CRISPR/Cas9 gene-editing strategies in cardiovascular cells.
Vermersch Eva,Jouve Charlène,Hulot Jean-Sébastien
Cardiovascular diseases are among the main causes of morbidity and mortality in Western countries and considered as a leading public health issue. Therefore, there is a strong need for new disease models to support the development of novel therapeutics approaches. The successive improvement of genome editing tools with zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and more recently with clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) has enabled the generation of genetically modified cells and organisms with much greater efficiency and precision than before. The simplicity of CRISPR/Cas9 technology made it especially suited for different studies, both in vitro and in vivo, and has been used in multiple studies evaluating gene functions, disease modelling, transcriptional regulation, and testing of novel therapeutic approaches. Notably, with the parallel development of human induced pluripotent stem cells (hiPSCs), the generation of knock-out and knock-in human cell lines significantly increased our understanding of mutation impacts and physiopathological mechanisms within the cardiovascular domain. Here, we review the recent development of CRISPR-Cas9 genome editing, the alternative tools, the available strategies to conduct genome editing in cardiovascular cells with a focus on its use for correcting mutations in vitro and in vivo both in germ and somatic cells. We will also highlight that, despite its potential, CRISPR/Cas9 technology comes with important technical and ethical limitations. The development of CRISPR/Cas9 genome editing for cardiovascular diseases indeed requires to develop a specific strategy in order to optimize the design of the genome editing tools, the manipulation of DNA repair mechanisms, the packaging and delivery of the tools to the studied organism, and the assessment of their efficiency and safety.
A short overview of the CRISPR-Cas adaptation stage.
Mosterd Cas,Rousseau Geneviève M,Moineau Sylvain
Canadian journal of microbiology
CRISPR research began over 30 years ago with the incidental discovery of an unusual nucleotide arrangement in the genome. It took 20 years to find the main function of CRISPR-Cas systems as an adaptive defence mechanism against invading nucleic acids, and our knowledge of their biology has steadily increased ever since. In parallel, the number of applications derived from CRISPR-Cas systems has risen spectacularly. The CRISPR-based genome editing tool is arguably the most exciting application in both basic and applied research. Lately, CRISPR-Cas research has partially shifted to the least understood aspect of its biology: the ability of CRISPR-Cas systems to acquire new immunities during the so-called adaptation step. To date, the most efficient natural system to readily acquire new spacers is the type II-A system of the gram-positive dairy bacterium . The discovery of additional systems able to acquire new spacers will hopefully draw more attention to this step of CRISPR-Cas biology. This review focuses on the breakthroughs that have helped to unravel the adaptation phase and on questions that remain to be answered.
Characterization and applications of Type I CRISPR-Cas systems.
Hidalgo-Cantabrana Claudio,Barrangou Rodolphe
Biochemical Society transactions
CRISPR-Cas constitutes the adaptive immune system of bacteria and archaea. This RNA-mediated sequence-specific recognition and targeting machinery has been used broadly for diverse applications in a wide range of organisms across the tree of life. The compact class 2 systems, that hinge on a single Cas effector nuclease have been harnessed for genome editing, transcriptional regulation, detection, imaging and other applications, in different research areas. However, most of the CRISPR-Cas systems belong to class 1, and the molecular machinery of the most widespread and diverse Type I systems afford tremendous opportunities for a broad range of applications. These highly abundant systems rely on a multi-protein effector complex, the CRISPR associated complex for antiviral defense (Cascade), which drives DNA targeting and cleavage. The complexity of these systems has somewhat hindered their widespread usage, but the pool of thousands of diverse Type I CRISPR-Cas systems opens new avenues for CRISPR-based applications in bacteria, archaea and eukaryotes. Here, we describe the features and mechanism of action of Type I CRISPR-Cas systems, illustrate how endogenous systems can be reprogrammed to target the host genome and perform genome editing and transcriptional regulation by co-delivering a minimal CRISPR array together with a repair template. Moreover, we discuss how these systems can also be used in eukaryotes. This review provides a framework for expanding the CRISPR toolbox, and repurposing the most abundant CRISPR-Cas systems for a wide range of applications.
Next-generation CRISPR-Cas for genome editing: focusing on the Cas protein and PAM.
Tang Lian Chao,Gu Feng
Yi chuan = Hereditas
The emergence of the gene editing technology, especial CRISPR-Cas (clustered regularly intersected short palindromic repeats and CRISPR associated proteins), has greatly promoted the ability of human beings to transform natural species. It has been widely harnessed for the engineering in the medical, industrial, agricultural and other fields. The key component of the CRISPR-Cas system, the Cas protein, possesses its specific features, including self-activity, recognition site, cutting end and guide RNA. PAM (protein assistant motif) is a number of nucleotides adjacent to the target site, which is very important for the Cas protein to recognize the target sequence and is also the key characteristic of CRISPR-Cas. There are several reported methods for identification of PAM. In this review, we summarize the searching for the Cas protein, the identification of Cas mutants with desired traits and the mapping of the PAM (including the extending of PAM spectrum), in order to provide a reference for the development and optimization of next-generation gene editing tools.
Neutrophil-to-lymphocyte ratio and platelet-to-lymphocyte ratio: novel markers for the diagnosis and prognosis in patients with restenosis following CAS.
Bao Xiang,Zhou Gezhi,Xu Wei,Liu Xiaobo,Ye Zhijun,Jiang Fengfeng
Biomarkers in medicine
In this study, we investigated the effect of neutrophil-to-lymphocyte ratio (NLR) and platelet-to-lymphocyte ratio on restenosis status in patients undergoing carotid angioplasty stenting (CAS). Clinical imageology and receiver operating characteristic analysis were utilized to study the prognostic significance of NLRs/platelet-to-lymphocyte ratios and their correlation with survival. NLR of restenosis (+) patients was evidently increased after the CAS procedures, while the NLR of restenosis (-) patients before the CAS procedures being the lowest. Area under the curve of pre-CAS NLR or/and post-CAS NLR were all evidently higher than 50%. Also, restenosis incidence was the highest in patients with both high pre-CAS and high post-CAS values. Therefore, NLR can be utilized as an independent prognostic indicator to predict the incidence of restenosis after CAS procedures.
Cellular apoptosis susceptibility protein (CAS) suppresses the proliferation of breast cancer cells by upregulated cyp24a1.
Ye Mei,Han Ruigang,Shi Jianwu,Wang Xunda,Zhao Allan Z,Li Fanghong,Chen Hao
Medical oncology (Northwood, London, England)
Breast cancer is the most common cancer in women. Although several studies demonstrated cellular apoptosis susceptibility protein (CAS) involved in the development of breast cancer, the underlying mechanisms of CAS regulating cell processes in the breast cancer remain elusive. In the present study, we explored the possible mechanism of CAS in contributing to the cell proliferation in the breast cancer cell line MCF-7. Knockdown of CAS led to the reduction of cell viability and proliferation. Furthermore, cell cycle was arrested in G0/G1 phase after knocking down CAS with the decrease of cyclinD1. In addition, RNA-seq analysis for the CAS knockdown cells demonstrated that total eleven genes were significantly altered (Fold changes > 2). Of note, the expression of cyp24a1 was dramatically increased in the shCAS cells compared to that of shNC cells as well as confirmed by quantitative real-time polymerase chain reaction (qPCR). These observations clarified the previous conflicting results on the cell fates of the breast cells regulated by CAS and provide new insight into the role of CAS in the development of breast cancer.
CRISPR-Cas systems in oral microbiome: From immune defense to physiological regulation.
Gong Tao,Zeng Jumei,Tang Boyu,Zhou Xuedong,Li Yuqing
Molecular oral microbiology
The clustered regularly interspaced short palindromic repeats with CRISPR-associated proteins (CRISPR-Cas) system, found in bacteria and archaea, provides sequence-based adaptive immunity against mobile genetic elements, including phages and plasmids. The oral cavity contains approximately 700 prokaryote species harboring known CRISPR-Cas systems, including type I, type II, type III, type V, and type VI, and unidentified CRISPR-Cas systems. There is increasing evidence to suggest that different CRISPR-Cas systems in the human oral microbiome can affect bacterial physiology through different mechanisms. Here, we review the canonical and novel functions of the CRISPR-Cas system, including defense against the invasion of foreign mobile elements, biofilm formation, acquisition of resistance genes, DNA repair, regulation of interspecific competition and intraspecific diversification, stress responses, and gene expression regulation. Overall, the mechanisms involved in CRISPR-Cas systems and their effects on bacterial physiology provide new insights into our understanding of the function and application of methods (including gene editing, modulation of CRISPR-Cas by anti-CRISPR, antimicrobials) on the oral microbiome.
CRISPRcasIdentifier: Machine learning for accurate identification and classification of CRISPR-Cas systems.
Padilha Victor A,Alkhnbashi Omer S,Shah Shiraz A,de Carvalho André C P L F,Backofen Rolf
BACKGROUND:CRISPR-Cas genes are extraordinarily diverse and evolve rapidly when compared to other prokaryotic genes. With the rapid increase in newly sequenced archaeal and bacterial genomes, manual identification of CRISPR-Cas systems is no longer viable. Thus, an automated approach is required for advancing our understanding of the evolution and diversity of these systems and for finding new candidates for genome engineering in eukaryotic models. RESULTS:We introduce CRISPRcasIdentifier, a new machine learning-based tool that combines regression and classification models for the prediction of potentially missing proteins in instances of CRISPR-Cas systems and the prediction of their respective subtypes. In contrast to other available tools, CRISPRcasIdentifier can both detect cas genes and extract potential association rules that reveal functional modules for CRISPR-Cas systems. In our experimental benchmark on the most recently published and comprehensive CRISPR-Cas system dataset, CRISPRcasIdentifier was compared with recent and state-of-the-art tools. According to the experimental results, CRISPRcasIdentifier presented the best Cas protein identification and subtype classification performance. CONCLUSIONS:Overall, our tool greatly extends the classification of CRISPR cassettes and, for the first time, predicts missing Cas proteins and association rules between Cas proteins. Additionally, we investigated the properties of CRISPR subtypes. The proposed tool relies not only on the knowledge of manual CRISPR annotation but also on models trained using machine learning.
Characterization of a Type II-A CRISPR-Cas System in .
Mosterd Cas,Moineau Sylvain
and its virulent phages are important members of the human oral microbiota. is also the primary causal agent of dental caries. To survive in this ecological niche, must encode phage defense mechanisms, which include CRISPR-Cas systems. Here, we describe the CRISPR-Cas type II-A system of strain P42S, which was found to display natural adaptation and interference activity in response to phage infection and plasmid transformation. Newly acquired spacers were integrated both at the 5' end of the CRISPR locus and ectopically. In comparisons of the genes of P42S to those of other strains of , , , and appear to be highly conserved within the species. However, more diversity was observed with While the nuclease domains of Cas9 (SmCas9) are conserved, the C terminus of the protein, including the protospacer adjacent motif (PAM) recognition domain, is less conserved. In support of these findings, we experimentally demonstrated that the PAMs associated with SmCas9 of strain P42S are NAA and NGAA. These PAMs are different from those previously reported for the CRISPR-Cas system of the model strain UA159. This study illustrates the diversity of CRISPR-Cas type II-A systems that can be found within the same bacterial species. CRISPR-Cas is one of the mechanisms used by bacteria to defend against viral predation. Increasing our knowledge of the biology and diversity of CRISPR-Cas systems will also improve our understanding of virus-bacterium interactions. As CRISPR-Cas systems acquiring novel immunities under laboratory conditions are rare, strain P42S provides an alternative model to study the adaptation step, which is still the least understood step in CRISPR-Cas biology. Furthermore, the availability of a natural Cas9 protein recognizing an AT-rich PAM opens up new avenues for genome editing purposes.
[Genome editing in plants directed by CRISPR/Cas ribonucleoprotein complexes].
Li Xia,Shi Wan,Geng Li Zhao,Xu Jian Ping
Yi chuan = Hereditas
The CRISPR/Cas system is the most popular genome editing technology in recent years and has been widely used in crop improvement. Compared with introducing the CRISPR/Cas system into plant cells with DNA constructs, introducing CRISPR/Cas ribonucleoprotein (RNP) to perform genome editing excels in rapid action, low off-target rates and is free of DNA insertions in editing plants. However, efficient delivery of CRISPR/Cas RNP into plant cells and achieving high editing frequency are still very challenging, which limits the extensive implementation of CRISPR/Cas RNP-mediated genome editing in plants. In this review, we summarize the progress of protein and RNP delivery methods in plant cells, and provide new perspectives of further development and future applications of the CRISPR/Cas RNP technology in plant genome editing.
Histone-like Nucleoid-Structuring Protein (H-NS) Paralogue StpA Activates the Type I-E CRISPR-Cas System against Natural Transformation in Escherichia coli.
Sun Dongchang,Mao Xudan,Fei Mingyue,Chen Ziyan,Zhu Tingheng,Qiu Juanping
Applied and environmental microbiology
Working mechanisms of CRISPR-Cas systems have been intensively studied. However, far less is known about how they are regulated. The histone-like nucleoid-structuring protein H-NS binds the promoter of genes (P ) and suppresses the type I-E CRISPR-Cas system in Although the H-NS paralogue StpA also binds P , its role in regulating the CRISPR-Cas system remains unidentified. Our previous work established that is able to take up double-stranded DNA during natural transformation. Here, we investigated the function of StpA in regulating the type I-E CRISPR-Cas system against natural transformation of We first documented that although the activated type I-E CRISPR-Cas system, due to deletion, interfered with CRISPR-Cas-targeted plasmid transfer, inactivation restored the level of natural transformation. Second, we showed that inactivating reduced the transcriptional activity of P Third, by comparing transcriptional activities of the intact P and the P with a disrupted H-NS binding site in the and null deletion mutants, we demonstrated that StpA activated transcription of genes by binding to the same site as H-NS in P Fourth, by expressing StpA with an arabinose-inducible promoter, we confirmed that StpA expressed at a low level stimulated the activity of P Finally, by quantifying the level of mature CRISPR RNA (crRNA), we demonstrated that StpA was able to promote the amount of crRNA. Taken together, our work establishes that StpA serves as a transcriptional activator in regulating the type I-E CRISPR-Cas system against natural transformation of StpA is normally considered a molecular backup of the nucleoid-structuring protein H-NS, which was reported as a transcriptional repressor of the type I-E CRISPR-Cas system in However, the role of StpA in regulating the type I-E CRISPR-Cas system remains elusive. Our previous work uncovered a new route for double-stranded DNA (dsDNA) entry during natural transformation of In this study, we show that StpA plays a role opposite to that of its paralogue H-NS in regulating the type I-E CRISPR-Cas system against natural transformation of Our work not only expands our knowledge on CRISPR-Cas-mediated adaptive immunity against extracellular nucleic acids but also sheds new light on understanding the complex regulation mechanism of the CRISPR-Cas system. Moreover, the finding that paralogues StpA and H-NS share a DNA binding site but play opposite roles in transcriptional regulation indicates that higher-order compaction of bacterial chromatin by histone-like proteins could switch prokaryotic transcriptional modes.
It is unclear how important CRISPR-Cas systems are for protecting natural populations of bacteria against infections by mobile genetic elements.
Westra Edze R,Levin Bruce R
Proceedings of the National Academy of Sciences of the United States of America
Articles on CRISPR commonly open with some variant of the phrase "these short palindromic repeats and their associated endonucleases (Cas) are an adaptive immune system that exists to protect bacteria and archaea from viruses and infections with other mobile genetic elements." There is an abundance of genomic data consistent with the hypothesis that CRISPR plays this role in natural populations of bacteria and archaea, and experimental demonstrations with a few species of bacteria and their phage and plasmids show that CRISPR-Cas systems can play this role in vitro. Not at all clear are the ubiquity, magnitude, and nature of the contribution of CRISPR-Cas systems to the ecology and evolution of natural populations of microbes and the strength of selection mediated by different types of phage and plasmids to the evolution and maintenance of CRISPR-Cas systems. In this perspective, with the aid of heuristic mathematical-computer simulation models, we explore the a priori conditions under which exposure to lytic and temperate phage and conjugative plasmids will select for and maintain CRISPR-Cas systems in populations of bacteria and archaea. We review the existing literature addressing these ecological and evolutionary questions and highlight the experimental and other evidence needed to fully understand the conditions responsible for the evolution and maintenance of CRISPR-Cas systems and the contribution of these systems to the ecology and evolution of bacteria, archaea, and the mobile genetic elements that infect them.
The type I-E CRISPR-Cas system influences the acquisition of -IncF plasmid in .
Zhou Ying,Tang Yu,Fu Pan,Tian Dongxing,Yu Lianhua,Huang Yunkun,Li Gang,Li Meng,Wang Yong,Yang Zehua,Xu Xiaogang,Yin Zhe,Zhou Dongsheng,Poirel Laurent,Jiang Xiaofei
Emerging microbes & infections
carbapenemase (KPC)-producing (KPC-KP) have disseminated worldwide and emerged as major threats to public health. Of epidemiological significance, the international pandemic of KPC-KP is primarily associated with CG258 isolates and -IncF plasmids. CRISPR-Cas system is an adaptive immune system that can hinder gene expansion driven by horizontal gene transfer. Because of -IncF plasmids are favored by CG258 it was of interest to examine the co-distribution of CRISPR and -IncF plasmids in such isolates. We collected 459 clinical in China and collected 203 global whole-genome sequences in GenBank to determine the prevalence of CRISPR-Cas systems. We observed that CRISPR-Cas system was significantly scarce in the CG258 lineage and -positive isolates. Furthermore, the results of conjugation and plasmid stability assay fully demonstrated the CRIPSR-Cas system in could effectively hindered -IncF plasmids invasion and existence. Notably, most -IncF plasmids were also proved to be good targets of CRISPR owing to carry matched and functional protospacers and PAMs. Overall, our work suggests that type I-E CRISPR-Cas systems could impact the spread of in populations, and the scarcity of CRISPR-Cas system was one of potential factors leading to the propagation of -IncF plasmids in CG258 .
Web-Based CRISPR Toolkits: Cas-OFFinder, Cas-Designer, and Cas-Analyzer.
Hwang Gue-Ho,Kim Jin-Soo,Bae Sangsu
Methods in molecular biology (Clifton, N.J.)
The CRISPR-Cas system facilitates highly efficient genome editing; thus, it has been applied in many research fields such as biological science, medicine, and gene therapy. However, CRISPR nucleases can cleave off-target sites as well as on-target sites, causing unwanted mutations. Furthermore, after CRISPR treatments are delivered into cells or organisms, it is important to estimate the resulting mutation rates and to determine the patterns of mutations, but these tasks can be difficult. To address these issues, we have developed a tool for identifying potential off-target sites (Cas-OFFinder), a tool for designing CRISPR targets (Cas-Designer), and an assessment tool (Cas-Analyzer). These programs are all implemented on our website so that researchers can easily design CRISPR guide RNAs and assess the resulting mutations by simply clicking on the appropriate buttons; no login process is required.
Approach for in vivo delivery of CRISPR/Cas system: a recent update and future prospect.
Chuang Yu-Fan,Phipps Andrew J,Lin Fan-Li,Hecht Valerie,Hewitt Alex W,Wang Peng-Yuan,Liu Guei-Sheung
Cellular and molecular life sciences : CMLS
The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system provides a groundbreaking genetic technology that allows scientists to modify genes by targeting specific genomic sites. Due to the relative simplicity and versatility of the CRISPR/Cas system, it has been extensively applied in human genetic research as well as in agricultural applications, such as improving crops. Since the gene editing activity of the CRISPR/Cas system largely depends on the efficiency of introducing the system into cells or tissues, an efficient and specific delivery system is critical for applying CRISPR/Cas technology. However, there are still some hurdles remaining for the translatability of CRISPR/Cas system. In this review, we summarized the approaches used for the delivery of the CRISPR/Cas system in mammals, plants, and aquacultures. We further discussed the aspects of delivery that can be improved to elevate the potential for CRISPR/Cas translatability.
Comparative Genomic Analysis of Reveals Horizontal Gene Transfer-Mediated Evolution of the CRISPR-Cas System in the Mycobacterium tuberculosis Complex.
Singh Anoop,Gaur Mohita,Sharma Vishal,Khanna Palak,Bothra Ankur,Bhaduri Asani,Mondal Anupam Kumar,Dash Debasis,Singh Yogendra,Misra Richa
Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) genes are conserved genetic elements in many prokaryotes, including , the causative agent of tuberculosis. Although knowledge of CRISPR locus variability has been utilized in strain genotyping, its evolutionary path in is not well understood. In this study, we have performed a comparative analysis of 141 mycobacterial genomes and identified the exclusive presence of the CRISPR-Cas type III-A system in complex (MTBC). Our global phylogenetic analysis of CRISPR repeats and Cas10 proteins offers evidence of horizontal gene transfer (HGT) of the CRISPR-Cas module in the last common ancestor of MTBC and from a -like environmental bacterium. Additionally, our results show that the variation of CRISPR-Cas organization in lineages, especially in the Beijing sublineage of lineage 2, is due to the transposition of insertion sequence IS The direct repeat (DR) region of the CRISPR-Cas locus acts as a hot spot for IS insertion. We show in H37Rv that the repeat at the 5' end of CRISPR1 of the forward strand is an atypical repeat made up partly of IS-terminal inverted repeat and partly CRISPR DR. By tracing an undetectable spacer sequence in the DR region, the two CRISPR loci could theoretically be joined to reconstruct the ancestral single CRISPR-Cas locus organization, as seen in This study retracing the evolutionary events of HGT and IS-driven genomic deletions helps us to better understand the strain-specific variations in lineages. Comparative genomic analysis of prokaryotes has led to a better understanding of the biology of several pathogenic microorganisms. One such clinically important pathogen is , the leading cause of bacterial infection worldwide. Recent evidence on the functionality of the CRISPR-Cas system in has brought back focus on these conserved genetic elements, present in many prokaryotes. Our study advances understanding of mycobacterial CRISPR-Cas origin and its diversity among the different species. We provide phylogenetic evidence of acquisition of CRISPR-Cas type III-A in the last common ancestor shared between MTBC and , by HGT-mediated events. The most likely source of HGT was an environmental bacterium. Genomic mapping of the CRISPR loci showed the IS transposition-driven variations in strains. Thus, this study offers insights into events related to the evolution of CRISPR-Cas in lineages.
Delivery of CRISPR/Cas systems for cancer gene therapy and immunotherapy.
Song Xiangrong,Liu Chao,Wang Ning,Huang Hai,He Siyan,Gong Changyang,Wei Yuquan
Advanced drug delivery reviews
The clustered, regularly interspaced, short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) systems are efficient and versatile gene editing tools, which offer enormous potential to treat cancer by editing genome, transcriptome or epigenome of tumor cells and/or immune cells. A large body of works have been done with CRISPR/Cas systems for genetic modification, and 16 clinical trials were conducted to treat cancer by ex vivo or in vivo gene editing approaches. Now, promising preclinical works have begun using CRISPR/Cas systems in vivo. However, efficient and safe delivery of CRISPR/Cas systems in vivo is still a critical challenge for their clinical applications. This article summarizes delivery of CRISPR/Cas systems by physical methods, viral vectors and non-viral vectors for cancer gene therapy and immunotherapy. The prospects for the development of physical methods, viral vectors and non-viral vectors for delivery of CRISPR/Cas systems are reviewed, and promising advances in cancer treatment using CRISPR/Cas systems are discussed.
Comprehensive Mining and Characterization of CRISPR-Cas Systems in .
Pan Meichen,Nethery Matthew A,Hidalgo-Cantabrana Claudio,Barrangou Rodolphe
The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas (CRISPR-associated ) systems constitute the adaptive immune system in prokaryotes, which provides resistance against bacteriophages and invasive genetic elements. The landscape of applications in bacteria and eukaryotes relies on a few Cas effector proteins that have been characterized in detail. However, there is a lack of comprehensive studies on naturally occurring CRISPR-Cas systems in beneficial bacteria, such as human gut commensal species. In this study, we mined 954 publicly available genomes and identified CRIPSR-Cas systems in 57% of these strains. A total of five CRISPR-Cas subtypes were identified as follows: Type I-E, I-C, I-G, II-A, and II-C. Among the subtypes, Type I-C was the most abundant (23%). We further characterized the CRISPR RNA (crRNA), tracrRNA, and PAM sequences to provide a molecular basis for the development of new genome editing tools for a variety of applications. Moreover, we investigated the evolutionary history of certain strains through visualization of acquired spacer sequences and demonstrated how these hypervariable CRISPR regions can be used as genotyping markers. This extensive characterization will enable the repurposing of endogenous CRISPR-Cas systems in for genome engineering, transcriptional regulation, genotyping, and screening of rare variants.
Application of different types of CRISPR/Cas-based systems in bacteria.
Liu Zhenquan,Dong Huina,Cui Yali,Cong Lina,Zhang Dawei
Microbial cell factories
As important genome editing tools, CRISPR/Cas systems, especially those based on type II Cas9 and type V Cas12a, are widely used in genetic and metabolic engineering of bacteria. However, the intrinsic toxicity of Cas9 and Cas12a-mediated CRISPR/Cas tools can lead to cell death in some strains, which led to the development of endogenous type I and III CRISPR/Cas systems. However, these systems are hindered by complicated development and limited applications. Thus, further development and optimization of CRISPR/Cas systems is needed. Here, we briefly summarize the mechanisms of different types of CRISPR/Cas systems as genetic manipulation tools and compare their features to provide a reference for selecting different CRISPR/Cas tools. Then, we show the use of CRISPR/Cas technology for bacterial strain evolution and metabolic engineering, including genome editing, gene expression regulation and the base editor tool. Finally, we offer a view of future directions for bacterial CRISPR/Cas technology.
Development of gene editing strategies for human β-globin (HBB) gene mutations.
Kalkan Batuhan Mert,Kala Ezgi Yagmur,Yuce Melek,Karadag Alpaslan Medine,Kocabas Fatih
Recent developments in gene editing technology have enabled scientists to modify DNA sequence by using engineered endonucleases. These gene editing tools are promising candidates for clinical applications, especially for treatment of inherited disorders like sickle cell disease (SCD). SCD is caused by a point mutation in human β-globin gene (HBB). Clinical strategies have demonstrated substantial success, however there is not any permanent cure for SCD available. CRISPR/Cas9 platform uses a single endonuclease and a single guide RNA (gRNA) to induce sequence-specific DNA double strand break (DSB). When this accompanies a repair template, it allows repairing the mutated gene. In this study, it was aimed to target HBB gene via CRISPR/Cas9 genome editing tool to introduce nucleotide alterations for efficient genome editing and correction of point mutations causing SCD in human cell line, by Homology Directed Repair (HDR). We have achieved to induce target specific nucleotide changes on HBB gene in the locus of mutation causing SCD. The effect of on-target activity of bone fide standard gRNA and newly developed longer gRNA were examined. It is observed that longer gRNA has higher affinity to target DNA while having the same performance for targeting and Cas9 induced DSBs. HDR mechanism was triggered by co-delivery of donor DNA repair templates in circular plasmid form. In conclusion, we have suggested methodological pipeline for efficient targeting with higher affinity to target DNA and generating desired modifications on HBB gene.
Multiplex gene editing and large DNA fragment deletion by the CRISPR/Cpf1-RecE/T system in Corynebacterium glutamicum.
Zhao Nannan,Li Lu,Luo Guangjuan,Xie Shan,Lin Ying,Han Shuangyan,Huang Yuanyuan,Zheng Suiping
Journal of industrial microbiology & biotechnology
Corynebacterium glutamicum is an essential industrial strain that has been widely harnessed for the production of all kinds of value-added products. Efficient multiplex gene editing and large DNA fragment deletion are essential strategies for industrial biotechnological research. Cpf1 is a robust and simple genome editing tool for simultaneous editing of multiplex genes. However, no studies on effective multiplex gene editing and large DNA fragment deletion by the CRISPR/Cpf1 system in C. glutamicum have been reported. Here, we developed a multiplex gene editing method by optimizing the CRISPR/Cpf1-RecT system and a large chromosomal fragment deletion strategy using the CRISPR/Cpf1-RecET system in C. glutamicum ATCC 14067. The CRISPR/Cpf1-RecT system exhibited a precise editing efficiency of more than 91.6% with the PAM sequences TTTC, TTTG, GTTG or CTTC. The sites that could be edited were limited due to the PAM region and the 1-7 nt at the 5' end of the protospacer region. Mutations in the PAM region increased the editing efficiency of the - 6 nt region from 0 to 96.7%. Using a crRNA array, two and three genes could be simultaneously edited in one step via the CRISPR/Cpf1-RecT system, and the efficiency of simultaneously editing two genes was 91.6%, but the efficiency of simultaneously editing three genes was below 10%. The editing efficiency for a deletion of 1 kb was 79.6%, and the editing efficiencies for 5- and 20 kb length DNA fragment deletions reached 91.3% and 36.4%, respectively, via the CRISPR/Cpf1-RecET system. This research provides an efficient and simple tool for C. glutamicum genome editing that can further accelerate metabolic engineering efforts and genome evolution.
Applying gene editing to tailor precise genetic modifications in plants.
Van Eck Joyce
The Journal of biological chemistry
The ability to tailor alterations in genomes, including plant genomes, in a site-specific manner has been greatly advanced through approaches that reduced the complexity and time of genome sequencing along with development of gene editing technologies. These technologies provide a valuable foundation for studies of gene function, metabolic engineering, and trait modification for crop improvement. Development of genome editing methodologies began ∼20 years ago, first with meganucleases and followed by zinc finger nucleases, transcriptional activator-like effector nucleases and, most recently, clustered regulatory interspaced short palindromic repeat (CRISPR)-associated protein (Cas) (CRISPR/Cas), which is by far the most utilized method. The premise of CRISPR/Cas centers on the cleaving of one or both DNA strands by a Cas protein, an endonuclease, followed by mending of the DNA by repair mechanisms inherent in cells. Its user-friendly construct design, greater flexibility in targeting genomic regions, and cost-effective attributes have resulted in it being widely adopted and revolutionizing precise modification of the genomes of many organisms. Indeed, the CRISPR/Cas system has been utilized for gene editing in many plant species, including important food crops, such as maize, wheat, rice, and potatoes. This review summarizes the various approaches, including the most recent designs being used to make modifications from as small as a single-base-pair change to insertion of DNA fragments. On the gene expression level, strategies are presented that make it possible to knock out or modulate through activation and repression. Also discussed are prerequisites necessary for CRISPR/Cas-mediated editing as well as the current challenges.
[Current development of gene editing].
Zhang Debin,Luo Yao,Chen Wenjin
Sheng wu gong cheng xue bao = Chinese journal of biotechnology
As the breakthrough in gene editing, represented by CRISPR/Cas9, gene manipulations now are more maneuverable, economically feasible and time saving. It is possible for China to catch an overtaking in researching and industrializing of downside sections (especially the application of plant gene editing), also the incubation of professional companies in gene editing fields. For this consideration, it is necessary and urgent to find the key demands and potential application for gene editing in China. Questionnaire and statistic analysis were carried out to find the key demands and the most potential application fields of the development for gene editing. Firstly, an ordered multi-classification Logistic regression model was established following with dependent variable analysis. Eight out of 24 questionnaires questions in 4 categories were regarded as independent variables with significance test. Then, regression model based on ordered multi-classification logistic method was established to analyze the specific impact of different options on the development of gene editing. The results showed that most researchers in the field of gene editing take the view that development of potential competitive advantages lies in the field of plant science. The results also showed that major gene editing experts believe more attention should be paid on how to carry out technology industrialization while focusing on basic technology development, as well as the development of potential competitive advantages of gene editing technology in plant field. To promote the development of gene editing in China, not only the participation of scientific research institution was needed, but also the synergy of various forces both universities and governments. It is urgent both properly guiding public opinion on gene editing and establishing a national safety standard system. At the same time, the key point of technology risk avoidance should be put on biological weapons and bioterrorism, gene editing related infectious disease, and the potential risk of species genetic change on the ecological environment, etc.