-treated cells. Estrogen promoted the malignant abilities of BCa cells. P2RY2e knockdown by CRISPR-Cas13a inhibit the cell multiplication, invasion and migration. Additionally, the cell apoptosis was facilitated. What's more, downregulation of P2RY2e could weaken the cancer-promoting effects of estrogen on BCa. Our study revealed that P2RY2e played a carcinogenic role in BCa and estrogen might promote the initiation of BCa by inducing P2RY2e. We provide a potential therapeutic target for BCa and a new perspective for the tumorigenesis of bladder cancer.

RNA is rarely used as a therapeutic target due to its flexible structure and instability. CRISPR‐Cas13a is a powerful tool for RNA knockdown, and the potential application of CRISPR‐Cas13a in cancer cells should be further studied. In this study, overexpression of LwCas13a by lentivirus in glioma cells reveals that crRNA‐EGFP induces a “collateral effect” after knocking down the target gene in EGFP‐expressing cells. EGFRvIII is a unique EGFR mutant subtype in glioma, and the CRISPR‐Cas13a system induces death in EGFRvIII‐overexpressing glioma cells. Bulk and single‐cell RNA sequencing analysis in U87‐Cas13a‐EGFRvIII cells confirm the collateral effect of the CRISPR‐Cas13a system. Furthermore, CRISPR‐Cas13a inhibits the formation of glioma intracranial tumors in mice. The results demonstrate the collateral effect of the CRISPR‐Cas13a system in cancer cells and the powerful tumor‐eliminating potential of this system., It is reported that a CRISPR/Cas13a system can exert a collateral effect after target recognition in glioma cells, indicating that the CRISPR/Cas13a system has great potential value for tumor elimination, especially for patients with specific oncogene mutations.