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Bibliography on: CRISPR-Cas

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Robert J. Robbins is a biologist, an educator, a science administrator, a publisher, an information technologist, and an IT leader and manager who specializes in advancing biomedical knowledge and supporting education through the application of information technology. More About:  RJR | OUR TEAM | OUR SERVICES | THIS WEBSITE

RJR: Recommended Bibliography 29 Sep 2022 at 01:52 Created: 

CRISPR-Cas

Clustered regularly interspaced short palindromic repeats (CRISPR, pronounced crisper) are segments of prokaryotic DNA containing short repetitions of base sequences. Each repetition is followed by short segments of "spacer DNA" from previous exposures to foreign DNA (e.g a virus or plasmid). The CRISPR/Cas system is a prokaryotic immune system that confers resistance to foreign genetic elements such as those present within plasmids and phages, and provides a form of acquired immunity. CRISPR associated proteins (Cas) use the CRISPR spacers to recognize and cut these exogenous genetic elements in a manner analogous to RNA interference in eukaryotic organisms. CRISPRs are found in approximately 40% of sequenced bacterial genomes and 90% of sequenced archaea. By delivering the Cas9 nuclease complexed with a synthetic guide RNA (gRNA) into a cell, the cell's genome can be cut at a desired location, allowing existing genes to be removed and/or new ones added. The Cas9-gRNA complex corresponds with the CAS III crRNA complex in the above diagram. CRISPR/Cas genome editing techniques have many potential applications, including altering the germline of humans, animals, and food crops. The use of CRISPR Cas9-gRNA complex for genome editing was the AAAS's choice for breakthrough of the year in 2015.

Created with PubMed® Query: "CRISPR.CAS" OR "crispr/cas" NOT pmcbook NOT ispreviousversion

Citations The Papers (from PubMed®)

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RevDate: 2022-09-28

Ortiz-Cartagena C, Fernández-García L, Blasco L, et al (2022)

Reverse Transcription-Loop-Mediated Isothermal Amplification-CRISPR-Cas13a Technology as a Promising Diagnostic Tool for SARS-CoV-2.

Microbiology spectrum [Epub ahead of print].

At the end of 2019, a new coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), caused a pandemic that persists to date and has resulted in more than 6.2 million deaths. In the last couple of years, researchers have made great efforts to develop a diagnostic technique that maintains high levels of sensitivity and specificity, since an accurate and early diagnosis is required to minimize the prevalence of SARS-CoV-2 infection. In this context, CRISPR-Cas systems are proposed as promising tools for development as diagnostic techniques due to their high specificity, highlighting that Cas13 endonuclease discriminates single nucleotide changes and displays collateral activity against single-stranded RNA molecules. With the aim of improving the sensitivity of diagnosis, this technology is usually combined with isothermal preamplification reactions (SHERLOCK, DETECTR). Based on this, we developed a reverse transcription-loop-mediated isothermal amplification (RT-LAMP)-CRISPR-Cas13a method for SARS-CoV-2 virus detection in nasopharyngeal samples without using RNA extraction that exhibits 100% specificity and 83% sensitivity, as well as a positive predictive value (PPV) of 100% and negative predictive values (NPVs) of 100%, 81%, 79.1%, and 66.7% for cycle threshold (CT) values of <20, 20 to 30, >30 and overall, respectively. IMPORTANCE The coronavirus disease 2019 (COVID-19) crisis has driven the development of innovative molecular diagnosis methods, including CRISPR-Cas technology. In this work, we performed a protocol, working with RNA extraction kit-free samples and using RT-LAMP-CRISPR-Cas13a technology; our results place this method at the forefront of rapid and specific diagnostic methods for COVID-19 due to the high specificity (100%), sensitivity (83%), PPVs (100%), and NPVs (81% for high viral loads) obtained with clinical samples.

RevDate: 2022-09-28
CmpDate: 2022-09-28

Carvalho J, M Peixoto (2022)

CT-205 CRISPR/Cas9 System: Use of Genetic Engineering in Healing Cancer and Other Genetic Diseases.

Clinical lymphoma, myeloma & leukemia, 22 Suppl 2:S438-S439.

CRISPR/Cas9 is an adaptive immune system of bacteria and archaea, also called archaea, against invading phages in a natural environment. Bacteria realize this system by capturing DNA sequences and using them as a memory to identify themselves as an enemy and destroy it in a future attack. Through this natural adaptive immunity of bacteria and archaea, a new way of redesigning an edit to achieve the desired genome was discovered, and more importantly, using this new design as a therapy against cancerous and genetic diseases. Thus, the present study was motivated by the fact that the CRISPR system is still a little-known topic. Thus, the objective was to carry out a literature review on the applicability of the CRISPR/Cas9 technology in the treatment of cancer, aiming to expand the knowledge of the technique for future therapeutic developments. The study is a literature review based on national and international journals. The primary consultations were carried out in the online libraries: NCIB, PubMed, Nature, Science, and Oncotarget. The secondary consultations were carried out through the reference articles of the primary consultation articles. The primary searches used the keyword "CRISPR". As for the secondary ones, no selection criteria were used, being limited only to the references of the primary articles. There were no restrictions on publication date and language. The CRISPR/Cas9 system, even with a short time of discovery, has become a very efficient tool, capable of repairing and modifying DNA sequences. In the field of genetic engineering, the ability to identify a specific location in DNA and modify it has always been a challenge, but this system has made it possible to manipulate genes to create a diversity of mutations in different organisms and cells. It is also worth mentioning its use in the discovery of possible targets for the treatment of pathologies and correction of phenotypes. CRISPR/Cas9 holds great promise for the development of diverse biotechnological therapies, with its easy genetic manipulation capability, efficiency, and wide application. However, we must not forget the potential weaknesses or limitations of this system until it becomes accepted by medical practice, and thus, its application for the benefit of society is possible.

RevDate: 2022-09-27

Hillary VE, SA Ceasar (2022)

A Review on the Mechanism and Applications of CRISPR/Cas9/Cas12/Cas13/Cas14 Proteins Utilized for Genome Engineering.

Molecular biotechnology [Epub ahead of print].

The clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein (CRISPR/Cas) system has altered life science research offering enormous options in manipulating, detecting, imaging, and annotating specific DNA or RNA sequences of diverse organisms. This system incorporates fragments of foreign DNA (spacers) into CRISPR cassettes, which are further transcribed into the CRISPR arrays and then processed to make guide RNA (gRNA). The CRISPR arrays are genes that encode Cas proteins. Cas proteins provide the enzymatic machinery required for acquiring new spacers targeting invading elements. Due to programmable sequence specificity, numerous Cas proteins such as Cas9, Cas12, Cas13, and Cas14 have been exploited to develop new tools for genome engineering. Cas variants stimulated genetic research and propelled the CRISPR/Cas tool for manipulating and editing nucleic acid sequences of living cells of diverse organisms. This review aims to provide detail on two classes (class 1 and 2) of the CRISPR/Cas system, and the mechanisms of all Cas proteins, including Cas12, Cas13, and Cas14 discovered so far. In addition, we also discuss the pros and cons and recent applications of various Cas proteins in diverse fields, including those used to detect viruses like severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). This review enables the researcher to gain knowledge on various Cas proteins and their applications, which have the potential to be used in next-generation precise genome engineering.

RevDate: 2022-09-27

Mukherjee IA, Gabel C, Noinaj N, et al (2022)

Structural basis of AcrIF24 as an anti-CRISPR protein and transcriptional suppressor.

Nature chemical biology [Epub ahead of print].

Anti-CRISPR (Acr) proteins are encoded by phages to inactivate CRISPR-Cas systems of bacteria and archaea and are used to enhance the CRISPR toolbox for genome editing. Here we report the structure and mechanism of AcrIF24, an Acr protein that inhibits the type I-F CRISPR-Cas system from Pseudomonas aeruginosa. AcrIF24 is a homodimer that associates with two copies of the surveillance complex (Csy) and prevents the hybridization between CRISPR RNA and target DNA. Furthermore, AcrIF24 functions as an anti-CRISPR-associated (Aca) protein to repress the transcription of the acrIF23-acrIF24 operon. Alone or in complex with Csy, AcrIF24 is capable of binding to the acrIF23-acrIF24 promoter DNA with nanomolar affinity. The structure of a Csy-AcrIF24-promoter DNA complex at 2.7 Å reveals the mechanism for transcriptional suppression. Our results reveal that AcrIF24 functions as an Acr-Aca fusion protein, and they extend understanding of the diverse mechanisms used by Acr proteins.

RevDate: 2022-09-28
CmpDate: 2022-09-28

Xin C, Yin J, Yuan S, et al (2022)

Comprehensive assessment of miniature CRISPR-Cas12f nucleases for gene disruption.

Nature communications, 13(1):5623.

Because of their small size, the recently developed CRISPR-Cas12f nucleases can be effectively packaged into adeno-associated viruses for gene therapy. However, a systematic evaluation of the editing outcomes of CRISPR-Cas12f is lacking. In this study, we apply a high-throughput sequencing method to comprehensively assess the editing efficiency, specificity, and safety of four Cas12f proteins in parallel with that of Cas9 and two Cas12a proteins at multiple genomic sites. Cas12f nucleases achieve robust cleavage at most of the tested sites and mainly produce deletional fragments. In contrast, Cas9 and Cas12a show relatively higher editing efficiency at the vast majority of the tested sites. However, the off-target hotspots identified in the Cas9- and Cas12a-edited cells are negligibly detected in the Cas12f-edited cells. Moreover, compared to Cas9 and Cas12a nucleases, Cas12f nucleases reduce the levels of chromosomal translocations, large deletions, and integrated vectors by 2- to 3-fold. Therefore, our findings confirm the editing capacity of Cas12f and reveal the ability of this nuclease family to preserve genome integrity during genome editing.

RevDate: 2022-09-26

Escobar M, Li J, Patel A, et al (2022)

Quantification of Genome Editing and Transcriptional Control Capabilities Reveals Hierarchies among Diverse CRISPR/Cas Systems in Human Cells.

ACS synthetic biology [Epub ahead of print].

CRISPR/Cas technologies have revolutionized the ability to redesign genomic information and tailor endogenous gene expression. Nevertheless, the discovery and development of new CRISPR/Cas systems has resulted in a lack of clarity surrounding the relative efficacies among these technologies in human cells. This deficit makes the optimal selection of CRISPR/Cas technologies in human cells unnecessarily challenging, which in turn hampers their adoption, and thus ultimately limits their utility. Here, we designed a series of endogenous testbed systems to methodically quantify and compare the genome editing, CRISPRi, and CRISPRa capabilities among 10 different natural and engineered Cas protein variants spanning Type II and Type V CRISPR/Cas families. We show that although all Cas protein variants are capable of genome editing and transcriptional control in human cells, hierarchies exist, particularly for genome editing and CRISPRa applications, wherein Cas9 ≥ Cas12a > Cas12e/Cas12j. Our findings also highlight the utility of our modular testbed platforms to rapidly and systematically quantify the functionality of practically any natural or engineered genomic-targeting Cas protein in human cells.

RevDate: 2022-09-28
CmpDate: 2022-09-28

Marciano S, Ionescu TM, Saw RS, et al (2022)

Combining CRISPR-Cas9 and brain imaging to study the link from genes to molecules to networks.

Proceedings of the National Academy of Sciences of the United States of America, 119(40):e2122552119.

Receptors, transporters, and ion channels are important targets for therapy development in neurological diseases, but their mechanistic role in pathogenesis is often poorly understood. Gene editing and in vivo imaging approaches will help to identify the molecular and functional role of these targets and the consequence of their regional dysfunction on the whole-brain level. We combine CRISPR-Cas9 gene editing with in vivo positron emission tomography (PET) and functional MRI (fMRI) to investigate the direct link between genes, molecules, and the brain connectome. The extensive knowledge of the Slc18a2 gene encoding the vesicular monoamine transporter (VMAT2), involved in the storage and release of dopamine, makes it an excellent target for studying the gene network relationships while structurally preserving neuronal integrity and function. We edited the Slc18a2 in the substantia nigra pars compacta of adult rats and used in vivo molecular imaging besides behavioral, histological, and biochemical assessments to characterize the CRISPR-Cas9-mediated VMAT2 knockdown. Simultaneous PET/fMRI was performed to investigate molecular and functional brain alterations. We found that stage-specific adaptations of brain functional connectivity follow the selective impairment of presynaptic dopamine storage and release. Our study reveals that recruiting different brain networks is an early response to the dopaminergic dysfunction preceding neuronal cell loss. Our combinatorial approach is a tool to investigate the impact of specific genes on brain molecular and functional dynamics, which will help to develop tailored therapies for normalizing brain function.

RevDate: 2022-09-28

Pandey PK, Bhowmik P, S Kagale (2022)

Optimized methods for random and targeted mutagenesis in field pea (Pisum sativum L.).

Frontiers in plant science, 13:995542.

Field pea is an important pulse crop for its dense nutritional profile and contribution to sustainable agricultural practices. Recently, it has received extensive attention as a potential leading source of plant-based proteins. However, the adoption of peas as a mainstream source of proteins is affected by a relatively moderate protein content, anti-nutritional factors and high levels of off-flavor components that reduce protein quality. Availability of genetic variation for desirable seed quality traits is the foundation for the sustainable development of pea varieties with improved protein content and quality. Mutagenesis has been an important tool in gene functional characterization studies and creating genetic variability for crop breeding. Large-scale mutagenesis of a crop using physical and chemical agents requires diligent selection of the mutagen and optimization of its dose to increase the frequency of mutations. In this study, we present detailed optimized protocols for physical and chemical mutagenesis of pea using gamma irradiation and ethyl methanesulfonate (EMS), respectively. Gamma radiation and EMS titration kill curves were established to identify optimal doses of the two mutagenic agents. Based on germination, survival rate and growth phenotypes, a gamma radiation dose of 225 Gy and EMS concentration of 5 mm were selected as optimal dosages for mutagenesis in field pea. The presented protocol has been modified from previously established mutagenesis protocols in other crop plants. Our results indicate that the optimal mutagen dosage is genotype dependent. CRISPR/Cas-based gene editing provides a precise and rapid method for targeted genetic manipulation in plants. With the recent success of gene editing in pea using CRISPR/Cas, this innovative technology is expected to become an integral component of the gene discovery and crop improvement toolkit in pea. Here, we describe an optimized methods for targeted mutagenesis of pea protoplasts, including mesophyll protoplast extraction, PEG-mediated transformation and gene editing of a LOX gene using CRISPR/Cas system. The general strategies and methods of mutagenesis described here provide an essential resource for mutation breeding and functional genomics studies in pea. These methods also provide a foundation for similar studies in other crops.

RevDate: 2022-09-28

Hossain MM (2022)

Wheat blast: A review from a genetic and genomic perspective.

Frontiers in microbiology, 13:983243.

The newly emerged wheat blast fungus Magnaporthe oryzae Triticum (MoT) is a severe threat to global wheat production. The fungus is a distinct, exceptionally diverse lineage of the M. oryzae, causing rice blast disease. Genome-based approaches employing MoT-specific markers are used to detect MoT field isolates. Sequencing the whole genome indicates the presence of core chromosome and mini-chromosome sequences that harbor effector genes and undergo divergent evolutionary routes. Significant genetic and pathotype diversity within the fungus population gives ample potential for evolutionary change. Identifying and refining genetic markers allows for tracking genomic regions with stable blast resistance. Introgression of quantitative and R gene resistance into popular cultivars is crucial to controlling disease in areas where the pathogen population is diverse and well established. Novel approaches such as CRISPR/Cas-9 genome editing could generate resistant varieties in wheat within a short time. This chapter provides an extensive summary of the genetic and genomic aspects of the wheat blast fungus MoT and offers an essential resource for wheat blast research in the affected areas.

RevDate: 2022-09-28

Li CZ, TY Hu (2022)

Nanotechnology Powered CRISPR-Cas Systems for Point of Care Diagnosis and Therapeutic.

Research (Washington, D.C.), 2022:9810237.

RevDate: 2022-09-26

Lau RK, Enustun E, Gu Y, et al (2022)

A conserved signaling pathway activates bacterial CBASS immune signaling in response to DNA damage.

The EMBO journal [Epub ahead of print].

To protect themselves from the constant threat of bacteriophage (phage) infection, bacteria have evolved diverse immune systems including restriction-modification, CRISPR-Cas, and many others. Here, we describe the discovery of a two-protein transcriptional regulator module associated with hundreds of CBASS immune systems and demonstrate that this module drives the expression of its associated CBASS system in response to DNA damage. We show that the helix-turn-helix transcriptional repressor CapH binds the promoter region of its associated CBASS system to repress transcription until it is cleaved by the metallopeptidase CapP. CapP is activated in vitro by single-stranded DNA, and in cells by DNA-damaging drugs. Together, CapH and CapP drive increased expression of their associated CBASS system in response to DNA damage. We identify CapH- and CapP-related proteins associated with diverse known and putative bacterial immune systems including DISARM and Pycsar antiphage operons. Overall, our data highlight a mechanism by which bacterial immune systems can sense and respond to a universal signal of cell stress, potentially enabling multiple immune systems to mount a coordinated defensive response against an invading pathogen.

RevDate: 2022-09-28

Kumar M, Maiti S, D Chakraborty (2022)

Capturing nucleic acid variants with precision using CRISPR diagnostics.

Biosensors & bioelectronics, 217:114712 pii:S0956-5663(22)00752-7 [Epub ahead of print].

CRISPR/Cas systems have the ability to precisely target nucleotide sequences and enable their rapid identification and modification. While nucleotide modification has enabled the therapeutic correction of diseases, the process of identifying the target DNA or RNA has greatly expanded the field of molecular diagnostics in recent times. CRISPR-based DNA/RNA detection through programmable nucleic acid binding or cleavage has been demonstrated for a large number of pathogenic and non-pathogenic targets. Combining CRISPR detection with nucleic acid amplification and a terminal signal readout step allowed the development of numerous rapid and robust nucleic acid platforms. Wherever the Cas effector can faithfully distinguish nucleobase variants in the target, the platform can also be extended for sequencing-free rapid variant detection. Some initial PAM disruption-based SNV detection reports were limited to finding or integrating mutated/mismatched nucleotides within the PAM sequences. In this review, we try to summarize the developments made in CRISPR diagnostics (CRISPRDx) to date emphasizing CRISPR-based SNV detection. We also discuss the applications where such diagnostic modalities can be put to use, covering various fields of clinical research, SNV screens, disease genotyping, primary surveillance during microbial infections, agriculture, food safety, and industrial biotechnology. The ease of rapid design and implementation of such multiplexable assays can potentially expand the applications of CRISPRDx in the domain of affinity-based target sequencing, with immense possibilities for low-cost, quick, and widespread usage. In the end, in combination with proximity assays and a suicidal gene approach, CRISPR-based in vivo SNV detection and cancer cell targeting can be formulated as personalized gene therapy.

RevDate: 2022-09-28
CmpDate: 2022-09-28

Konwar AN, Hazarika SN, Bharadwaj P, et al (2022)

Emerging Non-Traditional Approaches to Combat Antibiotic Resistance.

Current microbiology, 79(11):330.

An increasing number of bacterial pathogens are acquiring resistance to the commonly used antibiotics. This has spurred a global threat leading to a resistance era and has penetrated the consciousness of the common people and the clinicians alike. The delay in discovering new antibiotics has exacerbated the resistance problem, forcing researchers to focus on unconventional antimicrobial therapeutics that differ from conventional antibiotics. Alternative therapies have emerged in recent years, including antimicrobial peptides, phage therapy, efflux pump inhibitors, antibodies, and immunomodulatory agents, which have produced impressive results in both laboratory and in clinical trials. Additionally, ultra-narrow-spectrum therapeutics such as CRISPR-Cas system and peptide nucleic acids aided in the development of sequence-specific antimicrobials. Moreover, combinatorial therapies that combine these new approaches have been efficient enough to get approval for clinical use and have accelerated the discovery of novel combination approaches that enhance the performance of already in-use antibiotics. In this review, we provide an overview of these approaches along with studies that focus on the uncharted microbial territories that have been able to deliver some of the important new antibiotics of recent times. It is hoped that the information gathered in this article will provide an update on the current antibiotic resistance threat and encourage profound research.

RevDate: 2022-09-28
CmpDate: 2022-09-28

Du Y, Han D, An Z, et al (2022)

CRISPR/dCas9-surface-enhanced Raman scattering for the detection of drug resistance gene macB.

Mikrochimica acta, 189(10):394.

Antibiotics have brought many benefits to public health systems worldwide since their first use in the last century, yet with their overuse in clinical treatment and livestock farming, new public health issues have arisen. Previously, we found in our experiments that the levels of macB genes in bovine raw milk ranked among the top of many drug resistance genes. In this paper, we present an analysis of regularly interspaced clustered short palindromic repeats (CRISPR) combined with surface-enhanced Raman scattering (SERS) technology for the detection of the drug resistance gene macB. The analysis was accomplished through the collaboration of the CRISPR system's ability to specifically identify genes and the more sensitive performance of the SERS. The analysis detects the drug resistance gene macB and does not yet require complex steps such as nucleic acid amplification. This method may prove to be an effective method for accurate detection of the drug-resistant gene macB, thus enabling more effective prevention of contamination of drug-resistant genes in food hygiene.

RevDate: 2022-09-26

Abkhooie L, S Saberianpour (2022)

CRISPR/Cas9 Tool for MicroRNAs Editing in Cardiac Development, Function, and Disease.

MicroRNA (Shariqah, United Arab Emirates) pii:MIRNA-EPUB-126499 [Epub ahead of print].

CRISPR/Cas9 is a powerful gene-editing technology. Extensive scientific data exist that the CRISPR/Cas9 system can target small, non-coding, active RNA molecules including microRNAs [miRNAs]. miRNAs have been recognized as key regulators of different cell biological processes, such as modulation of fibrosis and cardiac hypertrophy, as well as the regulation of cardiomyocytes. Also, it has been demonstrated that miRNAs strongly affect organ evolution and the concentration of miRNAs can involve in the differentiation, development, and function of different organs. In addition, the current findings clearly indicate that miRNAs can select and control their targets based on their own concentrations. CRISPR/Cas9 genome-editing technology is a stronger system for stopping miRNAs than previous methods including antisense inhibitors. CRISPR/Cas9 tools can be used to eliminate small areas of DNA and determine miRNA in cases where similar groups of miRNAs are in the same strand. Herein, besides other emerging strategies we critically summarize the recent investigations linking miRNA-targeted therapeutics and CRISPR/Cas9 system, to precisely clarify and combine different delivery platforms and cell-fate engineering of miRNAs function and miRNA-based therapeutic intervention in cardiac development, function, and disease. Based on our findings from the literature, it appears that the use of the CRISPR/Cas technology provides new perspectives for understanding the molecular mechanism of cardiovascular disease and can be effective in the treatment and control of cardiac development, function, and disease in the future.

RevDate: 2022-09-28
CmpDate: 2022-09-28

Chan WF, Coughlan HD, Chen Y, et al (2022)

Activation of stably silenced genes by recruitment of a synthetic de-methylating module.

Nature communications, 13(1):5582.

Stably silenced genes that display a high level of CpG dinucleotide methylation are refractory to the current generation of dCas9-based activation systems. To counter this, we create an improved activation system by coupling the catalytic domain of DNA demethylating enzyme TET1 with transcriptional activators (TETact). We show that TETact demethylation-coupled activation is able to induce transcription of suppressed genes, both individually and simultaneously in cells, and has utility across a number of cell types. Furthermore, we show that TETact can effectively reactivate embryonic haemoglobin genes in non-erythroid cells. We anticipate that TETact will expand the existing CRISPR toolbox and be valuable for functional studies, genetic screens and potential therapeutics.

RevDate: 2022-09-28
CmpDate: 2022-09-26

Teng M, Zhou ZY, Yao Y, et al (2022)

A New Strategy for Efficient Screening and Identification of Monoclonal Antibodies against Oncogenic Avian Herpesvirus Utilizing CRISPR/Cas9-Based Gene-Editing Technology.

Viruses, 14(9):.

Marek's disease virus (MDV) is an important oncogenic α-herpesvirus that induces Marek's disease (MD), characterized by severe immunosuppression and rapid-onset T-cell lymphomas in its natural chicken hosts. Historically, MD is regarded as an ideal biomedical model for studying virally induced cancers. Monoclonal antibodies (mAbs) against viral or host antigenic epitopes are crucial for virology research, especially in the exploration of gene functions, clinical therapy, and the development of diagnostic reagents. Utilizing the CRISPR/Cas9-based gene-editing technology, we produced a pp38-deleted MDV-1 mutant-GX0101Δpp38-and used it for the rapid screening and identification of pp38-specific mAbs from a pool of MDV-specific antibodies from 34 hybridomas. The cross-staining of parental and mutated MDV plaques with hybridoma supernatants was first performed by immunofluorescence assay (IFA). Four monoclonal hybridomas-namely, 4F9, 31G7, 34F2, and 35G9-were demonstrated to secrete specific antibodies against MDV-1's pp38 protein, which was further confirmed by IFA staining and confocal analysis. Further experiments using Western blotting, immunoprecipitation (IP), liquid chromatography-tandem mass spectrometry (LC-MS/MS), and immunohistochemistry (IHC) analysis demonstrated that the pp38-specific mAb 31G7 has high specificity and wide application potential for further research in MD biology. To the best of our knowledge, this is the first demonstration of the use of CRISPR/Cas9-based gene-editing technology for efficient screening and identification of mAbs against a specific viral protein, and provides a meaningful reference for the future production of antibodies against other viruses-especially for large DNA viruses such as herpesviruses.

RevDate: 2022-09-28
CmpDate: 2022-09-26

Khanal S, Cao D, Zhang J, et al (2022)

Synthetic gRNA/Cas9 Ribonucleoprotein Inhibits HIV Reactivation and Replication.

Viruses, 14(9):.

The current antiretroviral therapy (ART) for human immunodeficiency virus (HIV) can halt viral replication but cannot eradicate HIV infection because proviral DNA integrated into the host genome remains genetically silent in reservoir cells and is replication-competent upon interruption or cessation of ART. CRISPR/Cas9-based technology is widely used to edit target genes via mutagenesis (i.e., nucleotide insertion/deletion and/or substitution) and thus can inactivate integrated proviral DNA. However, CRISPR/Cas9 delivery systems often require viral vectors, which pose safety concerns for therapeutic applications in humans. In this study, we used synthetic guide RNA (gRNA)/Cas9-ribonucleoprotein (RNP) as a non-viral formulation to develop a novel HIV gene therapy. We designed a series of gRNAs targeting different HIV genes crucial for HIV replication and tested their antiviral efficacy and cellular cytotoxicity in lymphoid and monocytic latent HIV cell lines. Compared with the scramble gRNA control, HIV-gRNA/Cas9 RNP-treated cells exhibited efficient viral suppression with no apparent cytotoxicity, as evidenced by the significant inhibition of latent HIV DNA reactivation and RNA replication. Moreover, HIV-gRNA/Cas9 RNP inhibited p24 antigen expression, suppressed infectious viral particle production, and generated specific DNA cleavages in the targeted HIV genes that are confirmed by DNA sequencing. Because of its rapid DNA cleavage, low off-target effects, low risk of insertional mutagenesis, easy production, and readiness for use in clinical application, this study provides a proof-of-concept that synthetic gRNA/Cas9 RNP drugs can be utilized as a novel therapeutic approach for HIV eradication.

RevDate: 2022-09-28
CmpDate: 2022-09-26

Takabayashi S, Iijima K, Tsujimura M, et al (2022)

Successful i-GONAD in Mice at Early Zygote Stage through In Vivo Electroporation Three Min after Intraoviductal Instillation of CRISPR-Ribonucleoprotein.

International journal of molecular sciences, 23(18):.

Improved genome editing via oviductal nucleic acids delivery (i-GONAD) is a new technology enabling in situ genome editing of mammalian zygotes exiting the oviductal lumen, which is now available in mice, rats, and hamsters. In this method, CRISPR/Cas9 genome-editing reagents are delivered directly to the oviducts of pregnant animals (corresponding to late zygote stage). After intraoviductal instillation, electric shock to the entire oviduct was provided with a specialized electroporation (EP) device to drive the genome editing reagents into the zygotes present in the oviductal lumen. i-GONAD toward early zygotes has been recognized as difficult, because they are tightly surrounded by a cumulus cell layer, which often hampers effective transfer of nucleic acids to zygotes. However, in vivo EP three min after intraoviductal instillation of the genome-editing reagents enabled genome editing of early zygotes with an efficiency of 70%, which was in contrast with the rate of 18% when in vivo EP was performed immediately after intraoviductal instillation at Day 0.5 of pregnancy (corresponding to 13:00-13:30 p.m. on the day when vaginal plug was recognized after natural mating). We also found that addition of hyaluronidase, an enzyme capable of removing cumulus cells from a zygote, slightly enhanced the efficiency of genome editing in early zygotes. These findings suggest that cumulus cells surrounding a zygote can be a barrier for efficient generation of genome-edited mouse embryos and indicate that a three-minute interval before in vivo EP is effective for achieving i-GONAD-mediated genome editing at the early zygote stage. These results are particularly beneficial for researchers who want to perform genome editing experiments targeting early zygotes.

RevDate: 2022-09-28
CmpDate: 2022-09-26

Song JH, Shin G, Kim HJ, et al (2022)

Mutation of GmIPK1 Gene Using CRISPR/Cas9 Reduced Phytic Acid Content in Soybean Seeds.

International journal of molecular sciences, 23(18):.

Phytic acid (PA) acts as an antinutrient substance in cereal grains, disturbing the bioavailability of micronutrients, such as iron and zinc, in humans, causing malnutrition. GmIPK1 encodes the inositol 1,3,4,5,6-pentakisphosphate 2-kinase enzyme, which converts myo-inopsitol-1,3,4,5,6-pentakisphosphate (IP5) to myo-inositol-1,2,3,4,5,6-hexakisphosphate (IP6) in soybean (Glycine max L.). In this study, for developing soybean with low PA levels, we attempted to edit the GmIPK1 gene using the CRISPR/Cas9 system to introduce mutations into the GmIPK1 gene with guide RNAs in soybean (cv. Kwangankong). The GmIPK1 gene was disrupted using the CRISPR/Cas9 system, with sgRNA-1 and sgRNA-4 targeting the second and third exon, respectively. Several soybean Gmipk1 gene-edited lines were obtained in the T0 generation at editing frequencies of 0.1-84.3%. Sequencing analysis revealed various indel patterns with the deletion of 1-9 nucleotides and insertions of 1 nucleotide in several soybean lines (T0). Finally, we confirmed two sgRNA-4 Gmipk1 gene-edited homozygote soybean T1 plants (line #21-2: 5 bp deletion; line #21-3: 1 bp insertion) by PPT leaf coating assay and PCR analysis. Analysis of soybean Gmipk1 gene-edited lines indicated a reduction in PA content in soybean T2 seeds but did not show any defects in plant growth and seed development.

RevDate: 2022-09-28
CmpDate: 2022-09-26

Wang M, He L, Chen B, et al (2022)

Transgenerationally Transmitted DNA Demethylation of a Spontaneous Epialleles Using CRISPR/dCas9-TET1cd Targeted Epigenetic Editing in Arabidopsis.

International journal of molecular sciences, 23(18):.

CRISPR/dCas9 is an important DNA modification tool in which a disarmed Cas9 protein with no nuclease activity is fused with a specific DNA modifying enzyme. A previous study reported that overexpression of the TET1 catalytic domain (TET1cd) reduces genome-wide methylation in Arabidopsis. A spontaneous naturally occurring methylation region (NMR19-4) was identified in the promoter region of the PPH (Pheophytin Pheophorbide Hydrolase) gene, which encodes an enzyme that can degrade chlorophyll and accelerate leaf senescence. The methylation status of NMR19-4 is associated with PPH expression and leaf senescence in Arabidopsis natural accessions. In this study, we show that the CRISPR/dCas9-TET1cd system can be used to target the methylation of hypermethylated NMR19-4 region to reduce the level of methylation, thereby increasing the expression of PPH and accelerating leaf senescence. Furthermore, hybridization between transgenic demethylated plants and hypermethylated ecotypes showed that the demethylation status of edited NMR19-4, along with the enhanced PPH expression and accelerated leaf senescence, showed Mendelian inheritance in F1 and F2 progeny, indicating that spontaneous epialleles are stably transmitted trans-generationally after demethylation editing. Our results provide a rational approach for future editing of spontaneously mutated epialleles and provide insights into the epigenetic mechanisms that control plant leaf senescence.

RevDate: 2022-09-28
CmpDate: 2022-09-26

Liu H, Chen W, Li Y, et al (2022)

CRISPR/Cas9 Technology and Its Utility for Crop Improvement.

International journal of molecular sciences, 23(18):.

The rapid growth of the global population has resulted in a considerable increase in the demand for food crops. However, traditional crop breeding methods will not be able to satisfy the worldwide demand for food in the future. New gene-editing technologies, the most widely used of which is CRISPR/Cas9, may enable the rapid improvement of crop traits. Specifically, CRISPR/Cas9 genome-editing technology involves the use of a guide RNA and a Cas9 protein that can cleave the genome at specific loci. Due to its simplicity and efficiency, the CRISPR/Cas9 system has rapidly become the most widely used tool for editing animal and plant genomes. It is ideal for modifying the traits of many plants, including food crops, and for creating new germplasm materials. In this review, the development of the CRISPR/Cas9 system, the underlying mechanism, and examples of its use for editing genes in important crops are discussed. Furthermore, certain limitations of the CRISPR/Cas9 system and potential solutions are described. This article will provide researchers with important information regarding the use of CRISPR/Cas9 gene-editing technology for crop improvement, plant breeding, and gene functional analyses.

RevDate: 2022-09-28
CmpDate: 2022-09-26

Mahdi AK, Medrano JF, PJ Ross (2022)

Single-Step Genome Editing of Small Ruminant Embryos by Electroporation.

International journal of molecular sciences, 23(18):.

We investigated the possibility of single-step genome editing in small ruminants by CRISPR-Cas9 zygote electroporation. We targeted SOCS2 and PDX1 in sheep embryos and OTX2 in goat embryos, utilizing a dual sgRNA approach. Gene editing efficiency was compared between microinjection and three different electroporation settings performed at four different times of embryo development. Electroporation of sheep zygotes 6 h after fertilization with settings that included short high-voltage (poring) and long low-voltage (transfer) pulses was efficient at producing SOCS2 knock-out blastocysts. The mutation rate after CRISPR/Cas9 electroporation was 95.6% ± 8%, including 95.4% ± 9% biallelic mutations; which compared favorably to 82.3% ± 8% and 25% ± 10%, respectively, when using microinjection. We also successfully disrupted the PDX1 gene in sheep and the OTX2 gene in goat embryos. The biallelic mutation rate was 81 ± 5% for PDX1 and 85% ± 6% for OTX2. In conclusion, using single-step CRISPR-Cas9 zygote electroporation, we successfully introduced biallelic deletions in the genome of small ruminant embryos.

RevDate: 2022-09-28
CmpDate: 2022-09-26

Li B, Fu C, Zhou J, et al (2022)

Highly Efficient Genome Editing Using Geminivirus-Based CRISPR/Cas9 System in Cotton Plant.

Cells, 11(18):.

Upland cotton (Gossypium hirsutum), an allotetraploid, contains At- and Dt- subgenome and most genes have multiple homologous copies, which pose a huge challenge to investigate genes' function due to the functional redundancy. Therefore, it is of great significance to establish effective techniques for the functional genomics in cotton. In this study, we tested two novel genome editing vectors and compared them with the CRISPR/Cas9 system (pRGEB32-GhU6.7) developed in our laboratory previously. In the first new vector, the sgRNA transcription unite was constructed into the replicon (LIR-Donor-SIR-Rep-LIR) of the bean yellow dwarf virus (BeYDV) and named as pBeYDV-Cas9-KO and in the second vector, the ubiquitin promoter that drives Cas9 protein was replaced with a constitutive CaMV 35S promoter and defined as pRGEB32-35S. The results from transgenic cotton calli/plants revealed that pBeYDV-Cas9-KO vector showed the highest editing efficiency of GhCLA1 in At and Dt subgenomes edited simultaneously up to 73.3% compared to the 44.6% of pRGEB32-GhU6.7 and 51.2% of pRGEB32-35S. The editing efficiency of GhCLA1 in At and Dt subgenome by pBeYDV-Cas9-KO was 85.7% and 97.2%, respectively, whereas the efficiency by pRGEB32-GhU6.7 and pRGEB32-35S vectors was 67.7%, 86.5%, 84%, and 87.2%, respectively. The editing profile of pBeYDV-Cas9-KO was mainly composed of fragment deletion, accounting for 84.0% and ranging 1-10 bp in length. The main editing sites are located at positions 11-17 upstream of PAM site. The off-target effects were not detected in all potential off-target sites. Taken together, the pBeYDV-Cas9-KO system has high editing efficiency and specificity with wide editing range than the traditional CRISPR/Cas9 system, which provides a powerful tool for cotton functional genomics research and molecular breeding.

RevDate: 2022-09-28
CmpDate: 2022-09-26

Chira S, Nutu A, Isacescu E, et al (2022)

Genome Editing Approaches with CRISPR/Cas9 for Cancer Treatment: Critical Appraisal of Preclinical and Clinical Utility, Challenges, and Future Research.

Cells, 11(18):.

The increasing burden on human malignant diseases became a major concern for healthcare practitioners, that must deal with tumor relapse and the inability to efficiently treat metastasis, in addition to side effects. Throughout the decades, many therapeutic strategies have been employed to improve the clinical outcomes of cancer patients and great efforts have been made to develop more efficient and targeted medicines. The malignant cell is characterized by genetic and epigenetic modifications, therefore targeting those specific drivers of carcinogenesis is highly desirable. Among the genome editing technologies, CRISPR/Cas9 stood as a promising candidate for cancer treatment alternatives, due to its low complexity design. First described as a defense mechanism of bacteria against invading foreign DNA, later it was shown that CRISPR components can be engineered to target specific DNA sequences in a test tube, a discovery that was awarded later with the Nobel Prize in chemistry for its rapid expansion as a reliable genome editing tool in many fields of research, including medicine. The present paper aims of describing CRISPR/Cas9 potential targets for malignant disorders, and the approaches used for achieving this goal. Aside from preclinical studies, we also present the clinical trials that use CRISPR-based technology for therapeutic purposes of cancer. Finally, a summary of the presented studies adds a more focused view of the therapeutic value CRISPR/Cas9 holds and the associated shortcomings.

RevDate: 2022-09-28
CmpDate: 2022-09-26

Nie D, Guo T, Yue M, et al (2022)

Research Progress on Nanoparticles-Based CRISPR/Cas9 System for Targeted Therapy of Tumors.

Biomolecules, 12(9):.

Cancer is a genetic mutation disease that seriously endangers the health and life of all human beings. As one of the most amazing academic achievements in the past decade, CRISPR/Cas9 technology has been sought after by many researchers due to its powerful gene editing capability. CRISPR/Cas9 technology shows great potential in oncology, and has become one of the most promising technologies for cancer genome-editing therapeutics. However, its efficiency and the safety issues of in vivo gene editing severely limit its widespread application. Therefore, developing a suitable delivery method for the CRISPR/Cas9 system is an urgent problem to be solved at present. Rapid advances in nanomedicine suggest nanoparticles could be a viable option. In this review, we summarize the latest research on the potential use of nanoparticle-based CRISPR/Cas9 systems in cancer therapeutics, in order to further their clinical application. We hope that this review will provide a novel insight into the CRISPR/Cas9 system and offer guidance for nanocarrier designs that will enable its use in cancer clinical applications.

RevDate: 2022-09-27
CmpDate: 2022-09-26

Kaiser J (2022)

CRISPR infusion eases symptoms in genetic disease.

Science (New York, N.Y.), 377(6613):1367.

Some patients no longer need drugs to prevent swelling.

RevDate: 2022-09-28
CmpDate: 2022-09-28

Friskes A, Koob L, Krenning L, et al (2022)

Double-strand break toxicity is chromatin context independent.

Nucleic acids research, 50(17):9930-9947.

Cells respond to double-strand breaks (DSBs) by activating DNA damage response pathways, including cell cycle arrest. We have previously shown that a single double-strand break generated via CRISPR/Cas9 is sufficient to delay cell cycle progression and compromise cell viability. However, we also found that the cellular response to DSBs can vary, independent of the number of lesions. This implies that not all DSBs are equally toxic, and raises the question if the location of a single double-strand break could influence its toxicity. To systematically investigate if DSB-location is a determinant of toxicity we performed a CRISPR/Cas9 screen targeting 6237 single sites in the human genome. Next, we developed a data-driven framework to design CRISPR/Cas9 sgRNA (crRNA) pools targeting specific chromatin features. The chromatin context was defined using ChromHMM states, Lamin-B1 DAM-iD, DNAseI hypersensitivity, and RNA-sequencing data. We computationally designed 6 distinct crRNA pools, each containing 10 crRNAs targeting the same chromatin state. We show that the toxicity of a DSB is highly similar across the different ChromHMM states. Rather, we find that the major determinants of toxicity of a sgRNA are cutting efficiency and off-target effects. Thus, chromatin features have little to no effect on the toxicity of a single CRISPR/Cas9-induced DSB.

RevDate: 2022-09-27
CmpDate: 2022-09-26

Li J, Tang L, Li T, et al (2022)

Tandem Cas13a/crRNA-Mediated CRISPR-FET Biosensor: A One-for-All Check Station for Virus without Amplification.

ACS sensors, 7(9):2680-2690.

The path toward field-effect transistor (FET) application from laboratory to clinic has delivered a compelling push in the biomedical domain, yet ultrasensitive and timely pathogen identification without PCR remains a long-lasting challenge. Herein, we create a generic check station termed "CRISPR-FET", first incorporating the CRISPR/Cas13a system within the FET modality, for accelerated and unamplified detection of viral RNA. Unlike conventional FETs bearing target-specific receptors, this sensor holds three unique advancements: (i) an ingenious sensing mechanism is used, which converts the signal of a large-sized analyte into an on-chip cleavage response of an immobilized CRISPR reporter, enabling signal generation events to occur all within the Debye length; (ii) the multipurpose inspection of the CoV ORF1ab, CoV N gene, and HCV RNA unveils the potential for "one-for-all" scalable FET-based molecular diagnostics; and (iii) it is shown that Cas13a-crRNAs targeting different sites of the viral genome can be deployed in tandem to amplify the FET response, empowering the detection limit down to 1.56 aM, which is a world-record level of sensitivity in the FET for direct viral gene sensing. Notably, a brilliant clinical applicability was made in distinguishing HCV-infected patients from normal controls. Overall, this study sheds new insights into FET-based nucleic acid sensing technology and invokes a vision for its possible future roles in diagnosis of various viral diseases.

RevDate: 2022-09-28
CmpDate: 2022-09-28

Chen Z, Du H, Tao Y, et al (2022)

Efficient breeding of low glutelin content rice germplasm by simultaneous editing multiple glutelin genes via CRISPR/Cas9.

Plant science : an international journal of experimental plant biology, 324:111449.

Chronic kidney disease (CKD) and phenylketonuria (PKU) patients need to eat rice with low glutelin content. Therefore, breeding low glutelin content rice varieties with high yield and delicious taste is one of the major goals of rice breeders due to the high demand for the product. In this study, we designed three sgRNAs targeting nine glutelin genes and generated nine T-DNA-free homozygous editing lines with reduced glutelin content compared with the wild-type due to simultaneous mutation(s) in 5-7 glutelin genes. The glutelin content of two lines is even significantly lower than that of the low glutelin content cultivar, LGC-1. Compared to the wild-type, these low glutelin lines showed similar agronomic traits, including yield components and viscosity properties, and can be used as new varieties or parental materials for further breeding.

RevDate: 2022-09-28
CmpDate: 2022-09-28

Yang Y, Zhang C, Song Y, et al (2022)

Small-molecule activators specific to adenine base editors through blocking the canonical TGF-β pathway.

Nucleic acids research, 50(17):9632-9646.

Adenine base editors (ABEs) catalyze A-to-G conversions, offering therapeutic options to treat the major class of human pathogenic single nucleotide polymorphisms (SNPs). However, robust and precise editing at diverse genome loci remains challenging. Here, using high-throughput chemical screening, we identified and validated SB505124, a selective ALK5 inhibitor, as an ABE activator. Treating cells with SB505124 enhanced on-target editing at multiple genome loci, including epigenetically refractory regions, and showed little effect on off-target conversion on the genome. Furthermore, SB505124 facilitated the editing of disease-associated genes in vitro and in vivo. Intriguingly, SB505124 served as a specific activator by selectively promoting ABE activity. Mechanistically, SB505124 promotes ABE editing, at least in part, by enhancing ABE expression and modulating DNA repair-associated genes. Our findings reveal the role of the canonical transforming growth factor-β pathway in gene editing and equip ABEs with precise chemical control.

RevDate: 2022-09-28
CmpDate: 2022-09-28

Meyerink BL, Kc P, Tiwari NK, et al (2022)

Breasi-CRISPR: an efficient genome-editing method to interrogate protein localization and protein-protein interactions in the embryonic mouse cortex.

Development (Cambridge, England), 149(18):.

In developing tissues, knowing the localization and interactors of proteins of interest is key to understanding their function. Here, we describe the Breasi-CRISPR approach (Brain Easi-CRISPR), combining Easi-CRISPR with in utero electroporation to tag endogenous proteins within embryonic mouse brains. Breasi-CRISPR enables knock-in of both short and long epitope tag sequences with high efficiency. We visualized epitope-tagged proteins with varied expression levels, such as ACTB, LMNB1, EMD, FMRP, NOTCH1 and RPL22. Detection was possible by immunohistochemistry as soon as 1 day after electroporation and we observed efficient gene editing in up to 50% of electroporated cells. Moreover, tagged proteins could be detected by immunoblotting in lysates from individual cortices. Next, we demonstrated that Breasi-CRISPR enables the tagging of proteins with fluorophores, allowing visualization of endogenous proteins by live imaging in organotypic brain slices. Finally, we used Breasi-CRISPR to perform co-immunoprecipitation mass-spectrometry analyses of the autism-related protein FMRP to discover its interactome in the embryonic cortex. Together, these data demonstrate that Breasi-CRISPR is a powerful tool with diverse applications that will propel the understanding of protein function in neurodevelopment.

RevDate: 2022-09-28
CmpDate: 2022-09-28

Mayta ML, Dotto M, Orellano EG, et al (2022)

An experimental protocol for teaching CRISPR/Cas9 in a post-graduate plant laboratory course: An analysis of mutant-edited plants without sequencing.

Biochemistry and molecular biology education : a bimonthly publication of the International Union of Biochemistry and Molecular Biology, 50(5):537-546.

The CRISPR/Cas9 system is widely used for editing genes in various organisms and is a very useful tool due to its versatility, simplicity, and efficiency. To teach its principles to post-graduate students we designed a laboratory activity to obtain and analyze PDS3 mutants in Arabidopsis thaliana plants consisting of: 1) Design of guide RNAs using bioinformatics tools; 2) plant transformation (which is optional depending on the length of the course); 3) observation and evaluation of the mutant's phenotypes in the Phytoene desaturase (PDS3) gene, which exhibit an albino phenotype and different degrees of mosaicism in the editing events we evaluated; 4) PCR amplification of a fragment that includes the mutated region followed by analysis of single-stranded DNA conformation polymorphisms (SSCP) using native polyacrylamide gel electrophoresis and silver nitrate staining to detect changes in the amplicon sequence due to gene editing. Through SSCP, the students were able to distinguish between homozygous and heterozygous edited plants. A highlight feature of this protocol is the visualization and detection of the mutation/edition without sequencing the edited fragment.

RevDate: 2022-09-28
CmpDate: 2022-09-28

Misra CS, Rangu SS, Phulsundar RD, et al (2022)

An improved, simple and field-deployable CRISPR-Cas12a assay for the detection of SARS-CoV-2.

Journal of applied microbiology, 133(4):2668-2677.

AIMS: The RT-PCR is the most popular confirmatory test for SARS-CoV-2. It is sensitive, but high instrumentation cost makes it difficult for use outside routine clinical setup. This has necessitated the development of alternative methods such as CRISPR-based DETECTR method which uses lateral flow technology. Although accurate and sensitive, this method is limited by complex steps and recurrent cost of high-quality lateral flow strips. The main goal of this study was to improve the Cas12a-based SARS-CoV-2 DETECTR method and develop a portable and field-deployable system to reduce the recurring consumable cost.

METHODS AND RESULTS: Specific regions of N and E genes from SARS-CoV-2 virus and human RNase P (internal control) were reverse transcribed (RT) and amplified by loop-mediated isothermal amplification (LAMP). The amplified products were detected by a Cas12a-based trans-cleavage reaction that generated a fluorescent signal which could be easily visualized by naked eye. Detection of internal control, RNase P gene was improved and optimized by redesigning RT-LAMP primers. A number of steps were reduced by combining the reagents related to the detection of Cas12a trans-cleavage reaction into a single ready-to-use mix. A portable, cost-effective battery-operated instrument, CRISPR-CUBE was developed to run the assay and visualize the outcome. The method and instrument were validated using both contrived and patient samples.

CONCLUSIONS: The simplified CRISPR-based SARS-CoV-2 detection and instrument developed in this study, along with improved design for internal control detection allows for easier, more definitive viral detection requiring only reagents, consumables and the battery operable CRISPR-CUBE.

Significant improvement in Cas12 method, coupled with simple visualization of end point makes the method and instrument deployable at the point-of-care (POC) for SARS-CoV-2 detection, without any recurrent cost for the lateral flow strips which is used in other POC methods.

RevDate: 2022-09-28
CmpDate: 2022-09-28

Pant S, Ritika , Nag P, et al (2022)

Employment of the CRISPR/Cas9 system to improve cellulase production in Trichoderma reesei.

Biotechnology advances, 60:108022.

Trichoderma reesei has been explored intensively in the laboratory and on an industrial scale for its highly potent cellulase secretion machinery since its characterization over 70 years ago. Emergence of new genetic tools over the past decade has strengthened the understanding of mechanism involved in transcription of cellulase genes in fungi and provided a boost to edit them at molecular level. Since several transcriptional factors work synergistically for cellulase expression in fungi; engineering of cellulase secretome for enhanced cellulase titer require combined manipulation of these factors. In the same context, CRISPR/Cas9 has emerged as a powerful, versatile genetic engineering tool for multiplex gene editing in fungi. It is true that considerable efforts with CRISPR technologies have largely developed fungal genetic engineering, but its application in fungi is still challenging and limited. The present review illustrates the precision, strengths and challenges of using CRISPR/Cas9 technology for cellulase engineering in T. reesei, highlighting key strategies that could be employed for strain improvement.

RevDate: 2022-09-28
CmpDate: 2022-09-28

Song Y, He S, Jopkiewicz A, et al (2022)

Development and application of CRISPR-based genetic tools in Bacillus species and Bacillus phages.

Journal of applied microbiology, 133(4):2280-2298.

Recently, the clustered regularly interspaced short palindromic repeats (CRISPR) system has been developed into a precise and efficient genome editing tool. Since its discovery as an adaptive immune system in prokaryotes, it has been applied in many different research fields including biotechnology and medical sciences. The high demand for rapid, highly efficient and versatile genetic tools to thrive in bacteria-based cell factories accelerates this process. This review mainly focuses on significant advancements of the CRISPR system in Bacillus subtilis, including the achievements in gene editing, and on problems still remaining. Next, we comprehensively summarize this genetic tool's up-to-date development and utilization in other Bacillus species, including B. licheniformis, B. methanolicus, B. anthracis, B. cereus, B. smithii and B. thuringiensis. Furthermore, we describe the current application of CRISPR tools in phages to increase Bacillus hosts' resistance to virulent phages and phage genetic modification. Finally, we suggest potential strategies to further improve this advanced technique and provide insights into future directions of CRISPR technologies for rendering Bacillus species cell factories more effective and more powerful.

RevDate: 2022-09-28
CmpDate: 2022-09-28

Awan MJA, Pervaiz K, Rasheed A, et al (2022)

Genome edited wheat- current advances for the second green revolution.

Biotechnology advances, 60:108006.

Common wheat is a major source of nutrition around the globe, but unlike maize and rice hybrids, no breakthrough has been made to enhance wheat yield since Green Revolution. With the availability of reference genome sequence of wheat and advancement of allied genomics technologies, understanding of genes involved in grain yield components and disease resistance/susceptibility has opened new avenues for crop improvement. Wheat has a huge hexaploidy genome of approximately 17 GB with 85% repetition, and it is a daunting task to induce any mutation across three homeologues that can be helpful for the enhancement of agronomic traits. The CRISPR-Cas9 system provides a promising platform for genome editing in a site-specific manner. In wheat, CRISPR-Cas9 is being used in the improvement of yield, grain quality, biofortification, resistance against diseases, and tolerance against abiotic factors. The promising outcomes of the CRISPR-based multiplexing approach circumvent the constraint of targeting merely one gene at a time. Deployment of clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) 9 endonuclease (CRISPR-Cas9) and Cas9 variant systems such as cytidine base editing, adenosine base editing, and prime editing in wheat has been used to induce point mutations more precisely. Scientists have acquired major events such as induction of male sterility, fertility restoration, and alteration of seed dormancy through Cas9 in wheat that can facilitate breeding programs for elite variety development. Furthermore, a recent discovery in tissue culturing enables scientists to significantly enhance regeneration efficiency in wheat by transforming the GRF4-GIF1 cassette. Rapid generation advancement by speed breeding technology provides the opportunity for the generation advancement of the desired plants to segregate out unwanted transgenes and allows rapid integration of gene-edited wheat into the breeding pipeline. The combination of these novel technologies addresses some of the most important limiting factors for sustainable and climate-smart wheat that should lead to the second "Green Revolution" for global food security.

RevDate: 2022-09-27
CmpDate: 2022-09-26

Yaish O, Asif M, Y Orenstein (2022)

A systematic evaluation of data processing and problem formulation of CRISPR off-target site prediction.

Briefings in bioinformatics, 23(5):.

CRISPR/Cas9 system is widely used in a broad range of gene-editing applications. While this editing technique is quite accurate in the target region, there may be many unplanned off-target sites (OTSs). Consequently, a plethora of computational methods have been developed to predict off-target cleavage sites given a guide RNA and a reference genome. However, these methods are based on small-scale datasets (only tens to hundreds of OTSs) produced by experimental techniques to detect OTSs with a low signal-to-noise ratio. Recently, CHANGE-seq, a new in vitro experimental technique to detect OTSs, was used to produce a dataset of unprecedented scale and quality (>200 000 OTS over 110 guide RNAs). In addition, the same study included in cellula GUIDE-seq experiments for 58 of the guide RNAs. Here, we fill the gap in previous computational methods by utilizing these data to systematically evaluate data processing and formulation of the CRISPR OTSs prediction problem. Our evaluations show that data transformation as a pre-processing phase is critical prior to model training. Moreover, we demonstrate the improvement gained by adding potential inactive OTSs to the training datasets. Furthermore, our results point to the importance of adding the number of mismatches between guide RNAs and their OTSs as a feature. Finally, we present predictive off-target in cellula models based on both in vitro and in cellula data and compare them to state-of-the-art methods in predicting true OTSs. Our conclusions will be instrumental in any future development of an off-target predictor based on high-throughput datasets.

RevDate: 2022-09-27
CmpDate: 2022-09-26

Hu WX, Rong Y, Guo Y, et al (2022)

ExsgRNA: reduce off-target efficiency by on-target mismatched sgRNA.

Briefings in bioinformatics, 23(5):.

Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene editing technology has been widely used to facilitate efficient genome editing. Current popular sgRNA design tools only consider the sgRNA perfectly matched to the target site and provide the results without any on-target mismatch. We suppose taking on-target gRNA-DNA mismatches into consideration might provide better sgRNA with similar binding activity and reduced off-target sites. Here, we trained a seq2seq-attention model with feedback-loop architecture, to automatically generate sgRNAs with on-target mismatches. Dual-luciferase reporter experiment showed that multiple sgRNAs with three mismatches could achieve the 80% of the relative activity of the perfect matched sgRNA. Meanwhile, it could reduce the number of off-target sites using sgRNAs with on-target mismatches. Finally, we provided a freely accessible web server sgRNA design tool named ExsgRNA. Users could submit their target sequence to this server and get optimal sgRNAs with less off-targets and similar on-target activity compared with the perfect-matched sgRNA.

RevDate: 2022-09-24

Zönnchen J, Gantner J, Lapin D, et al (2022)

EDS1 complexes are not required for PRR responses and execute TNL-ETI from the nucleus in Nicotiana benthamiana.

The New phytologist [Epub ahead of print].

Heterodimeric complexes incorporating the lipase-like proteins EDS1 with PAD4 or SAG101 are central hubs in plant innate immunity. EDS1 functions encompass signal relay from TIR domain-containing intracellular NLR-type immune receptors (TNLs) towards RPW8-type helper NLRs (RNLs) and, in A. thaliana, bolstering of signaling and resistance mediated by cell-surface pattern recognition receptors (PRRs). Increasing evidence points to the activation of EDS1 complexes by small molecule binding. We used CRISPR/Cas-generated mutant lines and agroinfiltration-based complementation assays to interrogate functions of EDS1 complexes in N. benthamiana. We do not detect impaired PRR signaling in N. benthamiana lines deficient in EDS1 complexes or RNLs. Intriguingly, in assays monitoring functions of SlEDS1-NbEDS1 complexes in N. benthamiana, mutations within the SlEDS1 catalytic triad can abolish or enhance TNL immunity. Furthermore, nuclear EDS1 accumulation is sufficient for N. benthamiana TNL (Roq1) immunity. Reinforcing PRR signaling in Arabidopsis might be a derived function of the TNL/EDS1 immune sector. Although Solanaceae EDS1 functionally depends on catalytic triad residues in some contexts, our data do not support binding of a TNL-derived small molecule in the triad environment. Whether and how nuclear EDS1 activity connects to membrane pore-forming RNLs remains unknown.

RevDate: 2022-09-24

Hai Y, Wang X, J Xie (2022)

[The role of bacterial toxin-antitoxin systems in phage abortive infections].

Sheng wu gong cheng xue bao = Chinese journal of biotechnology, 38(9):3291-3300.

Bacteria are often infected by large numbers of phages, and host bacteria have evolved diverse molecular strategies in the race with phages, with abortive infection (Abi) being one of them. The toxin-antitoxin system (TA) is expressed in response to bacterial stress, mediating hypometabolism and even dormancy, as well as directly reducing the formation of offspring phages. In addition, some of the toxins' sequences and structures are highly homologous to Cas, and phages even encode antitoxin analogs to block the activity of the corresponding toxins. This suggests that the failure of phage infection due to bacterial death in abortive infections is highly compatible with TA function, whereas TA may be one of the main resistance and defense forces for phage infestation of the host. This review summarized the TA systems involved in phage abortive infections based on classification and function. Moreover, TA systems with abortive functions and future use in antibiotic development and disease treatment were predicted. This will facilitate the understanding of bacterial-phage interactions as well as phage therapy and related synthetic biology research.

RevDate: 2022-09-23

Anonymous (2022)

Redirecting meiotic recombination by CRISPR-Cas-mediated chromosome engineering.

Nature plants [Epub ahead of print].

RevDate: 2022-09-23

Wienmeister A (2022)

Rereading Habermas in Times of CRISPR-cas: A Critique of and an Alternative to the Instrumentalist Interpretation of the Human Nature Argument.

Journal of bioethical inquiry [Epub ahead of print].

Habermas's argument from human nature, which speaks in favour of holding back the use of human germline editing for purposes of enhancement, has lately received criticism anew. Prominent are objections to its supposedly genetic essentialist and determinist framework, which underestimates social impacts on human development. I argue that this criticism originates from an instrumentalist reading of Habermas's argument, which wrongly focuses on empirical conditions and means-ends-relations. Drawing on Habermas's distinction of a threefold use of practical reason, I show how an alternative-the ethical-reading avoids essentialist and determinist objections by addressing an existential level of sense making. I present three reasons that speak in favour of the ethical reading and I demonstrate how it incorporates social aspects of character formation. Habermas's account therefore offers exactly what the critics claim is missing. The paper concludes with a conceptual challenge that the ethical reading has to face within Habermas's overall approach to genetic engineering.

RevDate: 2022-09-23

Salvi P, Varshney V, M Majee (2022)

Raffinose Family Oligosaccharides (RFOs): Role in seed vigor and longevity.

Bioscience reports pii:231854 [Epub ahead of print].

Seed vigor and longevity are important agronomic attributes, as they are essentially associated with crop yield and thus the global economy. Seed longevity is a measure of seed viability and the most essential property in gene bank management since it affects regeneration of seed recycling. Reduced seed life or storability is a serious issue in seed storage since germplasm conservation and agricultural enhancement initiatives rely on it. The irreversible and ongoing process of seed deterioration comprises a complex gene regulatory network and altered metabolism that results in membrane damage, DNA integrity loss, mitochondrial dysregulation, protein damage, and disrupted antioxidative machinery. Carbohydrates and/or sugars, primarily RFOs, have emerged as feasible components for boosting or increasing seed vigor and longevity in recent years. RFOs are known to perform diverse functions in plants, including abiotic and biotic stress tolerance, besides being involved in regulating seed germination, desiccation tolerance, vigor, and longevity. We emphasised and analysed the potential impact of RFOs on seed vigor and longevity in this review. Here, we comprehensively reviewed the molecular mechanisms involved in seed longevity, RFO metabolism, and how RFO content is critical and linked with seed vigor and longevity. Further molecular basis, biotechnological approaches, and CRISPR/Cas applications have been discussed briefly for the improvement of seed attributes and ultimately crop production. Likewise, we suggest advancements, challenges, and future possibilities in this area.

RevDate: 2022-09-23

Denner J (2022)

Virus Safety of Xenotransplantation.

Viruses, 14(9): pii:v14091926.

The practice of xenotransplantation using pig islet cells or organs is under development to alleviate the shortage of human donor islet cells or organs for the treatment of diabetes or organ failure. Multiple genetically modified pigs were generated to prevent rejection. Xenotransplantation may be associated with the transmission of potentially zoonotic porcine viruses. In order to prevent this, we developed highly sensitive PCR-based, immunologicals and other methods for the detection of numerous xenotransplantation-relevant viruses. These methods were used for the screening of donor pigs and xenotransplant recipients. Of special interest are the porcine endogenous retroviruses (PERVs) that are integrated in the genome of all pigs, which are able to infect human cells, and that cannot be eliminated by methods that other viruses can. We showed, using droplet digital PCR, that the number of PERV proviruses is different in different pigs (usually around 60). Furthermore, the copy number is different in different organs of a single pig, indicating that PERVs are active in the living animals. We showed that in the first clinical trials treating diabetic patients with pig islet cells, no porcine viruses were transmitted. However, in preclinical trials transplanting pig hearts orthotopically into baboons, porcine cytomegalovirus (PCMV), a porcine roseolovirus (PCMV/PRV), and porcine circovirus 3 (PCV3), but no PERVs, were transmitted. PCMV/PRV transmission resulted in a significant reduction of the survival time of the xenotransplant. PCMV/PRV was also transmitted in the first pig heart transplantation to a human patient and possibly contributed to the death of the patient. Transmission means that the virus was detected in the recipient, however it remains unclear whether it can infect primate cells, including human cells. We showed previously that PCMV/PRV can be eliminated from donor pigs by early weaning. PERVs were also not transmitted by inoculation of human cell-adapted PERV into small animals, rhesus monkey, baboons and cynomolgus monkeys, even when pharmaceutical immunosuppression was applied. Since PERVs were not transmitted in clinical, preclinical, or infection experiments, it remains unclear whether they should be inactivated in the pig genome by CRISPR/Cas. In summary, by using our sensitive methods, the safety of xenotransplantation can be ensured.

RevDate: 2022-09-23

Salman A, Kantor A, McClements ME, et al (2022)

Non-Viral Delivery of CRISPR/Cas Cargo to the Retina Using Nanoparticles: Current Possibilities, Challenges, and Limitations.

Pharmaceutics, 14(9): pii:pharmaceutics14091842.

The discovery of the CRISPR/Cas system and its development into a powerful genome engineering tool have revolutionized the field of molecular biology and generated excitement for its potential to treat a wide range of human diseases. As a gene therapy target, the retina offers many advantages over other tissues because of its surgical accessibility and relative immunity privilege due to its blood-retinal barrier. These features explain the large advances made in ocular gene therapy over the past decade, including the first in vivo clinical trial using CRISPR gene-editing reagents. Although viral vector-mediated therapeutic approaches have been successful, they have several shortcomings, including packaging constraints, pre-existing anti-capsid immunity and vector-induced immunogenicity, therapeutic potency and persistence, and potential genotoxicity. The use of nanomaterials in the delivery of therapeutic agents has revolutionized the way genetic materials are delivered to cells, tissues, and organs, and presents an appealing alternative to bypass the limitations of viral delivery systems. In this review, we explore the potential use of non-viral vectors as tools for gene therapy, exploring the latest advancements in nanotechnology in medicine and focusing on the nanoparticle-mediated delivery of CRIPSR genetic cargo to the retina.

RevDate: 2022-09-23

Schüller A, Studt-Reinhold L, J Strauss (2022)

How to Completely Squeeze a Fungus-Advanced Genome Mining Tools for Novel Bioactive Substances.

Pharmaceutics, 14(9): pii:pharmaceutics14091837.

Fungal species have the capability of producing an overwhelming diversity of bioactive substances that can have beneficial but also detrimental effects on human health. These so-called secondary metabolites naturally serve as antimicrobial "weapon systems", signaling molecules or developmental effectors for fungi and hence are produced only under very specific environmental conditions or stages in their life cycle. However, as these complex conditions are difficult or even impossible to mimic in laboratory settings, only a small fraction of the true chemical diversity of fungi is known so far. This also implies that a large space for potentially new pharmaceuticals remains unexplored. We here present an overview on current developments in advanced methods that can be used to explore this chemical space. We focus on genetic and genomic methods, how to detect genes that harbor the blueprints for the production of these compounds (i.e., biosynthetic gene clusters, BGCs), and ways to activate these silent chromosomal regions. We provide an in-depth view of the chromatin-level regulation of BGCs and of the potential to use the CRISPR/Cas technology as an activation tool.

RevDate: 2022-09-23

Kaewsapsak P, Chantaravisoot N, Nimsamer P, et al (2022)

In Silico Evaluation of CRISPR-Based Assays for Effective Detection of SARS-CoV-2.

Pathogens (Basel, Switzerland), 11(9): pii:pathogens11090968.

Coronavirus disease (COVID-19) caused by the SARS-CoV-2 has been an outbreak since late 2019 up to now. This pandemic causes rapid development in molecular detection technologies to diagnose viral infection for epidemic prevention. In addition to antigen test kit (ATK) and polymerase chain reaction (PCR), CRISPR-based assays for detection of SARS-CoV-2 have gained attention because it has a simple setup but still maintain high specificity and sensitivity. However, the SARS-CoV-2 has been continuing mutating over the past few years. Thus, molecular tools that rely on matching at the nucleotide level need to be reevaluated to preserve their specificity and sensitivity. Here, we analyzed how mutations in different variants of concern (VOC), including Alpha, Beta, Gamma, Delta, and Omicron strains, could introduce mismatches to the previously reported primers and crRNAs used in the CRISPR-Cas system. Over 40% of the primer sets and 15% of the crRNAs contain mismatches. Hence, primers and crRNAs in nucleic acid-based assays must be chosen carefully to pair up with SARS-CoV-2 variants. In conclusion, the data obtained from this study could be useful in selecting the conserved primers and crRNAs for effective detections against the VOC of SARS-CoV-2.

RevDate: 2022-09-23

Mori H, Kataoka M, X Yang (2022)

Past, Present, and Future of Genome Modification in Escherichia coli.

Microorganisms, 10(9): pii:microorganisms10091835.

Escherichia coli K-12 is one of the most well-studied species of bacteria. This species, however, is much more difficult to modify by homologous recombination (HR) than other model microorganisms. Research on HR in E. coli has led to a better understanding of the molecular mechanisms of HR, resulting in technical improvements and rapid progress in genome research, and allowing whole-genome mutagenesis and large-scale genome modifications. Developments using λ Red (exo, bet, and gam) and CRISPR-Cas have made E. coli as amenable to genome modification as other model microorganisms, such as Saccharomyces cerevisiae and Bacillus subtilis. This review describes the history of recombination research in E. coli, as well as improvements in techniques for genome modification by HR. This review also describes the results of large-scale genome modification of E. coli using these technologies, including DNA synthesis and assembly. In addition, this article reviews recent advances in genome modification, considers future directions, and describes problems associated with the creation of cells by design.

RevDate: 2022-09-23

Yoon S, Min Y, Park C, et al (2022)

Innate Immune Response Analysis in Meniscus Xenotransplantation Using Normal and Triple Knockout Jeju Native Pigs.

International journal of molecular sciences, 23(18): pii:ijms231810416.

Although allogenic meniscus grafting can be immunologically safe, it causes immune rejection due to an imbalanced tissue supply between donor and recipient. Pigs are anatomically and physiologically similar to adult humans and are, therefore, considered to be advantageous xenotransplantation models. However, immune rejection caused by genetic difference damages the donor tissue and can sometimes cause sudden death. Immune rejection is caused by genes; porcine GGTA1, CMAH, and B4GLANT2 are the most common. In this study, we evaluated immune cells infiltrating the pig meniscus transplanted subcutaneously into BALB/c mice bred for three weeks. We compared the biocompatibility of normal Jeju native black pig (JNP) meniscus with that of triple knockout (TKO) JNP meniscus (α-gal epitope, N-glycolylneuraminic acid (Neu5Gc), and Sd (a) epitope knockout using CRISPR-Cas 9). Mast cells, eosinophils, neutrophils, and macrophages were found to have infiltrated the transplant boundary in the sham (without transplantation), normal (normal JNP), and test (TKO JNP) samples after immunohistochemical analysis. When compared to normal and sham groups, TKO was lower. Cytokine levels did not differ significantly between normal and test groups. Because chronic rejection can occur after meniscus transplantation associated with immune cell infiltration, we propose studies with multiple genetic editing to prevent immune rejection.

RevDate: 2022-09-23

Kim JH, Yu J, Kim HK, et al (2022)

Genome Editing of Golden SNP-Carrying Lycopene Epsilon-Cyclase (LcyE) Gene Using the CRSPR-Cas9/HDR and Geminiviral Replicon System in Rice.

International journal of molecular sciences, 23(18): pii:ijms231810383.

Lycopene epsilon-cyclase (LcyE) is a key enzyme in the carotenoid biosynthetic pathway of higher plants. Using the CRSPR/Cas9 and the geminiviral replicon, we optimized a method for targeted mutagenesis and golden SNP replacement of the LcyE gene in rice. We have exploited the geminiviral replicon amplification as a means to provide a large amount of donor template for the repair of a CRISPR-Cas-induced DNA double-strand break (DSB) in the target gene via homology-directed repair (HDR). Mutagenesis experiments performed on the Donggin variety achieved precise modification of the LcyE loci with an efficiency of up to 90%. In HDR experiments, our target was the LcyE allele (LcyE-H523L) derived from anther culture containing a golden SNP replacement. The phenotype of the homologous recombination (HR) mutant obtained through the geminiviral replicon-based template delivery system was tangerine color, and the frequency was 1.32% of the transformed calli. In addition, the total carotenoid content of the LcyEsg2-HDR1 and LcyEsg2-HDR2 lines was 6.8-9.6 times higher than that of the wild-type (WT) calli, respectively. The reactive oxygen species content was lower in the LcyEsg2-HDR1 and LcyEsg2-HDR2 lines. These results indicate that efficient HDR can be achieved in the golden SNP replacement using a single and modular configuration applicable to different rice targets and other crops. This work demonstrates the potential to replace all genes with elite alleles within one generation and greatly expands our ability to improve agriculturally important traits.

RevDate: 2022-09-23

Zhang C, Liu S, Li X, et al (2022)

Virus-Induced Gene Editing and Its Applications in Plants.

International journal of molecular sciences, 23(18): pii:ijms231810202.

CRISPR/Cas-based genome editing technologies, which allow the precise manipulation of plant genomes, have revolutionized plant science and enabled the creation of germplasms with beneficial traits. In order to apply these technologies, CRISPR/Cas reagents must be delivered into plant cells; however, this is limited by tissue culture challenges. Recently, viral vectors have been used to deliver CRISPR/Cas reagents into plant cells. Virus-induced genome editing (VIGE) has emerged as a powerful method with several advantages, including high editing efficiency and a simplified process for generating gene-edited DNA-free plants. Here, we briefly describe CRISPR/Cas-based genome editing. We then focus on VIGE systems and the types of viruses used currently for CRISPR/Cas9 cassette delivery and genome editing. We also highlight recent applications of and advances in VIGE in plants. Finally, we discuss the challenges and potential for VIGE in plants.

RevDate: 2022-09-22

Zhang W, Xiao D, Li X, et al (2022)

SIRT1 inactivation switches reactive astrocytes to an anti-inflammatory phenotype in CNS autoimmunity.

The Journal of clinical investigation pii:151803 [Epub ahead of print].

Astrocytes are highly heterogenic in their phenotype and function, which contribute to CNS disease, repair and aging; however, the molecular mechanism of their functional states remains largely unknown. Here we show that activation of sirtuin 1 (SIRT1), a protein deacetylase, plays an important role in the detrimental actions of reactive astrocytes, whereas its inactivation endorsed these cells with anti-inflammatory functions that inhibited the production of proinflammatory mediators by myeloid cells/microglia and promoted the differentiation of oligodendrocyte progenitor cells. Mice with astrocyte-specific Sirt1 knockout had suppressed progression of experimental autoimmune encephalomyelitis (EAE), an animal model of CNS inflammatory demyelinating diseases. Ongoing EAE was also suppressed when Sirt1 expression in astrocytes was diminished by CRISPR/Cas vector, resulting in reduced demyelination, decreased numbers of T cells, and increased rate of IL-10-producing macrophages/microglia in the CNS, whereas peripheral immune response remained unaffected. Mechanistically, Sirt1-/- astrocytes expressed a range of nuclear factor erythroid-derived 2-like 2 (Nfe2l2) target genes, and Nfe2l2 deficiency shifted the beneficial action of Sirt1-/- astrocytes to a detrimental one. These findings identify a novel approach for switching functional state of reactive astrocytes and facilitate the development of astrocyte-targeting therapies for inflammatory neurodegenerative diseases such as multiple sclerosis.

RevDate: 2022-09-22

Kang K, Song Y, Kim I, et al (2022)

Therapeutic Applications of the CRISPR-Cas System.

Bioengineering (Basel, Switzerland), 9(9): pii:bioengineering9090477.

The clustered regularly interspaced palindromic repeat (CRISPR)-Cas system has revolutionized genetic engineering due to its simplicity, stability, and precision since its discovery. This technology is utilized in a variety of fields, from basic research in medicine and biology to medical diagnosis and treatment, and its potential is unbounded as new methods are developed. The review focused on medical applications and discussed the most recent treatment trends and limitations, with an emphasis on CRISPR-based therapeutics for infectious disease, oncology, and genetic disease, as well as CRISPR-based diagnostics, screening, immunotherapy, and cell therapy. Given its promising results, the successful implementation of the CRISPR-Cas system in clinical practice will require further investigation into its therapeutic applications.

RevDate: 2022-09-22

Lalonde ME, Sasseville M, Gélinas AM, et al (2022)

Genome-wide CRISPR screens identify ferroptosis as a novel therapeutic vulnerability in acute lymphoblastic leukemia.

Haematologica [Epub ahead of print].

Acute lymphoblastic leukemia (ALL) is the most frequent cancer diagnosed in children. Despite the great progress achieved over the last 40 years, with cure rates now exceeding 85%, refractory or relapsed ALL still exhibit a dismal prognosis. This poor outcome reflects the lack of treatment options specifically targeting relapsed or refractory ALL. To address this gap, we have performed whole-genome CRISPR/Cas drop-out screens on a panel of seven B-ALL cell lines. Our results demonstrate that while there was a significant overlap in gene essentiality between ALL cell lines and other cancer types survival of ALL cell lines was dependent on several unique metabolic pathways, including an exquisite sensitivity to GPX4 depletion and ferroptosis induction. Detailed molecular analysis of B-ALL cells suggest that they are primed to undergo ferroptosis as they exhibit high steady-state oxidative stress potential, a low buffering capacity, and a disabled GPX4-independent secondary lipid peroxidation detoxification pathway. Finally, we validated the sensitivity of B-ALL to ferroptosis induction using patient-derived B-ALL samples.

RevDate: 2022-09-23
CmpDate: 2022-09-23

Senger K, Akhmetzyanova I, Haley B, et al (2022)

Plasmid-Based Donor Templates for Nonviral CRISPR/Cas9-Mediated Gene Knock-In in Human T Cells.

Current protocols, 2(9):e538.

Effective and precise gene editing of T lymphocytes is critical for advancing the understanding of T cell biology and the development of next-generation cellular therapies. Although methods for effective CRISPR/Cas9-mediated gene knock-out in primary human T cells have been developed, complementary techniques for nonviral gene knock-in can be cumbersome and inefficient. Here, we report a simple and efficient method for nonviral CRISPR/Cas9-based gene knock-in utilizing plasmid-based donor DNA templates. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Purification of human CD4+ or CD8+ T cells from blood Basic Protocol 2: Activation of purified CD4+ or CD8+ T cells using TransAct CD3/CD28 agonist-conjugated nanomatrix Basic Protocol 3: Preparation of Cas9/sgRNA RNPs Basic Protocol 4: Transfection of CAS9-RNP and knock-in template into human T cells Support Protocol 1: Purity check following magnetic T cell isolation Support Protocol 2: Dextramer staining of TCR-edited T cells Support Protocol 3: Functional characterization of TCR knock-in T cells Support Protocol 4: Detection of knock-in reporter activity in CRISPR/CAS9-edited T cells.

RevDate: 2022-09-23
CmpDate: 2022-09-23

Shortt K, DP Heruth (2022)

Identification of Genes Regulating Hepatocyte Injury by a Genome-Wide CRISPR-Cas9 Screen.

Methods in molecular biology (Clifton, N.J.), 2544:227-251.

Gene editing introduces stable mutations into the genome and has powerful applications extending from research to clinical gene therapy. CRISPR-Cas9 gene editing can be employed to study directly the functional impact of stable gene knockout, activation, and knockdown. Here, we describe the end-to-end methodology by which we employ genome-wide CRISPR-Cas9 knockout to study drug toxicity using acetaminophen (APAP) in a hepatocellular carcinoma liver model as an example. This methodology can be extended to other proliferative cell types and chemical metabolic and toxicity models. By employing a massively parallelized genome-wide knockout model, the genes responsible for cellular toxicity and proliferation may be assayed concurrently. Resultant data are interrogated in the context of existing gene expression data, pathway analysis, drug-gene interactions, and orthogonal confirmatory assays to better understand the metabolic mechanisms.

RevDate: 2022-09-23
CmpDate: 2022-09-23

Hang XM, Wang HY, Liu PF, et al (2022)

Cas12a-assisted RTF-EXPAR for accurate, rapid and simple detection of SARS-CoV-2 RNA.

Biosensors & bioelectronics, 216:114683.

Developing highly accurate and simple approaches to rapidly identify and isolate SARS-CoV-2 infected patients is important for the control of the COVID-19 pandemic. We, herein, reported the performance of a Cas12a-assisted RTF-EXPAR strategy for the identification of SARS-CoV-2 RNA. This assay combined the advantages of RTF-EXPAR with CRISPR-Cas12a can detect SARS-CoV-2 within 40 min, requiring only isothermal control. Particularly, the simultaneous use of EXPAR amplification and CRISPR improved the detection sensitivity, thereby realizing ultrasensitive SARS-CoV-2 RNA detection with a detection limit of 3.77 aM (∼2 copies/μL) in an end-point fluorescence read-out fashion, and at 4.81 aM (∼3 copies/μL) level via a smartphone-assisted analysis system (RGB analysis). Moreover, Cas12a increases the specificity by intrinsic sequence-specific template recognition. Overall, this method is fast, sensitive, and accurate, needing minimal equipment, which holds great promise to meet the requirements of point-of-care molecular detection of SARS-CoV-2.

RevDate: 2022-09-23
CmpDate: 2022-09-23

Qiu F, Gan X, Yao J, et al (2022)

CRISPR/Cas12a-derived sensitive electrochemical biosensing of NF-κB p50 based on hybridization chain reaction and DNA hydrogel.

Biosensors & bioelectronics, 216:114665.

Transcription factors (TFs) are key substances in regulating the transcription, replication and expression of genes, and the detection of TFs can provide valuable information to diagnose a variety of diseases. By integrating hybridization chain reaction (HCR)-activated Cas12a enzyme with bio-responsive DNA hydrogels, we propose a dual amplification and label-free homogeneous electrochemical detection method to realize sensitive nuclear factor-kappa B p50 (NF-κB p50) detection. The presence of the target molecules protects the DNA duplex probes from digesting by exonuclease III and initiates HCR to generate long double stranded DNAs that can activate the activity of RNA-guided Cas12a enzymes. The single-stranded region of the DNA linkers that crosslink the DNA hydrogels can be cleaved by the activated Cas12a to release a large number of electroactive substances embedded in the gels, which exhibit highly enhanced electrochemical signals for detecting target molecules at the detection limit of 54.1 fM. In addition, the successful interrogation of NF-κB p50 spiked into lysate of HeLa cells by such method is also verified. The established method thus shows new opportunities for sensitive and convenient monitoring of other transcription factors and biomarkers.

RevDate: 2022-09-23
CmpDate: 2022-09-23

Liu JX, Sun XM, Liu D, et al (2022)

Smart NIR light-gated CRISPR/Cas12a fluorescent biosensor with boosted biological delivery and trans-cleavage activity for high-performance in vivo operation.

Biosensors & bioelectronics, 216:114646.

Despite the in vitro usage of CRISPR/Cas12a system in fluorescent biosensors has made remarkable achievements, many challenges such as poor biological delivery, insufficient sensitivity, and uncontrollable initiation compel them hard to conduct in vivo analysis. Thus, we propose here some fruitful sensing concepts. First, the multiple biomolecular components of CRISPR/Cas12a system are collectively carried by MnO2 nanosheets via a simple physical absorption to achieve a highly-efficient biological uptake. Under the reduction of widespread biothiols, not only the sensing frame is easily released but also sufficient Mn2+ is produced to serve as an effective trans-cleavage accelerator. Furthermore, a photocleavge-linker induced smart near-infrared (NIR) light-gated manner is designed to offer a spatiotemporal target recognition, for which a 808 nm NIR light transduced ultraviolet upconversion luminescence with weak thermal effect is employed to completely prevent the sensing flow from pre-initiating during the biological delivery. As a conceptual validation, this biosensor has satisfactory sensitivity and specificity to survivn messenger RNA (a broad-spectrum cancer biomarker). More importantly, it can work as a reliable imaging platform for differentiating cancers in live cellular level and also presents a high-performance operation ability for analyzing live mice, greatly promoting the CRISPR technology in biosensing field.

RevDate: 2022-09-22
CmpDate: 2022-09-22

Cao H, Mao K, Ran F, et al (2022)

Paper Device Combining CRISPR/Cas12a and Reverse-Transcription Loop-Mediated Isothermal Amplification for SARS-CoV-2 Detection in Wastewater.

Environmental science & technology, 56(18):13245-13253.

Wastewater-based surveillance of the COVID-19 pandemic holds great promise; however, a point-of-use detection method for SARS-CoV-2 in wastewater is lacking. Here, a portable paper device based on CRISPR/Cas12a and reverse-transcription loop-mediated isothermal amplification (RT-LAMP) with excellent sensitivity and specificity was developed for SARS-CoV-2 detection in wastewater. Three primer sets of RT-LAMP and guide RNAs (gRNAs) that could lead Cas12a to recognize target genes via base pairing were used to perform the high-fidelity RT-LAMP to detect the N, E, and S genes of SARS-CoV-2. Due to the trans-cleavage activity of CRISPR/Cas12a after high-fidelity amplicon recognition, carboxyfluorescein-ssDNA-Black Hole Quencher-1 and carboxyfluorescein-ssDNA-biotin probes were adopted to realize different visualization pathways via a fluorescence or lateral flow analysis, respectively. The reactions were integrated into a paper device for simultaneously detecting the N, E, and S genes with limits of detection (LODs) of 25, 310, and 10 copies/mL, respectively. The device achieved a semiquantitative analysis from 0 to 310 copies/mL due to the different LODs of the three genes. Blind experiments demonstrated that the device was suitable for wastewater analysis with 97.7% sensitivity and 82% semiquantitative accuracy. This is the first semiquantitative endpoint detection of SARS-CoV-2 in wastewater via different LODs, demonstrating a promising point-of-use method for wastewater-based surveillance.

RevDate: 2022-09-23
CmpDate: 2022-09-23

Gim GM, Uhm KH, Kwon DH, et al (2022)

Germline transmission of MSTN knockout cattle via CRISPR-Cas9.

Theriogenology, 192:22-27.

Although the production of several founder animals (F0) for gene editing in livestock has been reported in cattle, very few studies have assessed germline transmission to the next generation due to the long sexual maturation and gestation periods. The present study aimed to assess the germline transmission of MSTN mutations (-12bps deletion) in MSTN mutant F0 male and female cattle. For this purpose, oocytes and semen were collected after the sexual maturation of MSTN cattle, and embryos produced by in vitro fertilization were analyzed. In addition, the embryos were subjected to additional gene (PRNP) editing using electroporation. Embryos produced by in vitro fertilization with MSTN male and female cattle were transferred to a surrogate, and one calf was successfully born. MSTN heterozygous mutation was shown by sequencing of the F1 calf, which had no health issues. As a further experiment, using electroporation, additional gene-edited embryos fertilized with the MSTN male sperm showed a high mutation rate of PRNP (86.2 ± 3.4%). These data demonstrate that the cattle produced through gene editing matured without health issues and had transmitted MSTN mutation from the germ cells. Also, additional mutation of embryos fertilized with the MSTN male sperm could enable further mutagenesis using electroporation.

RevDate: 2022-09-23
CmpDate: 2022-09-23

Li Y, Pei Y, Huang D, et al (2022)

Direct visualization of living bacterial genotypes using CRISPR/Cas12a-circular reporter nanoprobes.

Biosensors & bioelectronics, 216:114641.

Bacterial genotyping is important for understanding the complex microbiota. Although fluorescence in situ hybridization (FISH) has enabled bacterial community identification with high spatial resolution, its unavoidable cell fixation steps and signal generation by multi-probe stacking greatly limit its application in living bacterial genotyping. Here, we designed polyethyleneimine-encapsulated CRISPR/Cas12a-circular reporter nanoprobes (CasCLR) for rapid and sensitive visualization of gene information in living bacteria. We found that, nanoprobe-based sequential delivery of Cas12a/crRNA and circular reporter into bacteria allowed single genomic loci to initiate trans-cleavage activity of Cas12a, thereby cleaving CLR to generate amplified fluorescent signals for imaging of target gene. Using CasCLR, we can sensitively analyze the percentage of target bacteria in co-culture experiments and directly detect pathogenic bacteria in uncultured mouse gut microbe. In addition, CasCLR has the ability to sensitively analyze specific genotype of microbial communities in vivo. This nanobiotechnology-based bacterial gene analysis is expected to advance understanding of in vivo bacterial cytogenetic information.

RevDate: 2022-09-22
CmpDate: 2022-09-22

Zhou C, Huang D, Wang Z, et al (2022)

CRISPR Cas12a-based "sweet" biosensor coupled with personal glucose meter readout for the point-of-care testing of Salmonella.

Journal of food science, 87(9):4137-4147.

Salmonella is a pathogen that comes from different animal-originated foods and poses a significant threat to human health. The present detection methods for Salmonella are time-consuming and labor-intensive and requires skilled workers and specialized instruments. In this study, the conservative invA was selected as the target gene, and a quantitative detection method for Salmonella with wide availability and user-friendliness was established based on CRISPR Cas12a and a personal glucose meter (PGM). The indirect signal transformation from the original target DNA to the final glucose signal was achieved through RAA, CRISPR Cas12a reaction, enzymic reaction, and glucose signal reading by a PGM (accu-chek type from Roche). This PGMs-CRISPR assay showed a detection sensitivity of Salmonella as low as 5 colony-forming units (CFU)/reaction in either pure culture or artificially contaminated food samples and exhibited specificity between Salmonella isolates and non-Salmonella isolates. Furthermore, quantitative detection of Salmonella in spiked milk samples was also achieved within the range from 1 to 1 × 103 CFU/reaction. Subsequently, the correlation and consistency between the PGMs-CRISPR assay and quantitative polymerase chain reaction (qPCR) in detection of Salmonella in spiked milk samples were achieved. Therefore, a highly sensitive, portable, quantitative, and user-friendly detection method based on CRISPR Cas12a and PGMs was developed in this study for Salmonella detection and identification. PRACTICAL APPLICATION: A sensitive, rapid, user-friendly, and quantitative detection method based on CRISPR Cas12a for Salmonella in food has been developed in this study, which is of great significance to food safety supervision and management.

RevDate: 2022-09-23
CmpDate: 2022-09-23

Fu K, Song W, Chen C, et al (2022)

Improving pre-harvest sprouting resistance in rice by editing OsABA8ox using CRISPR/Cas9.

Plant cell reports, 41(10):2107-2110.

KEY MESSAGE: Knock out OsABA8ox helps improve pre-harvest spouting resistance and do not affect rice yield. Pre-harvest sprouting(PHS) is a phenomenon that the seeds of crops germinate preharvest, which reduces the yield and quality of rice. Abscisic acid(ABA) is one of the phytohormones that promotes seed dormancy. ABA8' hydroxylase is the main enzyme that can catabolism ABA in plant. There are three genes that encode ABA8' hydroxylase in rice, named OsABA8ox1, OsABA8ox2 and OsABA8ox3. In this study, we use CRISPR/Cas9 gene editing technology to target these three genes in Ningjing6 and find that the knockout transgenic lines are all significantly strengthen in seed dormancy and have no effect on the yield. By a series of quantitative experiments, we consider that after knock out OsABA8ox, the high endogenous ABA level will influence the ABA signal which suppress the substantial and energy metabolism in the seeds, and finally led to higher dormancy.

RevDate: 2022-09-22
CmpDate: 2022-09-22

Pourrajab F, MR Zare-Khormizi (2022)

Extrachromosomal Circular DNAs, Amplified Oncogenes, and CRISPR-Cas9 System.

Molecular pharmacology, 102(4):209-215.

Structurally rearranged extrachromosomal circular DNAs (eccDNAs) have been identified in tumor cells, many of which carry regions related to recurrent cancer driver oncogenes (e.g., CCND1, EGFR, and MYC). In a tumor cell, eccDNAs are carrying regions associated with oncogene amplification (>10-fold amplified-copy numbers in human tumors) and poor outcome across multiple cancers. Even though dual-delivery of pairs of CRISPR and CRISPR-associated protein 9 (Cas9) guiding RNAs into normal human cells was reported to induce circularization of genes and chromosomes, in bacteria, the CRISPR-Cas9 system primarily targets extrachromosomal rearranged elements. Likewise, in cancer cells, it is expected that a designed CRISPR-Cas9 system would be able to target extrachromosomal copy number amplifications and produce double strand breaks detrimental to cellular fitness by dictating gene-independent copy number loss-of-fitness effects and antiproliferative responses. A system designed against amplified amplicons may provide a novel approach for cancer therapy and propose a practical implication for CRISPR-Cas9 pairs as a pathway in therapeutic strategies of cancer. SIGNIFICANCE STATEMENT: Structurally rearranged extrachromosomal circular DNAs (eccDNAs) have been identified in tumor cells. Many eccDNAs are carrying regions related to recurrent cancer driver oncogenes (e.g. CCND1, EGFR and MYC). It is expected that a designed CRISPR-Cas9 system would able to target extrachromosomal recurrent oncogenes.

RevDate: 2022-09-23
CmpDate: 2022-09-23

Wu L, Xiao H, Zhao L, et al (2022)

CRISPR/Cas9-mediated generation of fls2 mutant in Nicotiana benthamiana for investigating the flagellin recognition spectrum of diverse FLS2 receptors.

Plant biotechnology journal, 20(10):1853-1855.

RevDate: 2022-09-23
CmpDate: 2022-09-23

Segurado OG, Jiang R, SW Pipe (2022)

Challenges and opportunities when transitioning from in vivo gene replacement to in vivo CRISPR/Cas9 therapies - a spotlight on hemophilia.

Expert opinion on biological therapy, 22(9):1091-1098.

INTRODUCTION: Currently, a few in vivo gene replacement therapies are commercially available, with many in clinical development for the treatment of some inherited monogenic diseases. These disorders arise from mutations in genes encoding essential proteins with a well understood biological function. Wide adoption of gene replacement therapies requires solid safety and efficacy profiles with demonstrable long-term durability and cost-benefit advantages vs standard therapies.

AREAS COVERED: This expert review outlines the challenges and opportunities in treating hemophilia, including the progression from in vivo gene therapies toward in vivo gene editing, focusing on pre-clinical and emerging clinical data for gene editing and addressing the need for sustained and durable gene expression during hepatocyte proliferation when the liver is unable to maintain steady gene expression and protein production.

EXPERT OPINION: In vivo gene editing in liver tissues may be able to rescue patients younger than 18 years who are not eligible for gene replacement therapies, with hemophilia as a prime example.

RevDate: 2022-09-23
CmpDate: 2022-09-23

Cao T, Liu S, Qiu Y, et al (2022)

Generation of C-to-G transversion in mouse embryos via CG editors.

Transgenic research, 31(4-5):445-455.

Base editors (BEs) are efficient and precise tools for generating single base conversions in living organisms. While most BE systems are limited in mediating C-to-T or A-to-G conversions, recently developed C-to-G base editors (CGBEs) could produce C-to-G transversions. CGBEs convert cytosine within the editing window to abasic intermediates, which would be replaced with any base after base excision repair (BER). By far, though the efficiency and editing scope of CGBEs have been investigated in cultured cells via gRNA library and machine-learning, the viability of CGBEs in generating mouse models has not been adequately tested. In this study, we tested the C-to-G transversion efficiency of the CGBE1 and CGBE-XRCC1 systems in mouse embryos. Our results showed that both of the CGBE systems were able to mediate C-to-G transversion on 2 out of 3 targets tested, with up to 20% frequency within the editing window. Notably, most of the groups showed over 40% of other base conversions, predominantly C-to-T. Lastly, we successfully acquired the F1 mouse carrying a disease-causing mutation. In all, our study suggested that CGBEs systems held great potential in generating mouse models and indicated that XRCC1 based system is applicable in mouse embryos.

RevDate: 2022-09-23
CmpDate: 2022-09-23

Mao X, Zhao Y, Jiang J, et al (2022)

Sensitive and high-accuracy detection of Salmonella based on CRISPR/Cas12a combined with recombinase polymerase amplification.

Letters in applied microbiology, 75(4):899-907.

Salmonella is a crucial food-borne pathogen causing food poisoning, leading to severe public health events. Here, we developed a technique by integrating recombinase polymerase amplification with CRISPR-LbCas12a and employing two targets with engineered crRNA for detection of Salmonella (RPA-LbCas12a-TTECDS). Our findings revealed that this novel method rapidly detects trace Salmonella in food through fluorescence intensity and provides a template for other food-borne pathogen detection methods. Further, crRNA was optimized to increase detection sensitivity. Double targets were used to enhance the detection accuracy, reaching the level of qPCR, which was superior to fluorescent RPA. The RPA-LbCas12a-TTECDS system specifically detected Salmonella levels as low as 50 CFU per ml at 37°C in 1 h. In summary, a simple, rapid, sensitive and high accuracy detection technique based on CRISPR-Cas12a was created for Salmonella detection without complicated equipment.

RevDate: 2022-09-23
CmpDate: 2022-09-23

Statkute E, Wang ECY, RJ Stanton (2022)

An Optimized CRISPR/Cas9 Adenovirus Vector (AdZ-CRISPR) for High-Throughput Cloning of sgRNA, Using Enhanced sgRNA and Cas9 Variants.

Human gene therapy, 33(17-18):990-1001.

Recombinant adenovirus vectors enable highly efficient gene delivery in vitro and in vivo. As a result, they are widely used in gene therapy, vaccination, and anticancer applications. We have previously developed the AdZ vector system, which uses recombineering to permit high-throughput cloning of transgenes into Adenovirus vectors, simplifies alteration of the vector backbone, and enables rapid recovery of infectious virus, even if a transgene is incompatible with vector replication. In this study, we adapt this vector system to enable high-throughput cloning of sequences for CRISPR/Cas9 editing. Vectors were optimized to ensure efficient cloning, and high editing efficiency using spCas9 and single guide RNA (sgRNA) sequences in a single vector. Using a multiplicity of infection of 50, knockout efficiencies of up to 80% could be achieved with a single sgRNA. Vectors were further enhanced by altering the spCas9 sequence to match that of SniperCas9, which has reduced off-target activity, but maintains on-target efficiency, and by applying modifications to the sgRNA sequence that significantly enhance editing efficiency. Thus, the AdZ-CRISPR vectors offer highly efficient knockout, even in hard to transfect cells, and enables large-scale CRISPR/Cas9 projects to be undertaken easily and quickly.

RevDate: 2022-09-21

Tenjo-Castaño F, Montoya G, A Carabias (2022)

Transposons and CRISPR: Rewiring Gene Editing.

Biochemistry [Epub ahead of print].

CRISPR-Cas is driving a gene editing revolution because of its simple reprogramming. However, off-target effects and dependence on the double-strand break repair pathways impose important limitations. Because homology-directed repair acts primarily in actively dividing cells, many of the current gene correction/replacement approaches are restricted to a minority of cell types. Furthermore, current approaches display low efficiency upon insertion of large DNA cargos (e.g., sequences containing multiple gene circuits with tunable functionalities). Recent research has revealed new links between CRISPR-Cas systems and transposons providing new scaffolds that might overcome some of these limitations. Here, we comment on two new transposon-associated RNA-guided mechanisms considering their potential as new gene editing solutions. Initially, we focus on a group of small RNA-guided endonucleases of the IS200/IS605 family of transposons, which likely evolved into class 2 CRISPR effector nucleases (Cas9s and Cas12s). We explore the diversity of these nucleases (named OMEGA, obligate mobile element-guided activity) and analyze their similarities with class 2 gene editors. OMEGA nucleases can perform gene editing in human cells and constitute promising candidates for the design of new compact RNA-guided platforms. Then, we address the co-option of the RNA-guided activity of different CRISPR effector nucleases by a specialized group of Tn7-like transposons to target transposon integration. We describe the various mechanisms used by these RNA-guided transposons for target site selection and integration. Finally, we assess the potential of these new systems to circumvent some of the current gene editing challenges.

RevDate: 2022-09-21

Khurana A, Sayed N, Singh V, et al (2022)

A comprehensive overview of CRISPR/Cas 9 technology and application thereof in drug discovery.

Journal of cellular biochemistry [Epub ahead of print].

Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-Cas technology possesses revolutionary potential to positively affect various domains of drug discovery. It has initiated a rise in the area of genetic engineering and its advantages range from classical science to translational medicine. These genome editing systems have given a new dimension to our capabilities to alter, detect and annotate specified gene sequences. Moreover, the ease, robustness and adaptability of the CRISPR/Cas9 technology have led to its extensive utilization in research areas in such a short period of time. The applications include the development of model cell lines, understanding disease mechanisms, discovering disease targets, developing transgenic animals and plants, and transcriptional modulation. Further, the technology is rapidly growing; hence, an overlook of progressive success is crucial. This review presents the current status of the CRISPR-Cas technology in a tailor-made format from its discovery to several advancements for drug discovery alongwith future trends associated with possibilities and hurdles including ethical concerns.

RevDate: 2022-09-21

Wimmer E, Zink IA, C Schleper (2022)

Reprogramming CRISPR-Mediated RNA Interference for Silencing of Essential Genes in Sulfolobales.

Methods in molecular biology (Clifton, N.J.), 2522:177-201.

The manipulation of gene expression levels in vivo is often key to elucidating gene function and regulatory network interactions, especially when it comes to the investigation of essential genes that cannot be deleted from the model organism's genome. Several techniques have been developed for prokaryotes that allow to interfere with transcription initiation of specific genes by blocking or modifying promoter regions. However, a tool functionally similar to RNAi used in eukaryotes to efficiently degrade mRNA posttranscriptionally did not exist until recently. Type III CRISPR-Cas systems use small RNAs (crRNAs) that guide effector complexes (encoded by cas genes) which act as site-specific RNA endonuclease and can thus be harnessed for targeted posttranscriptional gene silencing. Guide RNAs complementary to the desired target mRNA that, in addition, exhibit complementarity to repeat sequences found in the CRISPR arrays, effectively suppress unspecific DNA and RNA activities of the CRISPR-Cas complexes. Here we describe the use of endogenous type III CRISPR-Cas systems in two model organisms of Crenarchaeota, Saccharolobus solfataricus and Sulfolobus acidocaldarius.

RevDate: 2022-09-21

Schwarz TS, Schreiber SS, A Marchfelder (2022)

CRISPR Interference as a Tool to Repress Gene Expression in Haloferax volcanii.

Methods in molecular biology (Clifton, N.J.), 2522:57-85.

To date, a plethora of tools for molecular biology have been developed on the basis of the CRISPR-Cas system. Almost all use the class 2 systems since here the setup is the simplest with only one protein and one guide RNA, allowing for easy transfer to and expression in other organisms. However, the CRISPR-Cas components harnessed for applications are derived from mesophilic bacteria and are not optimal for use in extremophilic archaea.Here, we describe the application of an endogenous CRISPR-Cas system as a tool for silencing gene expression in a halophilic archaeon. Haloferax volcanii has a CRISPR-Cas system of subtype I-B, which can be easily used to repress the transcription of endogenous genes, allowing to study the effects of their depletion. This article gives a step-by-step introduction on how to use the implemented system for any gene of interest in Haloferax volcanii. The concept of CRISPRi described here for Haloferax can be transferred to any other archaeon, that is genetically tractable and has an endogenous CRISPR-Cas I systems.

RevDate: 2022-09-20

Mitrofanov A, Ziemann M, Alkhnbashi OS, et al (2022)

CRISPRtracrRNA: robust approach for CRISPR tracrRNA detection.

Bioinformatics (Oxford, England), 38(Supplement_2):ii42-ii48.

MOTIVATION: The CRISPR-Cas9 system is a Type II CRISPR system that has rapidly become the most versatile and widespread tool for genome engineering. It consists of two components, the Cas9 effector protein, and a single guide RNA that combines the spacer (for identifying the target) with the tracrRNA, a trans-activating small RNA required for both crRNA maturation and interference. While there are well-established methods for screening Cas effector proteins and CRISPR arrays, the detection of tracrRNA remains the bottleneck in detecting Class 2 CRISPR systems.

RESULTS: We introduce a new pipeline CRISPRtracrRNA for screening and evaluation of tracrRNA candidates in genomes. This pipeline combines evidence from different components of the Cas9-sgRNA complex. The core is a newly developed structural model via covariance models from a sequence-structure alignment of experimentally validated tracrRNAs. As additional evidence, we determine the terminator signal (required for the tracrRNA transcription) and the RNA-RNA interaction between the CRISPR array repeat and the 5'-part of the tracrRNA. Repeats are detected via an ML-based approach (CRISPRidenify). Providing further evidence, we detect the cassette containing the Cas9 (Type II CRISPR systems) and Cas12 (Type V CRISPR systems) effector protein. Our tool is the first for detecting tracrRNA for Type V systems.

The implementation of the CRISPRtracrRNA is available on GitHub upon requesting the access permission, (https://github.com/BackofenLab/CRISPRtracrRNA). Data generated in this study can be obtained upon request to the corresponding person: Rolf Backofen (backofen@informatik.uni-freiburg.de).

SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

RevDate: 2022-09-19

Vassallo CN, Doering CR, Littlehale ML, et al (2022)

A functional selection reveals previously undetected anti-phage defence systems in the E. coli pangenome.

Nature microbiology [Epub ahead of print].

The ancient, ongoing coevolutionary battle between bacteria and their viruses, bacteriophages, has given rise to sophisticated immune systems including restriction-modification and CRISPR-Cas. Many additional anti-phage systems have been identified using computational approaches based on genomic co-location within defence islands, but these screens may not be exhaustive. Here we developed an experimental selection scheme agnostic to genomic context to identify defence systems in 71 diverse E. coli strains. Our results unveil 21 conserved defence systems, none of which were previously detected as enriched in defence islands. Additionally, our work indicates that intact prophages and mobile genetic elements are primary reservoirs and distributors of defence systems in E. coli, with defence systems typically carried in specific locations or hotspots. These hotspots encode dozens of additional uncharacterized defence system candidates. Our findings reveal an extended landscape of antiviral immunity in E. coli and provide an approach for mapping defence systems in other species.

RevDate: 2022-09-19

Suh S, Choi EH, Raguram A, et al (2022)

Precision genome editing in the eye.

Proceedings of the National Academy of Sciences of the United States of America, 119(39):e2210104119.

CRISPR-Cas-based genome editing technologies could, in principle, be used to treat a wide variety of inherited diseases, including genetic disorders of vision. Programmable CRISPR-Cas nucleases are effective tools for gene disruption, but they are poorly suited for precisely correcting pathogenic mutations in most therapeutic settings. Recently developed precision genome editing agents, including base editors and prime editors, have enabled precise gene correction and disease rescue in multiple preclinical models of genetic disorders. Additionally, new delivery technologies that transiently deliver precision genome editing agents in vivo offer minimized off-target editing and improved safety profiles. These improvements to precision genome editing and delivery technologies are expected to revolutionize the treatment of genetic disorders of vision and other diseases. In this Perspective, we describe current preclinical and clinical genome editing approaches for treating inherited retinal degenerative diseases, and we discuss important considerations that should be addressed as these approaches are translated into clinical practice.

RevDate: 2022-09-19

Sheikh BA, Bhat BA, MA Mir (2022)

Antimicrobial resistance: new insights and therapeutic implications.

Applied microbiology and biotechnology [Epub ahead of print].

Antimicrobial resistance has not been a new phenomenon. Still, the number of resistant organisms, the geographic areas affected by emerging drug resistance, and the magnitude of resistance in a single organism are enormous and mounting. Disease and disease-causing agents formerly thought to be contained by antibiotics are now returning in new forms resistant to existing therapies. Antimicrobial resistance is one of the most severe and complicated health issues globally, driven by interrelated dynamics in humans, animals, and environmental health sectors. Coupled with various epidemiological factors and a limited pipeline for new antimicrobials, all these misappropriations allow the transmission of drug-resistant organisms. The problem is likely to worsen soon. Antimicrobial resistance in general and antibiotic resistance in particular is a shared global problem. Actions taken by any single country can adversely or positively affect the other country. Targeted coordination and prevention strategies are critical in stopping the spread of antibiotic-resistant organisms and hence its overall management. This article has provided in-depth knowledge about various methods that can help mitigate the emergence and spread of antimicrobial resistance globally. KEY POINTS: • Overview of antimicrobial resistance as a global challenge and explain various reasons for its rapid progression. • Brief about the intrinsic and acquired resistance to antimicrobials and development of antibiotic resistance in bacteria. • Systematically organized information is provided on different strategies for tackling antimicrobial resistance for the welfare of human health.

RevDate: 2022-09-20
CmpDate: 2022-09-20

Mayran C, Henry S, Pinchon E, et al (2022)

CRISPR-Cas: the bacterial immunity that supports diagnostic in virology.

Virologie (Montrouge, France), 26(4):303-313.

CRISPR-Cas is an adaptive immune system that prevents bacteria and archea from nucleic acids invasion such as viral genomes. The ability of the CRISPR-Cas technology to effectively and precisely cut a targeted genomic DNA region was exploited to develop powerful genome editing tools that were adapted for a wide range of applications, revolutionizing biological sciences. The CRISPR-Cas system consists of a Cas endonuclease triggered by a RNA guide for highly specific cleavage of targeted DNA or RNA sequences. In addition to the target specific cleavage, some Cas enzymes, including Cas12a and Cas13a, display a collateral trans-cleavage activity that allows the cleavage of all surrounding single-stranded nucleic acids. These biosensing activities of CRISPR-Cas systems, based on target specific binding and cleavage, are promising tools to develop accurate diagnostic methods to detect specific nucleic acids. CRISPRCas could therefore be used to diagnose a wide variety of diseases. In the current review we propose to describe the more significant advances for virus detection based on CRISPR-Cas systems.

RevDate: 2022-09-20

Barragán-Álvarez CP, Flores-Fernandez JM, Hernández-Pérez OR, et al (2022)

Recent advances in the use of CRISPR/Cas for understanding the early development of molecular gaps in glial cells.

Frontiers in cell and developmental biology, 10:947769.

Glial cells are non-neuronal elements of the nervous system (NS) and play a central role in its development, maturation, and homeostasis. Glial cell interest has increased, leading to the discovery of novel study fields. The CRISPR/Cas system has been widely employed for NS understanding. Its use to study glial cells gives crucial information about their mechanisms and role in the central nervous system (CNS) and neurodegenerative disorders. Furthermore, the increasingly accelerated discovery of genes associated with the multiple implications of glial cells could be studied and complemented with the novel screening methods of high-content and single-cell screens at the genome-scale as Perturb-Seq, CRISP-seq, and CROPseq. Besides, the emerging methods, GESTALT, and LINNAEUS, employed to generate large-scale cell lineage maps have yielded invaluable information about processes involved in neurogenesis. These advances offer new therapeutic approaches to finding critical unanswered questions about glial cells and their fundamental role in the nervous system. Furthermore, they help to better understanding the significance of glial cells and their role in developmental biology.

RevDate: 2022-09-20

Lee S, Nam D, Park JS, et al (2022)

Highly Efficient DNA Reporter for CRISPR/Cas12a-Based Specific and Sensitive Biosensor.

Biochip journal [Epub ahead of print].

In addition to cis-cleavage activity that recognizes and cleaves nucleic acid sequences, a trans-cleavage activity that indiscriminately and non-specifically cleaves single-stranded DNA or RNA has been discovered in some Cas proteins, including Cas12a and Cas13a. Various detection methods using this activity have been widely reported. Herein, we describe a new highly efficient DNA reporter (5'-TTATT-CCCCC-3'; TTATT-5C) that outperformed the existing AT-rich DNA reporter (5'-TTATT-3') used in most Cas12a-based target nucleic detection assays. By systematically investigating the effect of DNA reporter length and sequence on the trans-cleavage activity of Cas12a, we achieved up to a 100-fold increase in fluorescence signal intensity derived from the trans-cleavage activity of Cas12a compared to that achieved using the existing AT-rich DNA reporter. The new DNA reporter was also applied, along with the existing AT-rich DNA reporter, for the detection of the Salmonella enterotoxin (stn) gene. Importantly, both detection speed and limit were significantly enhanced with the new DNA reporter. In addition, polymerase chain reaction (PCR) was adopted to the CRISR/Cas-Based system of the new DNA reporter, thereby confirming its practical applicability. The high-efficiency DNA reporter described herein can pave the way for further improving the trans-cleavage activity of other Cas proteins, as well as the sensitivity of CRISPR/Cas-Based systems.

Supplementary Information: The online version contains supplementary material available at 10.1007/s13206-022-00081-0.

RevDate: 2022-09-19

Avaro AS, JG Santiago (2022)

Uncertainty Quantification of Michaelis-Menten Kinetic Rates and Its Application to the Analysis of CRISPR-Based Diagnostics.

Angewandte Chemie (International ed. in English) [Epub ahead of print].

Michaelis-Menten kinetics is an essential model to rationalize enzyme reactions. The quantification of Michaelis-Menten parameters can be very challenging as it is sensitive to even small experimental errors. We here present a quantification of the uncertainty inherent to the experimental determination of kinetic rate parameters for enzymatic reactions. We study the influence of several sources of uncertainty and bias, including the inner filter effect, pipetting errors, number of points in the Michaelis-Menten curve, and flat-field correction. Using Monte Carlo simulations and analyses of experimental data, we compute typical uncertainties of [[EQUATION]] , [[EQUATION]] , and catalytic efficiency [[EQUATION]] . As a salient example, we analyze the extraction of such parameters for CRISPR-Cas systems. CRISPR diagnostics have recently attracted much interest and yet reports of these enzymatic kinetic rates have been highly unreliable and inconsistent.

RevDate: 2022-09-18

Mahler M, Costa AR, van Beljouw SPB, et al (2022)

Approaches for bacteriophage genome engineering.

Trends in biotechnology pii:S0167-7799(22)00226-8 [Epub ahead of print].

In recent years, bacteriophage research has been boosted by a rising interest in using phage therapy to treat antibiotic-resistant bacterial infections. In addition, there is a desire to use phages and their unique proteins for specific biocontrol applications and diagnostics. However, the ability to manipulate phage genomes to understand and control gene functions, or alter phage properties such as host range, has remained challenging due to a lack of universal selectable markers. Here, we discuss the state-of-the-art techniques to engineer and select desired phage genomes using advances in cell-free methodologies and clustered regularly interspaced short palindromic repeats-CRISPR associated protein (CRISPR-Cas) counter-selection approaches.

RevDate: 2022-09-21
CmpDate: 2022-09-20

Qian W, Huang J, Wang T, et al (2022)

Ultrasensitive and visual detection of human norovirus genotype GII.4 or GII.17 using CRISPR-Cas12a assay.

Virology journal, 19(1):150.

BACKGROUND: Integrating CRISPR-Cas12a sensors with isothermal signal amplification can be exploited to develop low-cost, disposable, and ultrasensitive assays for the diagnostics of human pathogens.

METHODS: RT-RAA-Cas12a-mediated real-time or end-point fluorescent and lateral flow strip (LFS) assays for direct detection of norovirus (NOV) genotype GII.4 or GII.17 were explored.

RESULTS: The results showed that our RT-RAA-Cas12a-mediated fluorescent and LFS assay could detect NOV GII.4 or GII.17 by targeting the viral protein 1 gene. Our RT-RAA-Cas12a-mediated fluorescent and LFS assay can specifically detect NOV GII.4 or GII.17 with no cross-reactivity for other related viruses. The low limit of detection could reach 0.1 copies/μL within approximately 30-40 min, and the results were visualized using an ultraviolet light illuminator or on a LFS without complex equipment. In addition, our RT-RAA-Cas12a-mediated fluorescent and LFS assay provided a visual and faster alternative to real-time RT-PCR assay, with 95.7% and 94.3% positive predictive agreement and 100% negative predictive agreement.

CONCLUSIONS: Together, our RT-RAA-Cas12a-mediated approach would have a great potential for point-of-care diagnostics of NOV GII.4 and/or GII.17 in resource-limited settings.

RevDate: 2022-09-19

Bot JF, van der Oost J, N Geijsen (2022)

The double life of CRISPR-Cas13.

Current opinion in biotechnology, 78:102789 pii:S0958-1669(22)00123-9 [Epub ahead of print].

Since the discovery of RNA-programmable nucleases from the prokaryotic adaptive immune system CRISPR-Cas, these proteins have seen rapid and widespread adoption for biotechnological and clinical research. A recently discovered system, CRISPR-Cas13, uses CRISPR RNA guides to target RNA. Interestingly, RNA targeting by Cas13 results in cleavage of both target RNA and bystander RNA. This feature has been used to develop innovative diagnostic tools for the detection of specific RNAs. Unlike in vitro detection of RNA using collateral RNA cleavage, however, initial studies of mammalian cells only revealed highly specific target RNA-knockdown activity. Although these findings have been confirmed subsequently, several recent publications do report Cas13-mediated toxicity and collateral RNA cleavage when using Cas13 in eukaryotes. Here, we review these conflicting observations and discuss its potential molecular basis.

RevDate: 2022-09-20
CmpDate: 2022-09-20

Trakarnsanga K, Thongsin N, Metheetrairut C, et al (2022)

Genetic correction of haemoglobin E in an immortalised haemoglobin E/beta-thalassaemia cell line using the CRISPR/Cas9 system.

Scientific reports, 12(1):15551.

β-thalassaemia is one of the most common genetic blood diseases worldwide with over 300 mutations in the HBB gene affecting red blood cell functions. Recently, advances in genome editing technology have provided a powerful tool for precise genetic correction. Generation of patient-derived induced pluripotent stem cells (iPSCs) followed by genetic correction of HBB mutations and differentiation into haematopoietic stem/progenitor cells (HSPCs) offers a potential therapy to cure the disease. However, the biggest challenge is to generate functional HSPCs that are capable of self-renewal and transplantable. In addition, functional analyses of iPSC-derived erythroid cells are hampered by poor erythroid expansion and incomplete erythroid differentiation. Previously, we generated an immortalised erythroid cell line (SiBBE) with unique properties, including unlimited expansion and the ability to differentiate into mature erythrocytes. In this study, we report a highly efficient genetic correction of HbE mutation in the SiBBE cells using the CRISPR/Cas9 system. The HbE-corrected clones restored β-globin production with reduced levels of HbE upon erythroid differentiation. Our approach provides a sustainable supply of corrected erythroid cells and represents a valuable model for validating the therapeutic efficacy of gene editing systems.

RevDate: 2022-09-20
CmpDate: 2022-09-20

Altgilbers S, Dierks C, Klein S, et al (2022)

Quantitative analysis of CRISPR/Cas9-mediated provirus deletion in blue egg layer chicken PGCs by digital PCR.

Scientific reports, 12(1):15587.

Primordial germ cells (PGCs), the precursors of sperm and oocytes, pass on the genetic material to the next generation. The previously established culture system of chicken PGCs holds many possibilities for functional genomics studies and the rapid introduction of desired traits. Here, we established a CRISPR/Cas9-mediated genome editing protocol for the genetic modification of PGCs derived from chickens with blue eggshell color. The sequence targeted in the present report is a provirus (EAV-HP) insertion in the 5'-flanking region of the SLCO1B3 gene on chromosome 1 in Araucana chickens, which is supposedly responsible for the blue eggshell color. We designed pairs of guide RNAs (gRNAs) targeting the entire 4.2 kb provirus region. Following transfection of PGCs with the gRNA, genomic DNA was isolated and analyzed by mismatch cleavage assay (T7EI). For absolute quantification of the targeting efficiencies in homozygous blue-allele bearing PGCs a digital PCR was established, which revealed deletion efficiencies of 29% when the wildtype Cas9 was used, and 69% when a high-fidelity Cas9 variant was employed. Subsequent single cell dilutions of edited PGCs yielded 14 cell clones with homozygous deletion of the provirus. A digital PCR assay proved the complete absence of this provirus in cell clones. Thus, we demonstrated the high efficiency of the CRISPR/Cas9 system in introducing a large provirus deletion in chicken PGCs. Our presented workflow is a cost-effective and rapid solution for screening the editing success in transfected PGCs.

RevDate: 2022-09-20
CmpDate: 2022-09-20

Crone MA, MacDonald JT, Freemont PS, et al (2022)

gDesigner: computational design of synthetic gRNAs for Cas12a-based transcriptional repression in mammalian cells.

NPJ systems biology and applications, 8(1):34.

Synthetic networks require complex intertwined genetic regulation often relying on transcriptional activation or repression of target genes. CRISPRi-based transcription factors facilitate the programmable modulation of endogenous or synthetic promoter activity and the process can be optimised by using software to select appropriate gRNAs and limit non-specific gene modulation. Here, we develop a computational software pipeline, gDesigner, that enables the automated selection of orthogonal gRNAs with minimized off-target effects and promoter crosstalk. We next engineered a Lachnospiraceae bacterium Cas12a (dLbCas12a)-based repression system that downregulates target gene expression by means of steric hindrance of the cognate promoter. Finally, we generated a library of orthogonal synthetic dCas12a-repressed promoters and experimentally demonstrated it in HEK293FT, U2OS and H1299 cells lines. Our system expands the toolkit of mammalian synthetic promoters with a new complementary and orthogonal CRISPRi-based system, ultimately enabling the design of synthetic promoter libraries for multiplex gene perturbation that facilitate the understanding of complex cellular phenotypes.

RevDate: 2022-09-16

Zhang X, Shi Y, Chen G, et al (2022)

CRISPR/Cas Systems-Inspired Nano/Biosensors for Detecting Infectious Viruses and Pathogenic Bacteria.

Small methods [Epub ahead of print].

Infectious pathogens cause severe human illnesses and great deaths per year worldwide. Rapid, sensitive, and accurate detection of pathogens is of great importance for preventing infectious diseases caused by pathogens and optimizing medical healthcare systems. Inspired by a microbial defense system (i.e., CRISPR/ CRISPR-associated proteins (Cas) system, an adaptive immune system for protecting microorganisms from being attacked by invading species), a great many new biosensors have been successfully developed and widely applied in the detection of infectious viruses and pathogenic bacteria. Moreover, advanced nanotechnologies have also been integrated into these biosensors to improve their detection stability, sensitivity, and accuracy. In this review, the recent advance in CRISPR/Cas systems-based nano/biosensors and their applications in the detection of infectious viruses and pathogenic bacteria are comprehensively reviewed. First of all, the categories and working principles of CRISPR/Cas systems for establishing the nano/biosensors are simply introduced. Then, the design and construction of CRISPR/Cas systems-based nano/biosensors are comprehensively discussed. In the end, attentions are focused on the applications of CRISPR/Cas systems-based nano/biosensors in the detection of infectious viruses and pathogenic bacteria. Impressively, the remaining opportunities and challenges for the further design and development of CRISPR/Cas system-based nano/biosensors and their promising applications are proposed.

RevDate: 2022-09-20
CmpDate: 2022-09-20

Chirgwin ME, Schroeder EA, ER Derbyshire (2022)

Cut it out! A CRISPR-Cas9 screen identifies host regulators of the Plasmodium liver stage.

Cell chemical biology, 29(9):1365-1367.

In this issue of Cell Chemical Biology, Vijayan and colleagues identify host factors integral for Plasmodium liver-stage infection using a whole-genome CRISPR-Cas9 knockout screen. Their efforts reveal that liver-stage parasites redistribute host microtubules to the parasite membrane in a process dependent on the host Golgi.

RevDate: 2022-09-21
CmpDate: 2022-09-20

Hänggeli KPA, Hemphill A, Müller N, et al (2022)

Single- and duplex TaqMan-quantitative PCR for determining the copy numbers of integrated selection markers during site-specific mutagenesis in Toxoplasma gondii by CRISPR-Cas9.

PloS one, 17(9):e0271011.

Herein, we developed a single and a duplex TaqMan quantitative PCR (qPCR) for absolute quantification of copy numbers of integrated dihydrofolate reductase-thymidylate synthase (mdhfr-ts) drug selectable marker for pyrimethamine resistance in Toxoplasma gondii knockouts (KOs). The single TaqMan qPCR amplifies a 174 bp DNA fragment of the inserted mdhfr-ts and of the wild-type (WT) dhfr-ts (wtdhfr-ts) which is present as single copy gene in Toxoplasma and encodes a sensitive enzyme to pyrimethamine. Thus, the copy number of the dhfr-ts fragment in a given DNA quantity from KO parasites with a single site-specific integration should be twice the number of dhfr-ts copies recorded in the same DNA quantity from WT parasites. The duplex TaqMan qPCR allows simultaneous amplification of the 174 bp dhfr-ts fragment and the T. gondii 529-bp repeat element. Accordingly, for a WT DNA sample, the determined number of tachyzoites given by dhfr-ts amplification is equal to the number of tachyzoites determined by amplification of the Toxoplasma 529-bp, resulting thus in a ratio of 1. However, for a KO clone having a single site-specific integration of mdhfr-ts, the calculated ratio is 2. We then applied both approaches to test T. gondii RH mutants in which the major surface antigen (SAG1) was disrupted through insertion of mdhfr-ts using CRISPR-Cas9. Results from both assays were in correlation showing a high accuracy in detecting KOs with multiple integrated mdhfr-ts. Southern blot analyses using BsaBI and DraIII confirmed qPCRs results. Both TaqMan qPCRs are needed for reliable diagnostic of T. gondii KOs following CRISPR-Cas9-mediated mutagenesis, particularly with respect to off-target effects resulting from multiple insertions of mdhfr-ts. The principle of the duplex TaqMan qPCR is applicable for other selectable markers in Toxoplasma. TaqMan qPCR tools may contribute to more frequent use of WT Toxoplasma strains during functional genomics.

RevDate: 2022-09-16

Shen B, Schmidt MA, Collet KH, et al (2022)

RNAi and CRISPR-Cas silencing E3-ring ubiquitin ligase AIP2 enhances soybean seed protein content.

Journal of experimental botany pii:6702279 [Epub ahead of print].

The majority of plant protein in the world's food supply is derived from soybean. Soybean (Glycine max) is a key protein source for global animal feed and in its incorporation into plant-based foods for people, including meat alternatives. Soybean protein content is genetically variable and is usually inversely related to seed oil content. AIP2 (ABI3-interacting protein 2) is an E3-ring ubiquitin ligase that targets the seed-specific transcription factor, ABI3. Silencing both soybean AIP2 genes (AIP2a/AIP2b) by RNAi enhanced seed protein content by up to seven percentage-points, with no significant decrease in seed oil content. The protein content enhancement did not alter the composition of the seed storage proteins. Inactivation of either AIP2a or AIP2b by a CRISPR Cas 9-mediated mutation increased seed protein content, this effect was greater when both genes were inactivated. Transactivation assays in transfected soybean hypocotyl protoplasts indicated that ABI3 changes the expression of glycinin, conglycinin, 2S albumin, and oleosin genes, indicating that AIP2 depletion increased seed protein content by regulating activity of the ABI3 transcription factor protein. These results provide an example of a gene-editing prototype directed to improve global food security and protein availability in soybean which may also be applicable to other protein-source crops.

RevDate: 2022-09-19
CmpDate: 2022-09-19

Shi H, Guo J, Yu Q, et al (2022)

CRISPR/Cas9 based blockade of IL-10 signaling impairs lipid and tissue homeostasis to accelerate atherosclerosis.

Frontiers in immunology, 13:999470.

Interleukin-10 (IL-10) is a widely recognized immunosuppressive factor. Although the concept that IL-10 executes an anti-inflammatory role is accepted, the relationship between IL-10 and atherosclerosis is still unclear, thus limiting the application of IL-10-based therapies for this disease. Emerging evidence suggests that IL-10 also plays a key role in energy metabolism and regulation of gut microbiota; however, whether IL-10 can affect atherosclerotic lesion development by integrating lipid and tissue homeostasis has not been investigated. In the present study, we developed a human-like hamster model deficient in IL-10 using CRISPR/Cas9 technology. Our results showed that loss of IL-10 changed the gut microbiota in hamsters on chow diet, leading to an increase in lipopolysaccharide (LPS) production and elevated concentration of LPS in plasma. These changes were associated with systemic inflammation, lipodystrophy, and dyslipidemia. Upon high cholesterol/high fat diet feeding, IL-10-deficient hamsters exhibited abnormal distribution of triglyceride and cholesterol in lipoprotein particles, impaired lipid transport in macrophages and aggravated atherosclerosis. These findings show that silencing IL-10 signaling in hamsters promotes atherosclerosis by affecting lipid and tissue homeostasis through a gut microbiota/adipose tissue/liver axis.

RevDate: 2022-09-15

Rönspies M, Schmidt C, Schindele P, et al (2022)

Massive crossover suppression by CRISPR-Cas-mediated plant chromosome engineering.

Nature plants [Epub ahead of print].

Recent studies have demonstrated that not only genes but also entire chromosomes can be engineered using clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPER-associated protein 9 (Cas9)1-5. A major objective of applying chromosome restructuring in plant breeding is the manipulation of genetic exchange6. Here we show that meiotic recombination can be suppressed in nearly the entire chromosome using chromosome restructuring. We were able to induce a heritable inversion of a >17 Mb-long chromosome fragment that contained the centromere and covered most of chromosome 2 of the Arabidopsis ecotype Col-0. Only the 2 and 0.5 Mb-long telomeric ends remained in their original orientation. In single-nucleotide polymorphism marker analysis of the offspring of crosses with the ecotype Ler-1, we detected a massive reduction of crossovers within the inverted chromosome region, coupled with a shift of crossovers to the telomeric ends. The few genetic exchanges detected within the inversion all originated from double crossovers. This not only indicates that heritable genetic exchange can occur by interstitial chromosome pairing, but also that it is restricted to the production of viable progeny.

RevDate: 2022-09-19
CmpDate: 2022-09-19

Kupcinskaite E, Tutkus M, Kopūstas A, et al (2022)

Disarming of type I-F CRISPR-Cas surveillance complex by anti-CRISPR proteins AcrIF6 and AcrIF9.

Scientific reports, 12(1):15548.

CRISPR-Cas systems are prokaryotic adaptive immune systems that protect against phages and other invading nucleic acids. The evolutionary arms race between prokaryotes and phages gave rise to phage anti-CRISPR (Acr) proteins that act as a counter defence against CRISPR-Cas systems by inhibiting the effector complex. Here, we used a combination of bulk biochemical experiments, X-ray crystallography and single-molecule techniques to explore the inhibitory activity of AcrIF6 and AcrIF9 proteins against the type I-F CRISPR-Cas system from Aggregatibacter actinomycetemcomitans (Aa). We showed that AcrIF6 and AcrIF9 proteins hinder Aa-Cascade complex binding to target DNA. We solved a crystal structure of Aa1-AcrIF9 protein, which differ from other known AcrIF9 proteins by an additional structurally important loop presumably involved in the interaction with Cascade. We revealed that AcrIF9 association with Aa-Cascade promotes its binding to off-target DNA sites, which facilitates inhibition of CRISPR-Cas protection.

RevDate: 2022-09-15

Nidhi S, Tripathi P, V Tripathi (2022)

Phylogenetic Analysis of Anti-CRISPR and Member Addition in the Families.

Molecular biotechnology [Epub ahead of print].

CRISPR-Cas is a widespread anti-viral adaptive immune system in the microorganisms. Viruses living in bacteria or some phages carry anti-CRISPR proteins to evade immunity by CRISPR-Cas. The anti-CRISPR proteins are prevalent in phages capable of lying dormant in a CRISPR-carrying host, while their orthologs frequently found in virulent phages. Here, we propose a probabilistic strategy of ancestral sequence reconstruction (ASR) and Hidden Markov Model (HMM) profile search to fish out sequences of anti-CRISPR proteins from environmental metagenomic, human microbiome metagenomic, human microbiome reference genome, and NCBI's non-redundant databases. Our results revealed that the metagenome database dark matter might contain anti-CRISPR encoding genes.

RevDate: 2022-09-15

Wörle E, Newman A, D'Silva J, et al (2022)

Allosteric activation of CRISPR-Cas12a requires the concerted movement of the bridge helix and helix 1 of the RuvC II domain.

Nucleic acids research pii:6701592 [Epub ahead of print].

Nucleases derived from the prokaryotic defense system CRISPR-Cas are frequently re-purposed for gene editing and molecular diagnostics. Hence, an in-depth understanding of the molecular mechanisms of these enzymes is of crucial importance. We focused on Cas12a from Francisella novicida (FnCas12a) and investigated the functional role of helix 1, a structural element that together with the bridge helix (BH) connects the recognition and the nuclease lobes of FnCas12a. Helix 1 is structurally connected to the lid domain that opens upon DNA target loading thereby activating the active site of FnCas12a. We probed the structural states of FnCas12a variants altered in helix 1 and/or the bridge helix using single-molecule FRET measurements and assayed the pre-crRNA processing, cis- and trans-DNA cleavage activity. We show that helix 1 and not the bridge helix is the predominant structural element that confers conformational stability of FnCas12a. Even small perturbations in helix 1 lead to a decrease in DNA cleavage activity while the structural integrity is not affected. Our data, therefore, implicate that the concerted remodeling of helix 1 and the bridge helix upon DNA binding is structurally linked to the opening of the lid and therefore involved in the allosteric activation of the active site.

RevDate: 2022-09-15

Aksoy E, Yildirim K, Kavas M, et al (2022)

General guidelines for CRISPR/Cas-based genome editing in plants.

Molecular biology reports [Epub ahead of print].

CRISPR (clustered regularly interspaced short palindromic repeats)/Cas (CRISPR-associated) technology is a versatile genome editing tool that has been used to improve agriculturally important plant traits. Due to its precision, CRISPR/Cas9 is more effective than either conventional plant breeding methods or standard genetic engineering approaches for the rapid development of new varieties resilient to climate change. In addition to knowledge in tissue culture-based plant transformation, effective gene-specific single guide RNA (sgRNA) design, prediction of its off-target effect and utilization of vectors, promoters, Cas proteins and terminators is required for CRISPR/Cas9. Various bioinformatics tools are available for the best sgRNA design and screening of the off-targets. Various tools are used in the delivery of CRISPR/Cas components into cells and the genome. Moreover, some recent studies proved the simultaneous silencing of different paralogs in the same family or several genes working in the same pathway by using multiple-target sgRNA designs. This review summarizes the type of promoters, Cas proteins, recognition sequences, and terminators available for the development of knock-out and overexpression plant lines. It also provides a general guideline for the development of genome-edited plants from the design of sgRNAs to the selection of non-transgenic genome-edited T2 generation.

RevDate: 2022-09-20
CmpDate: 2022-09-19

Tamura R, D Kamiyama (2023)

CRISPR-Cas9-Mediated Knock-In Approach to Insert the GFP11 Tag into the Genome of a Human Cell Line.

Methods in molecular biology (Clifton, N.J.), 2564:185-201.

The protocol in this chapter describes a method to label endogenous proteins using a self-complementing split green fluorescent protein (split GFP1-10/11) in a human cell line. By directly delivering Cas9/sgRNA ribonucleoprotein (RNP) complexes through nucleofection, this protocol allows for the efficient integration of GFP11 into a specific genomic locus via CRISPR-Cas9-mediated homology-directed repair (HDR). We use the GFP11 sequence in the form of a single-stranded DNA (ssDNA) as an HDR template. Because the ssDNA with less than 200 nucleotides used here is commercially synthesized, this approach remains cloning-free. The integration of GFP11 is performed in cells stably expressing GFP1-10, thereby inducing fluorescence reconstitution. Subsequently, such a reconstituted signal is analyzed using fluorescence flow cytometry for estimating knock-in efficiencies and enriching the GFP-positive cell population. Finally, the enriched cells can be visualized using fluorescence microscopy.

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RJR Experience and Expertise

Researcher

Robbins holds BS, MS, and PhD degrees in the life sciences. He served as a tenured faculty member in the Zoology and Biological Science departments at Michigan State University. He is currently exploring the intersection between genomics, microbial ecology, and biodiversity — an area that promises to transform our understanding of the biosphere.

Educator

Robbins has extensive experience in college-level education: At MSU he taught introductory biology, genetics, and population genetics. At JHU, he was an instructor for a special course on biological database design. At FHCRC, he team-taught a graduate-level course on the history of genetics. At Bellevue College he taught medical informatics.

Administrator

Robbins has been involved in science administration at both the federal and the institutional levels. At NSF he was a program officer for database activities in the life sciences, at DOE he was a program officer for information infrastructure in the human genome project. At the Fred Hutchinson Cancer Research Center, he served as a vice president for fifteen years.

Technologist

Robbins has been involved with information technology since writing his first Fortran program as a college student. At NSF he was the first program officer for database activities in the life sciences. At JHU he held an appointment in the CS department and served as director of the informatics core for the Genome Data Base. At the FHCRC he was VP for Information Technology.

Publisher

While still at Michigan State, Robbins started his first publishing venture, founding a small company that addressed the short-run publishing needs of instructors in very large undergraduate classes. For more than 20 years, Robbins has been operating The Electronic Scholarly Publishing Project, a web site dedicated to the digital publishing of critical works in science, especially classical genetics.

Speaker

Robbins is well-known for his speaking abilities and is often called upon to provide keynote or plenary addresses at international meetings. For example, in July, 2012, he gave a well-received keynote address at the Global Biodiversity Informatics Congress, sponsored by GBIF and held in Copenhagen. The slides from that talk can be seen HERE.

Facilitator

Robbins is a skilled meeting facilitator. He prefers a participatory approach, with part of the meeting involving dynamic breakout groups, created by the participants in real time: (1) individuals propose breakout groups; (2) everyone signs up for one (or more) groups; (3) the groups with the most interested parties then meet, with reports from each group presented and discussed in a subsequent plenary session.

Designer

Robbins has been engaged with photography and design since the 1960s, when he worked for a professional photography laboratory. He now prefers digital photography and tools for their precision and reproducibility. He designed his first web site more than 20 years ago and he personally designed and implemented this web site. He engages in graphic design as a hobby.

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CRISPR-Cas

By delivering the Cas9 nuclease, complexed with a synthetic guide RNA (gRNA) into a cell, the cell's genome can be precisely cut at any desired location, allowing existing genes to be removed and/or new ones added. That is, the CRISPR-Cas system provides a tool for the cut-and-paste editing of genomes. Welcome to the brave new world of genome editing. R. Robbins

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Collection of publications by R J Robbins

Reprints and preprints of publications, slide presentations, instructional materials, and data compilations written or prepared by Robert Robbins. Most papers deal with computational biology, genome informatics, using information technology to support biomedical research, and related matters.

Research Gate page for R J Robbins

ResearchGate is a social networking site for scientists and researchers to share papers, ask and answer questions, and find collaborators. According to a study by Nature and an article in Times Higher Education , it is the largest academic social network in terms of active users.

Curriculum Vitae for R J Robbins

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Curriculum Vitae for R J Robbins

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