Logic-gated tumor-microenvironment nanoamplifier enables targeted delivery of CRISPR/Cas9 for multimodal cancer therapy

Recent innovations in nanomaterials inspire abundant novel tumor-targeting CRISPR-based gene therapies. However, the therapeutic efficiency of traditional targeted nanotherapeutic strategies is limited by that the biomarkers vary in a spatiotemporal-dependent manner with tumor progression. Here, we propose a self-amplifying logic-gated gene editing strategy for gene/H2O2-mediated/starvation multimodal cancer therapy. In this approach, a hypoxia-degradable covalent-organic framework (COF) is synthesized to coat a-ZIF-8 in which glucose oxidase (GOx) and CRISPR system are packaged. To intensify intracellular redox dyshomeostasis, DNAzymes which can cleave catalase mRNA are loaded as well. When the nanosystem gets into the tumor, the weakly acidic and hypoxic microenvironment degrades the ZIF-8@COF to activate GOx, which amplifies intracellular H+ and hypoxia, accelerating the nanocarrier degradation to guarantee available CRISPR plasmid and GOx release in target cells. These tandem reactions deplete glucose and oxygen, leading to logic-gated-triggered gene editing as well as synergistic gene/H2O2-mediated/starvation therapy. Overall, this approach highlights the biocomputing-based CRISPR delivery and underscores the great potential of precise cancer therapy.

CRISPR/Cas9 genome editing approaches for psychiatric research

Currently, genome editing technologies, such as clustered regularly interspaced short palindromic repeats (CRISPR/Cas9), are predominantly used to model genetic diseases. This genome editing system can correct point or frameshift mutations in risk genes. Here, we analyze and discuss the advantages of genome editing, its current applications, and the feasibility of the CRISPR/Cas9 system in research on psychiatric disorders. These disorders produce cognitive and behavioral alterations and their etiology is associated with polygenetic and environmental factors. CRISPR/Cas9 may reveal the biological mechanisms of psychiatric disorders at a basic research level, translating a suitable clinical approach for use in the diagnosis and treatment of psychiatric disorders. Genetic diagnosis and treatment for these disorders have not yet been fully established in psychiatry due to the limited understanding of their heterogeneity and polygenicity. We discuss the challenges and ethical issues in using CRISPR/Cas9 as a tool for diagnosis or gene therapy.

Editing the Human Genome with CRISPR/Cas: A Review of its Molecular Basis, Current Clinical Applications, and Bioethical Implications

ABSTRACT CRISPR/Cas genes evolved in prokaryotic organisms as a mechanism of defense designed to identify and destroy genetic material from threatening viruses. A breakthrough discovery is that CRISPR/Cas system can be used in eukaryotic cells to edit almost any desired gene. This comprehensive review addresses the most relevant work in the CRISPR/Cas field, including its history, molecular biology, gene editing capability, ongoing clinical trials, and bioethics. Although the science involved is complex, we intended to describe it in a concise manner that could be of interest to diverse readers, including anyone dedicated to the treatment of patients who could potentially benefit from gene editing, molecular biologists, and bioethicists. CRISPR/Cas has the potential to correct inherited diseases caused by single point mutations, to knock-in the promoter of a gene whose expression is highly desirable or knockout the gene coding for a deleterious protein. CRISPR/Cas technique can also be used to edit ex vivo immune cells and reinsert them in patients, improving their efficiency in attacking malignant cells, limiting the infectious potential of viruses or modulating xenotransplant rejection. Very important bioethical considerations on this topic include the need to internationally regulate its use by ad hoc expert committees and to limit its use until safety and bioethical issues are satisfactorily resolved.

Evidence of a synthetic lethality interaction between SETDB1 histone methyltransferase and CHD4 chromatin remodeling protein in a triple negative breast cancer cell line

During the tumorigenic process, cancer cells may become overly dependent on the activity of backup cellular pathways for their survival, representing vulnerabilities that could be exploited as therapeutic targets. Certain molecular vulnerabilities manifest as a synthetic lethality relationship, and the identification and characterization of new synthetic lethal interactions may pave the way for the development of new therapeutic approaches for human cancer. Our goal was to investigate a possible synthetic lethal interaction between a member of the Chromodomain Helicase DNA binding proteins family (CHD4) and a member of the histone methyltransferases family (SETDB1) in the molecular context of a cell line (Hs578T) representing the triple negative breast cancer (TNBC), a subtype of breast cancer lacking validated molecular targets for treatment. Therefore, we employed the CRISPR-Cas9 gene editing tool to individually or simultaneously introduce indels in the genomic loci corresponding to the catalytic domains of SETDB1 and CHD4 in the Hs578T cell line. Our main findings included: a) introduction of indels in exon 22 of SETDB1 sensitized Hs578T to the action of the genotoxic chemotherapy doxorubicin; b) by sequentially introducing indels in exon 22 of SETDB1 and exon 23 of CHD4 and tracking the percentage of the remaining wild-type sequences in the mixed cell populations generated, we obtained evidence of the existence of a synthetic lethality interaction between these genes. Considering the lack of molecular targets in TNBC, our findings provided valuable insights for development of new therapeutic approaches not only for TNBC but also for other cancer types.

Current advances of CRISPR/Cas system in antibacterial field / 药学学报

A breakthrough in molecular biology for the twenty-first century is CRISPR/Cas gene editing, which has been used in a variety of fields due to its simplicity, adaptability, and targeting. Given the current global challenge of severe bacterial resistance, difficulties in detecting antimicrobial resistance, and slow development of antimicrobial drugs, CRISPR/Cas gene-editing technology offers a promising avenue for the development of antibacterial treatments. On the one hand, CRISPR/Cas gene editing technology helps advance the study of bacterial functions and serves as a toolbox. For instance, Cas proteins and exogenous repair systems enable efficient and precise gene editing, nCas proteins and deaminase systems facilitate template-free and single base precision editing, dCas proteins and reverse transcriptase allow for repair-free gene editing, and dCas proteins and modified sgRNA enable gene expression level regulation and gene function analysis. On the other hand, its specific gene recognition and targeted DNA cleavage characteristics can be used for pathogen detection, elimination of drug-resistant bacteria and genes, and hold promise as a new strategy for clinical diagnosis and treatment.

CRISPR/Cas9-mediated Knockout of KIFC1 Inhibits Proliferation and Induces Apoptosis of Cervical Cancer Cells / 肿瘤防治研究

Objective To investigate the functions of the KIFC1 gene in tumor cells and its effect on the proliferation of cervical cancer cells. Methods We designed sgRNAs targeting the KIFC1 gene and constructed a recombinant plasmid based on the pSpCas9 (BB)-2A-GFP vector, which was co-transfected into HeLa cells. We screened monoclonal knockout cell lines through flow cytometry sorting, limited dilution inoculation of cells, and sequencing. RT-qPCR, Western blot, and immunofluorescence were used to detect the transcription and protein expression levels of KIFC1 in knockout cells. Cell phenotypes such as nucleus and microtubule cytoskeleton were observed using phase-contrast microscopy and fluorescence confocal microscopy. Cell proliferation, cell cycle, and apoptosis were analyzed by growth curve plotting, EdU labeling, and acridine orange staining. Results The deletion of the KIFC1 gene resulted in the abnormal phenotypes of HeLa cells, with increased numbers of multinuclei, micronucleus, and disordered microtubules. The cell cycle was disrupted, accompanied with a significant increase in the ratio of late apoptotic cells and a decrease in cell proliferation (all P < 0.05). Conclusion KIFC1 gene deletion affects the assembly of microtubules and cell division in HeLa cells, leading to abnormal nuclear morphology, chromatin elimination, cell cycle arrest, and increased cell apoptosis.

Construction of CRISPR/Cas9 system targeting mouse Tsc1 and Tsc2 gene and verification of targeting effectiveness / 中国生物制品学杂志

@#ObjectiveTo design and construct CRISPR/Cas9 gene editing system targeting Tsc1 and Tsc2 genes,and verify the effectiveness of gene editing at cellular level.MethodsThree sgRNA guide sequences were designed for mouse Tsc1 and Tsc2 genes respectively. The sgRNA expression vector was constructed and co-transfected with the Cas9 expression plasmid into mouse N2a cells. After the positive cells were obtained through drug screening,the DNA fragments at the targeting site were amplified by PCR,and the targeting efficiency was verified by TA clone sequencing.ResultsThe five targets of Tsc1-M-sgRNA2 and Tsc1-M-sgRNA3 of Tsc1 gene and Tsc2-M-sgRNA1,Tsc2-M-sgRNA2 and Tsc2-M-sgRNA3 of Tsc2 gene were all edited,and the editing efficiency was 40%,80%,30%,30% and 20%,respectively.ConclusionA CRISPR-Cas9 gene editing system with editing efficiency targeting mouse Tsc1 and Tsc2 genes was successfully constructed.

High expression of MYH9 inhibits apoptosis of non-small cell lung cancer cells through activating the AKT/c-Myc pathway / 南方医科大学学报

OBJECTIVE@#To investigate the role of myosin heavy chain 9 (MYH9) in regulation of cell proliferation, apoptosis, and cisplatin sensitivity of non-small cell lung cancer (NSCLC).@*METHODS@#Six NSCLC cell lines (A549, H1299, H1975, SPCA1, H322, and H460) and a normal bronchial epithelial cell line (16HBE) were examined for MYH9 expression using Western blotting. Immunohistochemical staining was used to detect MYH9 expression in a tissue microarray containing 49 NSCLC and 43 adjacent tissue specimens. MYH9 knockout cell models were established in H1299 and H1975 cells using CRISPR/Cas9 technology, and the changes in cell proliferation cell were assessed using cell counting kit-8 (CCK8) and clone formation assays; Western blotting and flow cytometry were used to detect apoptosis of the cell models, and cisplatin sensitivity of the cells was evaluated using IC50 assay. The growth of tumor xenografts derived from NSCLC with or without MYH9 knockout was observed in nude mice.@*RESULTS@#MYH9 expression was significantly upregulated in NSCLC (P < 0.001), and the patients with high MYH9 expression had a significantly shorter survival time (P=0.023). In cultured NSCLC cells, MYH9 knockout obviously inhibited cell proliferation (P < 0.001), promoted cell apoptosis (P < 0.05), and increased their chemosensitivity of cisplatin. In the tumor-bearing mouse models, the NSCLC cells with MYH9 knockout showed a significantly lower growth rate (P < 0.05). Western blotting showed that MYH9 knockout inactivated the AKT/c- Myc axis (P < 0.05) to inhibit the expression of BCL2- like protein 1 (P < 0.05), promoted the expression of BH3- interacting domain death agonist and the apoptosis regulator BAX (P < 0.05), and activated apoptosis-related proteins caspase-3 and caspase-9 (P < 0.05).@*CONCLUSION@#High expression of MYH9 contributes to NSCLC progression by inhibiting cell apoptosis via activating the AKT/c-Myc axis.

Rapid detection and genotyping of SARS-CoV-2 Omicron BA.4/5 variants using a RT-PCR and CRISPR-Cas12a-based assay / 南方医科大学学报

OBJECTIVE@#To establish a rapid detection and genotyping method for SARS-CoV-2 Omicron BA.4/5 variants using CRISPPR-Cas12a gene editing technology.@*METHODS@#We combined reverse transcription-polymerase chain reaction (RT-PCR) and CRISPR gene editing technology and designed a specific CRISPPR RNA (crRNA) with suboptimal protospacer adjacent motifs (PAM) for rapid detection and genotyping of SARS- CoV-2 Omicron BA.4/5 variants. The performance of this RT- PCR/ CRISPPR-Cas12a assay was evaluated using 43 clinical samples of patients infected by wild-type SARS-CoV-2 and the Alpha, Beta, Delta, Omicron BA. 1 and BA. 4/5 variants and 20 SARS- CoV- 2-negative clinical samples infected with 11 respiratory pathogens. With Sanger sequencing method as the gold standard, the specificity, sensitivity, concordance (Kappa) and area under the ROC curve (AUC) of RT-PCR/CRISPPR-Cas12a assay were calculated.@*RESULTS@#This assay was capable of rapid and specific detection of SARS- CoV-2 Omicron BA.4/5 variant within 30 min with the lowest detection limit of 10 copies/μL, and no cross-reaction was observed in SARS-CoV-2-negative clinical samples infected with 11 common respiratory pathogens. The two Omicron BA.4/5 specific crRNAs (crRNA-1 and crRNA-2) allowed the assay to accurately distinguish Omicron BA.4/5 from BA.1 sublineage and other major SARS-CoV-2 variants of concern. For detection of SARS-CoV-2 Omicron BA.4/5 variants, the sensitivity of the established assay using crRNA-1 and crRNA-2 was 97.83% and 100% with specificity of 100% and AUC of 0.998 and 1.000, respectively, and their concordance rate with Sanger sequencing method was 92.83% and 96.41%, respectively.@*CONCLUSION@#By combining RT-PCR and CRISPPR-Cas12a gene editing technology, we successfully developed a new method for rapid detection and identification of SARS-CoV-2 Omicron BA.4/5 variants with a high sensitivity, specificity and reproducibility, which allows rapid detection and genotyping of SARS- CoV-2 variants and monitoring of the emerging variants and their dissemination.

Advances in AAV-CRISPR/Cas9-Mediated Hemophilia A Gene Therapy --Review / 中国实验血液学杂志

Hemophilia A(HA) is an X-linked recessive bleeding disorder caused by mutations in coagulation factor VIII. Nowadays, exogenous coagulation factor replacement therapy is the main treatment. With the continuous development of gene therapy, new research directions have been provided for the treatment of hemophilia A. CRISPR-Cas9 technology was applied to select suitable target sites, and mediate the targeted knock-in and efficient expression of exogenous B-domain-deleted FⅧ variant gene through corresponding vectors for the treatment of hemophilia A.CRISPR-Cas9 technology is an emerging gene editing tool with great efficiency, safety and effectiveness, and has been widely used in hemophilia gene therapy research. This paper reviews the vector selection, construction of therapeutic genes, gene editing technology and selection of expression target sites for hemophilia A gene therapy at this stage.

Application of CRISPR-Cas9 gene editing technology in crop breeding / 生物工程学报

The CRISPR-Cas9 system is composed of a clustered regularly interspaced short palindromic repeat (CRISPR) and its associated proteins, which are widely present in bacteria and archaea, serving as a specific immune protection against viral and phage secondary infections. CRISPR-Cas9 technology is the third generation of targeted genome editing technologies following zinc finger nucleases (ZFNs) and transcription activator like effector nucleases (TALENs). The CRISPR-Cas9 technology is now widely used in various fields. Firstly, this article introduces the generation, working mechanism and advantages of CRISPR-Cas9 technology; secondly, it reviews the applications of CRISPR-Cas9 technology in gene knockout, gene knock-in, gene regulation and genome in breeding and domestication of important food crops such as rice, wheat, maize, soybean and potato. Finally, the article summarizes the current problems and challenges encountered by CRISPR-Cas9 technology and prospects future development and application of CRISPR-Cas9 technology.

Identification of the target site of antimicrobial peptide AMP-17 against Candida albicans / 生物工程学报

Candida albicans is one of the major causes of invasive fungal infections and a serious opportunistic pathogen in immunocompromised individuals. The antimicrobial peptide AMP-17 has prominent anti-Candida activity, and proteomic analysis revealed significant differences in the expression of cell wall (XOG1) and oxidative stress (SRR1) genes upon the action of AMP-17 on C. albicans, suggesting that AMP-17 may exert anti-C. albicans effects by affecting the expression of XOG1 and SRR1 genes. To further investigate whether XOG1 and SRR1 genes were the targets of AMP-17, C. albicans xog1Δ/Δ and srr1Δ/Δ mutants were constructed using the clustered regulatory interspaced short palindromic repeats-associated protein 9 (CRISPR/Cas9) system. Phenotypic observations revealed that deletion of two genes had no significant effect on C. albicans growth and biofilm formation, whereas XOG1 gene deletion affected in vitro stress response and mycelium formation of C. albicans. Drug sensitivity assay showed that the MIC80 values of AMP-17 against xog1Δ/Δ and srr1Δ/Δ mutants increased from 8 μg/mL (for the wild type C. albicans SC5314) to 16 μg/mL, while the MIC80 values against srr1Δ/Δ: : srr1 revertants decreased to the level of the wild type SC5314. In addition, the ability of AMP-17 to inhibit biofilm formation of both deletion strains was significantly reduced compared to that of wild type SC5314, indicating that the susceptibility of the deletion mutants to AMP-17 was reduced in both the yeast state and during biofilm formation. These results suggest that XOG1 and SRR1 genes are likely two of the potential targets for AMP-17 to exert anti-C. albicans effects, which may facilitate further exploration of the antibacterial mechanism of novel peptide antifungal drugs.

Development of porcine induced pluripotent stem cells with a CD163 reporter system / 生物工程学报

As main recipient cells for porcine reproductive and respiratory syndrome virus (PRRSV), porcine alveolar macrophage (PAM) are involved in the progress of several highly pathogenic virus infections. However, due to the fact that the PAM cells can only be obtained from primary tissues, research on PAM-based virus-host interactions remains challenging. The improvement of induced pluripotent stem cells (iPSCs) technology provides a new strategy to develop IPSCs-derived PAM cells. Since the CD163 is a macrophage-specific marker and a validated receptor essential for PRRSV infection, generation of stable porcine induced pluripotent stem cells lines containing CD163 reporter system play important roles in the investigation of IPSCs-PAM transition and PAM-based virus-host interaction. Based on the CRISPR/Cas9- mediated gene editing system, we designed a sgRNA targeting CD163 locus and constructed the corresponding donor vectors. To test whether this reporter system has the expected function, the reporter system was introduced into primary PAM cells to detect the expression of RFP. To validate the low effect on stem cell pluripotency, we generated porcine iPSC lines containing CD163 reporter and assessed the pluripotency through multiple assays such as alkaline phosphatase staining, immunofluorescent staining, and EdU staining. The red-fluorescent protein (RFP) expression was detected in CD163-edited PAM cells, suggesting that our reporter system indeed has the ability to reflect the expression of gene CD163. Compared with wild-type (WT) iPSCs, the CD163 reporter-iPSCs display similar pluripotency-associated transcription factors expression. Besides, cells with the reporter system showed consistent cell morphology and proliferation ability as compared to WT iPSCs, indicating that the edited-cells have no effect on stem cell pluripotency. In conclusion, we generated porcine iPSCs that contain a CD163 reporter system. Our results demonstrated that this reporter system was functional and safe. This study provides a platform to investigate the iPS-PAM development and virus-host interaction in PAM cells.

Application of cell-free transcription and translation system in CRISPR technologies and the associated biosensors / 生物工程学报

Cell-free transcription and translation (TXTL) system is a cell extract-based system for rapid in vitro protein expression. The system bypasses routine laboratory processes such as bacterial transformation, clonal screening and cell lysis, which allows more precise and convenient control of reaction substrates, reduces the impact of bacteria on protein production, and provides a high degree of versatility and flexibility. In recent years, TXTL has been widely used as an emerging platform in clusterd regularly interspaced short palindromic repeat (CRISPR) technologies, enabling more rapid and convenient characterization of CRISPR/Cas systems, including screening highly specific gRNAs as well as anti-CRISPR proteins. Furthermore, TXTL-based CRISPR biosensors combined with biological materials and gene circuits are able to detect pathogens through validation of related antibiotics and nucleic acid-based markers, respectively. The reagents can be freeze-dried to improve portability and achieve point-of-care testing with high sensitivity. In addition, combinations of the sensor with programmable circuit elements and other technologies provide a non-biological alternative to whole-cell biosensors, which can improve biosafety and accelerate its application for approval. Here, this review discusses the TXTL-based characterization of CRISPR and their applications in biosensors, to facilitate the development of TXTL-based CRISPR/Cas systems in biosensors.

CRISPR-based molecular diagnostics: a review / 生物工程学报

Rapid and accurate detection technologies are crucial for disease prevention and control. In particular, the COVID-19 pandemic has posed a great threat to our society, highlighting the importance of rapid and highly sensitive detection techniques. In recent years, CRISPR/Cas-based gene editing technique has brought revolutionary advances in biotechnology. Due to its fast, accurate, sensitive, and cost-effective characteristics, the CRISPR-based nucleic acid detection technology is revolutionizing molecular diagnosis. CRISPR-based diagnostics has been applied in many fields, such as detection of infectious diseases, genetic diseases, cancer mutation, and food safety. This review summarized the advances in CRISPR-based nucleic acid detection systems and its applications. Perspectives on intelligent diagnostics with CRISPR-based nucleic acid detection and artificial intelligence were also provided.

CRISPR/Cas9-mediated endogenous TCR knockout enhances TCR-T cells targeted killing HPV16-positive cervical cancer cells / 中国肿瘤生物治疗杂志

@#[摘 要] 目的：基于CRISPR/Cas9基因编辑技术制备无内源TCR的TCR-T细胞并鉴定其在体外杀伤HPV16阳性宫颈癌SiHa细胞的功能。方法：培养健康志愿者外周血CD8+ T细胞和Jurkat细胞，CRISPR/Cas9基因编辑技术敲除CD8+ T、Jurkat细胞的TCR基因，制备过表达转基因TCR的重组慢病毒，在敲除内源性TCR的CD8+ T和Jurkat细胞中用慢病毒过表达转基因TCR制备TCR-T细胞，多色FCM检测TCR-T细胞中TCR和CD3的表达水平，荧光素酶活性实验检测TCR-T细胞对HPV16阳性SiHa细胞的杀伤效率。结果：CRIPSR/Cas9基因编辑技术高效地敲除了外周血CD8+ T细胞和Jurkat细胞中的TRAC和TRBC基因，敲除效率分别为（81.4±4.5）%、（98.5±0.07）%，制备的无内源TCR的TCR-T细胞高效表达转基因TCR，在外周血CD8+ T和Jurkat细胞中表达率为（66.0±17.8）%、（97.3±2.6）%，敲除内源TRAC和TRBC基因有效增强CD8+ T和Jurkat细胞膜表达转基因TCR（均P<0.01），敲除内源TCR增强TCR-T细胞特异性杀伤HPV16阳性的SiHa细胞[（71.4±1.0）% vs （35.1±2.0）%，P<0.01）]。结论：无内源TCR的TCR-T细胞显著增强转基因TCR的表达和对HPV16阳性宫颈癌SiHa细胞的靶向杀伤能力，为提高TCR-T细胞的临床疗效提供了实验依据。

Precision gene editing technologies based on CRISPR/Cas9: a review / 生物工程学报

Gene editing technology is a genetic operation technology that can modify the DNA sequence at the genomic level. The precision gene editing technology based on CRISPR/Cas9 system is a gene editing technology that is easy to operate and widely used. Unlike the traditional CRISPR/Cas9 system, the precision gene editing technology can perform site-directed mutation of genes without DNA template. This review summarizes the recent development of precision gene editing technology based on CRISPR/Cas9, and prospects the challenges and opportunities of this technology.

Multiplex gene editing and regulation techniques based on CRISPR/Cas system / 生物工程学报

The CRISPR/Cas systems comprising the clustered regularly interspaced short palindromic repeats (CRISPR) and its associated Cas protein is an acquired immune system unique to archaea or bacteria. Since its development as a gene editing tool, it has rapidly become a popular research direction in the field of synthetic biology due to its advantages of high efficiency, precision, and versatility. This technique has since revolutionized the research of many fields including life sciences, bioengineering technology, food science, and crop breeding. Currently, the single gene editing and regulation techniques based on CRISPR/Cas systems have been increasingly improved, but challenges still exist in the multiplex gene editing and regulation. This review focuses on the development and application of multiplex gene editing and regulation techniques based on the CRISPR/Cas systems, and summarizes the techniques for multiplex gene editing or regulation within a single cell or within a cell population. This includes the multiplex gene editing techniques developed based on the CRISPR/Cas systems with double-strand breaks; or with single-strand breaks; or with multiple gene regulation techniques, etc. These works have enriched the tools for the multiplex gene editing and regulation and contributed to the application of CRISPR/Cas systems in the multiple fields.

Generation of Mlk3 KO mice by CRISPR/Cas9 and its effect on blood pressure / 生物工程学报

To explore the effect of Mlk3 (mixed lineage kinase 3) deficiency on blood pressure, Mlk3 gene knockout (Mlk3KO) mice were generated. Activities of sgRNAs targeted Mlk3 gene were evaluated by T7 endonuclease I (T7E1) assay. CRISPR/Cas9 mRNA and sgRNA were obtained by in vitro transcription, microinjected into zygote, followed by transferring into a foster mother. Genotyping and DNA sequencing confirmed the deletion of Mlk3 gene. Real- time PCR (RT-PCR), Western blotting or immunofluorescence analysis showed that Mlk3KO mice had an undetectable expression of Mlk3 mRNA or Mlk3 protein. Mlk3KO mice exhibited an elevated systolic blood pressure compared with wild-type mice as measured by tail-cuff system. Immunohistochemistry and Western blotting analysis showed that the phosphorylation of MLC (myosin light chain) was significantly increased in aorta isolated from Mlk3KO mice. Together, Mlk3KO mice was successfully generated by CRISPR/Cas9 system. MLK3 functions in maintaining blood pressure homeostasis by regulating MLC phosphorylation. This study provides an animal model for exploring the mechanism by which Mlk3 protects against the development of hypertension and hypertensive cardiovascular remodeling.

Advances in the RNA-targeting CRISPR-Cas systems / 生物工程学报

The CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR associated proteins) system is an adaptive immune system of bacteria and archaea against phages, plasmids and other exogenous genetic materials. The system uses a special RNA (CRISPR RNA, crRNA) guided endonuclease to cut the exogenous genetic materials complementary to crRNA, thus blocking the infection of exogenous nucleic acid. According to the composition of the effector complex, CRISPR-Cas system can be divided into two categories: class 1 (including type Ⅰ, Ⅳ, and Ⅲ) and class 2 (including type Ⅱ, Ⅴ, and Ⅵ). Several CRISPR-Cas systems have been found to have very strong ability to specifically target RNA editing, such as type Ⅵ CRISPR-Cas13 system and type Ⅲ CRISPR-Cas7-11 system. Recently, several systems have been widely used in the field of RNA editing, making them a powerful tool for gene editing. Understanding the composition, structure, molecular mechanism and potential application of RNA-targeting CRISPR-Cas systems will facilitate the mechanistic research of this system and provide new ideas for developing gene editing tools.