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Bibliography on: CRISPR-Cas

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ESP: PubMed Auto Bibliography 08 Dec 2022 at 11:46 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: 2022-12-08

Cui N, Zhang JT, Li Z, et al (2022)

Structural basis for the non-self RNA-activated protease activity of the type III-E CRISPR nuclease-protease Craspase.

Nature communications, 13(1):7549.

The RNA-targeting type III-E CRISPR-gRAMP effector interacts with a caspase-like protease TPR-CHAT to form the CRISPR-guided caspase complex (Craspase), but their functional mechanism is unknown. Here, we report cryo-EM structures of the type III-E gRAMP[crRNA] and gRAMP[crRNA]-TPR-CHAT complexes, before and after either self or non-self RNA target binding, and elucidate the mechanisms underlying RNA-targeting and non-self RNA-induced protease activation. The associated TPR-CHAT adopted a distinct conformation upon self versus non-self RNA target binding, with nucleotides at positions -1 and -2 of the CRISPR-derived RNA (crRNA) serving as a sensor. Only binding of the non-self RNA target activated the TPR-CHAT protease, leading to cleavage of Csx30 protein. Furthermore, TPR-CHAT structurally resembled eukaryotic separase, but with a distinct mechanism for protease regulation. Our findings should facilitate the development of gRAMP-based RNA manipulation tools, and advance our understanding of the virus-host discrimination process governed by a nuclease-protease Craspase during type III-E CRISPR-Cas immunity.

RevDate: 2022-12-08

Hiramoto T, T Ohmori (2022)

[Application of genome editing technology in gene therapy].

[Rinsho ketsueki] The Japanese journal of clinical hematology, 63(11):1558-1565.

Genome editing has been attracting increasing attention as a new treatment for several refractory diseases since the CRISPR-Cas discovery has facilitated easy modification of target chromosomal DNA. The concept of treating refractory diseases by genome editing has been achieved in various animal models, and genome editing has been applied to human clinical trials for β-thalassemia, sickle cell disease, mucopolysaccharidosis, transthyretin amyloidosis, HIV infection, and CAR-T therapy. The genome editing technology targets the germline in industrial applications in animals and plants and is directed at the chromosomal DNA of the somatic cells in human therapeutic applications. Genome editing therapy for germline cells is currently forbidden due to ethical and safety concerns. Concerns regarding genome editing technology include safety (off-target effects) as well as technical aspects (low homologous recombination). Various technological innovations for genome editing are expected to expand its clinical application to various diseases in the future.

RevDate: 2022-12-08

Lu Y, Yang H, Bai J, et al (2022)

CRISPR-Cas based molecular diagnostics for foodborne pathogens.

Critical reviews in food science and nutrition [Epub ahead of print].

Foodborne pathogenic infection has brought multifaceted issues to human life, leading to an urgent demand for advanced detection technologies. CRISPR/Cas-based biosensors have the potential to address various challenges that exist in conventional assays such as insensitivity, long turnaround time and complex pretreatments. In this perspective, we review the relevant strategies of CRISPR/Cas-assisted diagnostics on foodborne pathogens, focusing on biosensing platforms for foodborne pathogens based on fluorescence, colorimetric, (electro)chemiluminescence, electrochemical, and surface-enhanced Raman scattering detection. It summarizes their detection principles by the clarification of foodborne pathogenic bacteria, fungi, and viruses. Finally, we discuss the current challenges or technical barriers of these methods against broad application, and put forward alternative solutions to improve CRISPR/Cas potential for food safety.

RevDate: 2022-12-07

Kuluev BR, Mikhailova EV, Kuluev AR, et al (2022)

[Genome Editing in Species of the Tribe Triticeae with the CRISPR/Cas System].

Molekuliarnaia biologiia, 56(6):949-968.

The tribe Triticeae includes important agricultural crops, such as bread wheat, durum wheat, barley, rye, and triticale. Research in the field of reverse genetics and genetic engineering of Triticeae received a new impetus as the CRISPR/Cas genome editing system came into broad use. The review describes and analyzes the data on recent advances in genomic editing of cultivated plants of the tribe Triticeae and tools used in the field. The tools most commonly used for genome editing in Triticeae include the codon-optimized Cas9 gene under the control of the maize ubiquitin gene promoter and guide RNAs under the control of Pol III promoters U6 and U3 in one or more binary vectors. Phosphinothricin and hygromycin resistance genes are used as selectable genes. Agrobacterium-mediated transformation and biolistics are performed to obtain genome-edited plants, and immature embryos are used as explants. Approaches developed to overcome the problem of low regenerative capacity of Triticeae include in planta transformation of shoot apical meristems, transformation of microspores and pollen grains, and the use of haploinductors. Bread wheat and barley were subject to genomic editing in the majority of studies published to date, and durum wheat and triticale were recently used in CRISPR/Cas knockout studies of target genes. Further progress in the development of genome editing of cultivated plants of the tribe Triticeae should be aimed at expanding the range of species and varieties involved and overcoming the problems of low regenerative capacity. This will allow genetic modification of elite varieties, which will be in demand in agricultural production.

RevDate: 2022-12-07

Spasskaya DS, Davletshin AI, Tutyaeva VV, et al (2022)

[A Test System for Assessment of the Activity of Mutant Cas9 Variants in Saccharomyces cerevisiae].

Molekuliarnaia biologiia, 56(6):937-948.

The key component of the revolutionary Streptococcus pyogenes CRISPR/Cas genome editing technology is the multidomain protein Cas9. However, the specificity of wild type Cas9 is not sufficiently high for editing large genomes of higher eukaryotes, which limits the realization of the potential of genomic editing both in fundamental investigations and in the therapy of genetic diseases. The main way to obtain more specific variants of Cas9 is through mutagenesis followed by characterization of mutant proteins in in vitro or in vivo test systems. The in vitro and some in vivo test systems described in the literature are often labor-intensive and have scaling limitations, which makes it challenging to screen SpCas9 mutant variant libraries. In order to develop a simple method for high-throughput screening of Cas9 mutants in vivo, we characterized three test systems using CRISPR/Cas9-mediated inactivation of the reporter genes, tsPurple, ADE2, and URA3, in the Saccharomyces cerevisiae yeast as a model subject. We measured the activities of high-precision forms of Cas9, evoCas9, and HiFiCas9, and compared them with the wild-type form. ADE2 gene inactivation was found to be the most valid method for the evaluation of Cas9 activity. In the test-system developed, the sensitivity to chromatin structure was demonstrated for the high-fidelity variant of Cas9, HiFiCas9. The proposed test-system can be used for the development of new generation genome editors.

RevDate: 2022-12-07

Armianinova DK, Karpov DS, Kotliarova MS, et al (2022)

[Genetic Engineering in Mycobacteria].

Molekuliarnaia biologiia, 56(6):900-913.

Genetic tools for targeted modification of the mycobacterial genome contribute to the understanding of the physiology and virulence mechanisms of mycobacteria. Human and animal pathogens, such as the Mycobacterium tuberculosis complex, which causes tuberculosis, and M. leprae, which causes leprosy, are of particular importance. Genetic research opens up novel opportunities to identify and validate new targets for antibacterial drugs and to develop improved vaccines. Although mycobacteria are difficult to work with due to their slow growth rate and a limited possibility to transfer genetic information, significant progress has been made in developing genetic engineering methods for mycobacteria. The review considers the main approaches to changing the mycobacterial genome in a targeted manner, including homologous and site-specific recombination and use of the CRISPR/Cas system.

RevDate: 2022-12-07

Nadolinskaia NI, AV Goncharenko (2022)

[CRISPR Interference in Regulation of Bacterial Gene Expression].

Molekuliarnaia biologiia, 56(6):892-899.

The review is devoted to the use of the CRISPR/Cas system for obtaining knockdowns of target bacterial genes by CRISPR-mediated interference (CRISPRi). CRISPRi is based on the preservation of the ability of the inactivated dCas nuclease in complex with guide RNA to bind a target, which leads to reversible repression of the selected genes. The review describes the principle of operation of CRISPR/Cas and CRIS-PRi/dCas and provides examples of various approaches to the use of CRISPRi with the most popular inactivated nucleases dCas9 and dCas12a. Also, attention is paid to the use of CRISPRi screening for genome-wide studies and the modular system for identifying many important patterns at the Mobile-CRISPRi genome level. In addition, we discuss the use of CRISPRi to optimize biotechnological production, such as the synthesis of malonyl-CoA, L-lysine, L-glutamate, and other significant products.

RevDate: 2022-12-07

Isaev A, Andriianov A, Znobishcheva E, et al (2022)

[Editing of Phage Genomes - Recombineering-Assisted SpCas9 Modification of Model Coliphages T7, T5, and T3].

Molekuliarnaia biologiia, 56(6):883.

Bacteriophages-viruses that infect bacterial cells - are the most abundant biological entities on Earth. The use of phages in fundamental research and industry requires tools for precise manipulation of their genomes. Yet, compared to bacterial genome engineering, modification of phage genomes is challenging because of the lack of selective markers and thus requires laborious screenings of recombinant/mutated phage variants. The development of the CRISPR-Cas technologies allowed to solve this issue by the implementation of negative selection that eliminates the parental phage genomes. In this manuscript, we summarize current methods of phage genome engineering and their coupling with CRISPR-Cas technologies. We also provide examples of our successful application of these methods for introduction of specific insertions, deletions, and point mutations in the genomes of model Escherichia coli lytic phages T7, T5, and T3.

RevDate: 2022-12-07

Tao S, Chen H, Li N, et al (2022)

Association of CRISPR-Cas System with the Antibiotic Resistance and Virulence Genes in Nosocomial Isolates of Enterococcus.

Infection and drug resistance, 15:6939-6949 pii:388354.

PURPOSE: This study aimed to investigate the prevalence of the CRISPR-Cas system in nosocomial isolates of Enterococcus and their possible association with antibiotic resistance and virulence genes.

MATERIALS AND METHODS: Identification and antimicrobial susceptibility of the microorganism were performed by the automatized VITEK 2 Compact system (bioMerieux, France). A total of 100 Enterococcus isolates were collected and identified by VITEK 2 Compact automatic microbial identification drug susceptibility analyzer. The prevalence of various CRISPR-Cas systems, antibiotic resistance genes and virulence genes were investigated by polymerase chain reaction (PCR). The prevalence of CRISPR-Cas systems associated with antibiotic resistance and virulence genes was performed by appropriate statistical tests.

RESULTS: A total of 100 isolates of Enterococcus were identified and there were 62/100(62.0%) Enterococcus faecalis isolates and 38/100(38.0%) Enterococcus faecalis isolates. In total, 46 (46.0%) of 100 isolates had at least one CRISPR-Cas locus. CRISPR elements were more prevalent in Enterococcus faecalis isolates. The results of PCR demonstrated that CRISPR1-Cas, orphan CRISPR2, and CRISPR3-Cas were present in 23 (23.0%), 42 (42.0%) and 5 (5.0%) Enterococcus isolates, respectively. Compared with CRISPR-Casnegative isolates, the CRISPR-Cas positive isolates showed significant lower resistance rates against ampicillin, erythromycin, levofloxacin, tetracycline, vancomycin, gentamicin, streptomycin, and rifampicin. Presumably consistent with drug susceptibility, fewer CRISPR loci were identified in vanA, tetM, ermB, aac6'-aph(2"), aadE, and ant(6) positive isolates. There was a significant negative correlation between the CRISPR-Cas locus and the enterococcal virulence factors enterococcal surface protein (esp) gene.

CONCLUSION: In conclusion, the results indicated that the absence of the CRISPR-Cas system was negatively associated with some antibiotic resistance in clinical isolates of Enterococcus faecalis and Enterococcus faecium. Also, there was a negative correlation with the carriage of antibiotic resistance genes. Furthermore, CRISPR-Cas may prevent some isolates from acquiring certain virulence factors.

RevDate: 2022-12-06

Selma S, Ceulemans E, Goossens A, et al (2022)

Clustered regularly interspaced short palindromic repeats tools for plant metabolic engineering: achievements and perspectives.

Current opinion in biotechnology, 79:102856 pii:S0958-1669(22)00190-2 [Epub ahead of print].

The plant kingdom represents the biggest source of feedstock, food, and added-value compounds. Engineering plant metabolic pathways to increase the phytochemical production or improve the nutraceutical value of crops is challenging because of the intricate interaction networks that link multiple genes, enzymatic steps, and metabolites, even when pathways are fully elucidated. The development of clustered regularly interspaced short palindromic repeats - CRISPR-associated (CRISPR-Cas) technologies has helped to overcome limitations in metabolic engineering, providing efficient and versatile tools for multigene editing. CRISPR approaches in plants were shown to have a remarkable efficiency in genome editing of different species to improve agronomic and metabolic traits. Here, we give an overview of the different achievements and perspectives of CRISPR technology in plant metabolic engineering.

RevDate: 2022-12-07
CmpDate: 2022-12-07

Gumustop I, F Ortakci (2022)

Comparative Genomics of Lentilactobacillus parabuchneri isolated from dairy, KEM complex, Makgeolli, and Saliva Microbiomes.

BMC genomics, 23(1):803.

BACKGROUND: Lentilactobacillus parabuchneri is of particular concern in fermented food bioprocessing due to causing unwanted gas formation, cracks, and off-flavor in fermented dairy foods. This species is also a known culprit of histamine poisonings because of decarboxylating histidine to histamine in ripening cheese. Twenty-eight genomes in NCBI GenBank were evaluated via comparative analysis to determine genomic diversity within this species and identify potential avenues for reducing health associated risks and economic losses in the food industry caused by these organisms.

RESULT: Core genome-based phylogenetic analysis revealed four distinct major clades. Eight dairy isolates, two strains from an unknown source, and a saliva isolate formed the first clade. Three out of five strains clustered on clade 2 belonged to dairy, and the remaining two strains were isolated from the makgeolli and Korean effective microorganisms (KEM) complex. The third and fourth clade members were isolated from Tete de Moine and dairy-associated niches, respectively. Whole genome analysis on twenty-eight genomes showed ~ 40% of all CDS were conserved across entire strains proposing a considerable diversity among L. parabuchneri strains analyzed. After assigning CDS to their corresponding function, ~ 79% of all strains were predicted to carry putative intact prophages, and ~ 43% of the strains harbored at least one plasmid; however, all the strains were predicted to encode genomic island, insertion sequence, and CRISPR-Cas system. A type I-E CRISPR-Cas subgroup was identified in all the strains, with the exception of DSM15352, which carried a type II-A CRISPR-Cas system. Twenty strains were predicted to encode histidine decarboxylase gene cluster that belongs to not only dairy but also saliva, KEM complex, and unknown source. No bacteriocin-encoding gene(s) or antibiotic resistome was found in any of the L. parabuchneri strains screened.

CONCLUSION: The findings of the present work provide in-depth knowledge of the genomics of L. parabuchneri by comparing twenty-eight genomes available to date. For example, the hdc gene cluster was generally reported in cheese isolates; however, our findings in the current work indicated that it could also be encoded in those strains isolated from saliva, KEM complex, and unknown source. We think prophages are critical mobile elements of L. parabuchneri genomes that could pave the way for developing novel tools to reduce the occurrence of this unwanted species in the food industry.

RevDate: 2022-12-07
CmpDate: 2022-12-07

Blomme J, Ribera JA, Develtere W, et al (2022)

A Simple and Low-Tech Heat-Shock Method to Increase Genome Editing Efficiency in Plants.

Current protocols, 2(12):e608.

CRISPR/Cas is now the standard technique to generate novel plant genotypes. However, optimizing the efficiency of the system continues to be an aspect of research and development. One of the improvements for increasing mutagenesis efficiency in different species is the application of heat stress. However, many experimental setups are limited by the requirement of using dedicated climate chambers to impose heat stress and by difficulties in the phenotyping of soil-grown plants. Here, we describe a simplified heat stress assay for in vitro-grown plants that can be completed in 6 days using commonly available laboratory equipment. We show that three 24-hr heat shocks (3×HS) at 37°C alternated with 24 hr of recovery at 21°C efficiently increases indel rates of LbCas12a and Cas9. We illustrate how visual mutant phenotypes (pds3 and gl1) can assist in quantifying genome editing efficiency, and describe how to quantify genome editing efficiency using genotyping by Sanger sequencing. We also provide a support protocol to efficiently clone a CRISPR expression vector in a single step. Together, our methods allow researchers to increase CRISPR-induced mutations using a low-tech setup in plants. © 2022 Wiley Periodicals LLC. Basic Protocol 1: 3×HS protocol Basic Protocol 2: Genotyping by Sanger sequencing Support Protocol: One-step cloning of a CRISPR expression vector.

RevDate: 2022-12-06
CmpDate: 2022-12-06

Chien Y, Hsiao YJ, Chou SJ, et al (2022)

Nanoparticles-mediated CRISPR-Cas9 gene therapy in inherited retinal diseases: applications, challenges, and emerging opportunities.

Journal of nanobiotechnology, 20(1):511.

Inherited Retinal Diseases (IRDs) are considered one of the leading causes of blindness worldwide. However, the majority of them still lack a safe and effective treatment due to their complexity and genetic heterogeneity. Recently, gene therapy is gaining importance as an efficient strategy to address IRDs which were previously considered incurable. The development of the clustered regularly-interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) system has strongly empowered the field of gene therapy. However, successful gene modifications rely on the efficient delivery of CRISPR-Cas9 components into the complex three-dimensional (3D) architecture of the human retinal tissue. Intriguing findings in the field of nanoparticles (NPs) meet all the criteria required for CRISPR-Cas9 delivery and have made a great contribution toward its therapeutic applications. In addition, exploiting induced pluripotent stem cell (iPSC) technology and in vitro 3D retinal organoids paved the way for prospective clinical trials of the CRISPR-Cas9 system in treating IRDs. This review highlights important advances in NP-based gene therapy, the CRISPR-Cas9 system, and iPSC-derived retinal organoids with a focus on IRDs. Collectively, these studies establish a multidisciplinary approach by integrating nanomedicine and stem cell technologies and demonstrate the utility of retina organoids in developing effective therapies for IRDs.

RevDate: 2022-12-03

Chen Y, Zeng Z, She Q, et al (2022)

The abortive infection functions of CRISPR-Cas and Argonaute.

Trends in microbiology pii:S0966-842X(22)00313-4 [Epub ahead of print].

CRISPR-Cas and prokaryotic Argonaute (pAgo) are nucleic acid (NA)-guided defense systems that protect prokaryotes against the invasion of mobile genetic elements. Previous studies established that they are directed by NA fragments (guides) to recognize invading complementary NA (targets), and that they cleave the targets to silence the invaders. Nevertheless, growing evidence indicates that many CRISPR-Cas and pAgo systems exploit the abortive infection (Abi) strategy to confer immunity. The CRISPR-Cas and pAgo Abi systems typically sense invaders using the NA recognition ability and activate various toxic effectors to kill the infected cells to prevent the invaders from spreading. This review summarizes the diverse mechanisms of these CRISPR-Cas and pAgo systems, and highlights their critical roles in the arms race between microbes and invaders.

RevDate: 2022-12-06
CmpDate: 2022-12-06

Rutkauskas M, Songailiene I, Irmisch P, et al (2022)

A quantitative model for the dynamics of target recognition and off-target rejection by the CRISPR-Cas Cascade complex.

Nature communications, 13(1):7460.

CRISPR-Cas effector complexes recognise nucleic acid targets by base pairing with their crRNA which enables easy re-programming of the target specificity in rapidly emerging genome engineering applications. However, undesired recognition of off-targets, that are only partially complementary to the crRNA, occurs frequently and represents a severe limitation of the technique. Off-targeting lacks comprehensive quantitative understanding and prediction. Here, we present a detailed analysis of the target recognition dynamics by the Cascade surveillance complex on a set of mismatched DNA targets using single-molecule supercoiling experiments. We demonstrate that the observed dynamics can be quantitatively modelled as a random walk over the length of the crRNA-DNA hybrid using a minimal set of parameters. The model accurately describes the recognition of targets with single and double mutations providing an important basis for quantitative off-target predictions. Importantly the model intrinsically accounts for observed bias regarding the position and the proximity between mutations and reveals that the seed length for the initiation of target recognition is controlled by DNA supercoiling rather than the Cascade structure.

RevDate: 2022-12-06
CmpDate: 2022-12-06

Steens JA, van der Oost J, RHJ Staals (2022)

Compact but mighty: Biology and applications of type III-E CRISPR-Cas systems.

Molecular cell, 82(23):4405-4406.

In this issue, Liu et al. present an in-depth study aiming to unravel the structural, biochemical, and physiological aspects of how type III-E CRISPR-Cas systems trigger abortive infection by activating a protease upon target RNA recognition.[1].

RevDate: 2022-12-06
CmpDate: 2022-12-06

Mahata T, U Qimron (2022)

Thou shalt not cleave DNA-only repress transcription: A compact Cas protein representing a new CRISPR-Cas subtype.

Molecular cell, 82(23):4403-4404.

Wu et al.[1] characterize Cas12m, a compact Cas protein that silences transcription without cleaving DNA and is a prototype protein of the novel CRISPR-Cas subtype V-M.

RevDate: 2022-12-06
CmpDate: 2022-12-06

Chen E, Lin-Shiao E, Trinidad M, et al (2022)

Decorating chromatin for enhanced genome editing using CRISPR-Cas9.

Proceedings of the National Academy of Sciences of the United States of America, 119(49):e2204259119.

CRISPR-associated (Cas) enzymes have revolutionized biology by enabling RNA-guided genome editing. Homology-directed repair (HDR) in the presence of donor templates is currently the most versatile method to introduce precise edits following CRISPR-Cas-induced double-stranded DNA cuts, but HDR efficiency is generally low relative to end-joining pathways that lead to insertions and deletions (indels). We tested the hypothesis that HDR could be increased using a Cas9 construct fused to PRDM9, a chromatin remodeling factor that deposits histone methylations H3K36me3 and H3K4me3 to mediate homologous recombination in human cells. Our results show that the fusion protein contacts chromatin specifically at the Cas9 cut site in the genome to increase the observed HDR efficiency by threefold and HDR:indel ratio by fivefold compared with that induced by unmodified Cas9. HDR enhancement occurred in multiple cell lines with no increase in off-target genome editing. These findings underscore the importance of chromatin features for the balance between DNA repair mechanisms during CRISPR-Cas genome editing and provide a strategy to increase HDR efficiency.

RevDate: 2022-12-07
CmpDate: 2022-12-05

Guo X, Steinman RA, Sheng Y, et al (2022)

An AGS-associated mutation in ADAR1 catalytic domain results in early-onset and MDA5-dependent encephalopathy with IFN pathway activation in the brain.

Journal of neuroinflammation, 19(1):285.

BACKGROUND: Aicardi-Goutières syndrome (AGS) is a severe neurodegenerative disease with clinical features of early-onset encephalopathy and progressive loss of intellectual abilities and motor control. Gene mutations in seven protein-coding genes have been found to be associated with AGS. However, the causative role of these mutations in the early-onset neuropathogenesis has not been demonstrated in animal models, and the mechanism of neurodegeneration of AGS remains ambiguous.

METHODS: Via CRISPR/Cas-9 technology, we established a mutant mouse model in which a genetic mutation found in AGS patients at the ADAR1 coding gene (Adar) loci was introduced into the mouse genome. A mouse model carrying double gene mutations encoding ADAR1 and MDA-5 was prepared using a breeding strategy. Phenotype, gene expression, RNA sequencing, innate immune pathway activation, and pathologic studies including RNA in situ hybridization (ISH) and immunohistochemistry were used for characterization of the mouse models to determine potential disease mechanisms.

RESULTS: We established a mouse model bearing a mutation in the catalytic domain of ADAR1, the D1113H mutation found in AGS patients. With this mouse model, we demonstrated a causative role of this mutation for the early-onset brain injuries in AGS and determined the signaling pathway underlying the neuropathogenesis. First, this mutation altered the RNA editing profile in neural transcripts and led to robust IFN-stimulated gene (ISG) expression in the brain. By ISH, the brains of mutant mice showed an unusual, multifocal increased expression of ISGs that was cell-type dependent. Early-onset astrocytosis and microgliosis and later stage calcification in the deep white matter areas were observed in the mutant mice. Brain ISG activation and neuroglial reaction were completely prevented in the Adar D1113H mutant mice by blocking RNA sensing through deletion of the cytosolic RNA receptor MDA-5.

CONCLUSIONS: The Adar D1113H mutation in the ADAR1 catalytic domain results in early-onset and MDA5-dependent encephalopathy with IFN pathway activation in the mouse brain.

RevDate: 2022-12-05
CmpDate: 2022-12-05

Bhowmik R, B Chaubey (2022)

CRISPR/Cas9: a tool to eradicate HIV-1.

AIDS research and therapy, 19(1):58.

The development of antiretroviral therapy (ART) has been effective in suppressing HIV replication. However, severe drug toxicities due to the therapy and its failure in targeting the integrated proviral genome have led to the introduction of a new paradigm of gene-based therapies. With its effective inhibition and high precision, clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein-9 nuclease (Cas9) or CRISPR/Cas9 has emerged as an effective genome editing tool in the last decade. Mediated by guide RNAs (gRNAs), Cas9 endonuclease acts like genetic scissors that can modify specific target sites. With this concept, CRISPR/Cas9 has been used to target the integrated proviral HIV-1 genome both in in vitro as well as in vivo studies including non-human primates. The CRISPR has also been tested for targeting latent HIV-1 by modulating the proviral transcription with the help of a specialized Cas9 mutant. Overcoming the limitations of the current therapy, CRISPR has the potential to become the primary genome editing tool for eradicating HIV-1 infection. In this review, we summarize the recent advancements of CRISPR to target the proviral HIV-1 genome, the challenges and future prospects.

RevDate: 2022-12-06
CmpDate: 2022-12-06

Arias CF, Acosta FJ, Bertocchini F, et al (2022)

The coordination of anti-phage immunity mechanisms in bacterial cells.

Nature communications, 13(1):7412.

Bacterial cells are equipped with a variety of immune strategies to fight bacteriophage infections. Such strategies include unspecific mechanisms directed against any phage infecting the cell, ranging from the identification and cleavage of the viral DNA by restriction nucleases (restriction-modification systems) to the suicidal death of infected host cells (abortive infection, Abi). In addition, CRISPR-Cas systems generate an immune memory that targets specific phages in case of reinfection. However, the timing and coordination of different antiviral systems in bacterial cells are poorly understood. Here, we use simple mathematical models of immune responses in individual bacterial cells to propose that the intracellular dynamics of phage infections are key to addressing these questions. Our models suggest that the rates of viral DNA replication and cleavage inside host cells define functional categories of phages that differ in their susceptibility to bacterial anti-phage mechanisms, which could give raise to alternative phage strategies to escape bacterial immunity. From this viewpoint, the combined action of diverse bacterial defenses would be necessary to reduce the chances of phage immune evasion. The decision of individual infected cells to undergo suicidal cell death or to incorporate new phage sequences into their immune memory would be determined by dynamic interactions between the host's immune mechanisms and the phage DNA. Our work highlights the importance of within-cell dynamics to understand bacterial immunity, and formulates hypotheses that may inspire future research in this area.

RevDate: 2022-12-01

Capdeville N, Schindele P, H Puchta (2022)

Getting better all the time - recent progress in the development of CRISPR/Cas-based tools for plant genome engineering.

Current opinion in biotechnology, 79:102854 pii:S0958-1669(22)00188-4 [Epub ahead of print].

Since their first adaptation for plant genome editing, clustered regularly interspaced short palindromic repeats/CRISPR-associated system nucleases and tools have revolutionized the field. While early approaches focused on targeted mutagenesis that relies on mutagenic repair of induced double-strand breaks, newly developed tools now enable the precise induction of predefined modifications. Constant efforts to optimize these tools have led to the generation of more efficient base editors with enlarged editing windows and have enabled previously unachievable C-G transversions. Prime editors were also optimized for the application in plants and now allow to accurately induce substitutions, insertions, and deletions. Recently, great progress was made through precise restructuring of chromosomes, which enables not only the breakage or formation of genetic linkages but also the swapping of promoters.

RevDate: 2022-12-06
CmpDate: 2022-12-06

Leslie M (2022)

CRISPR is so popular even viruses may use it.

Science (New York, N.Y.), 378(6623):935-936.

Thousands of phages appear to have stolen the gene-cutting mechanism.

RevDate: 2022-12-05
CmpDate: 2022-12-05

Ilahibaks NF, Hulsbos MJ, Lei Z, et al (2023)

Enabling Precision Medicine with CRISPR-Cas Genome Editing Technology: A Translational Perspective.

Advances in experimental medicine and biology, 1396:315-339.

Genome editing technologies, particularly CRISPR-Cas (clustered regularly interspaced short palindromic repeats (CRISPR) associated nucleases), are redefining the boundaries of therapeutic gene therapy. CRISPR-Cas is a robust, straightforward, and programmable genome editing tool capable of mediating site-specific DNA modifications. The rapid advancements from discovery to clinical adaptation have expanded the therapeutic landscape to treat genetically defined diseases. Together with the technical developments in human DNA and RNA sequencing, CRISPR-directed gene therapy enables a new era to realize precision medicine where pathogenic mutations underlying monogenic disorders can potentially be corrected. Also, protective or therapeutic genomic alterations can be introduced as preventative or curative therapy. Despite its high therapeutic potential, CRISPR-Cas´ clinical translation is still in its infancy and is highly dependent on its efficiency, specificity in gene corrections, and cell-specific delivery. Therefore, this chapter focuses on the challenges and opportunities the CRISPR-Cas toolbox offers together with delivery vehicles to realize its use for therapeutic gene editing. Furthermore, we discuss the obstacles the CRISPR-Cas system faces for successful clinical translation and summarize its current clinical progress.

RevDate: 2022-12-05
CmpDate: 2022-12-05

Giallongo S, Lo Re O, Resnick I, et al (2023)

Gene Editing and Human iPSCs in Cardiovascular and Metabolic Diseases.

Advances in experimental medicine and biology, 1396:275-298.

The incidence and the burden of cardiovascular disease (CVD), coronary heart disease (CHD), type 2 diabetes mellitus (T2DM), and the metabolic syndrome are greatly increasing in our societies. Together, they account for 31% of all deaths worldwide. This chapter focuses on the role of two revolutionary discoveries that are changing the future of medicine, induced pluripotent stem cells (iPSCs) and CRISPR/Cas9 technology, in the study, and the cure of cardiovascular and metabolic diseases.We summarize the state-of-the-art knowledge about the possibility of editing iPSC genome for therapeutic applications without hampering their pluripotency and differentiation, using CRISPR/Cas technology, in the field of cardiovascular and metabolic diseases.

RevDate: 2022-12-05
CmpDate: 2022-12-05

Lu D, Cushman S, Thum T, et al (2023)

Gene Therapy and Cardiovascular Diseases.

Advances in experimental medicine and biology, 1396:235-254.

Cardiovascular diseases (CVDs) are the leading causes of death globally and urgently require new novel therapeutic strategies. Gene therapy is the application of gene modulation technology to treat abnormal gene expression under disease conditions. Viral- and nonviral-based gene delivery systems are the foundation of gene modulation in target cells. Moreover, plasmid- or oligo-based gene modulation tools as well as new advancements in gene editing using CRISPR/Cas technology are currently being tested in a variety of clinical trials. Here, we summarized state-of-the-art gene therapy technologies as well as recent clinical trials and discuss the applications and lessons of gene therapy in CVDs.

RevDate: 2022-12-05
CmpDate: 2022-12-05

Chen Z, Lehertshuber C, H Schunkert (2023)

Genome Editing in Dyslipidemia and Atherosclerosis.

Advances in experimental medicine and biology, 1396:139-156.

Despite successive advancement of genome editing technology with zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), the recent breakthrough in the field has been related to clustered regularly interspaced short palindromic repeats/associated proteins (CRISPR/Cas). The high efficiency and convenience of CRIPSR/Cas systems dramatically accelerate pre- and clinical experimentations of dyslipidemia and atherosclerosis. In this chapter, we review the latest state of genome editing in translational research of dyslipidemia and atherosclerosis. We highlight recent progress in therapeutic development for familial dyslipidemia by genome editing. We point to the challenges in maximizing efficacy and minimizing safety issues related to the once-and-done therapy focusing on CRISPR/Cas systems. We give an outlook on the potential gene targets prioritized by large-scale genetic studies of cardiovascular diseases and genome editing in precision medicine of dyslipidemia and atherosclerosis.

RevDate: 2022-12-05
CmpDate: 2022-12-05

Kato T (2023)

Genome Editing and Pathological Cardiac Hypertrophy.

Advances in experimental medicine and biology, 1396:87-101.

Three major genome editing tools, transcription activator-like effector nucleases (TALENs), zinc finger nucleases (ZFNs), and clustered regularly interspaced short palindromic repeat (CRISPR) systems, are increasingly important technologies used in the study and treatment of hereditary myocardial diseases. Germ cell genome editing and modification can permanently eliminate monogenic cardiovascular disease from the offspring of affected families and the next generation, although ethically controversial. Somatic genome editing may be a promising method for the treatment of hereditary cardiomyopathy various diseases for which gene knockout is favorable and can also treat people who are already ill, although there are currently some technical challenges. This chapter describes the application of genome editing in the experimental studies and treatment of hypertrophic cardiomyopathy as well as other cardiomyopathies.

RevDate: 2022-12-05
CmpDate: 2022-12-05

Carrillo-Rodriguez P, Martinez-Lopez J, Gómez-Hernández L, et al (2023)

Online Databases of Genome Editing in Cardiovascular and Metabolic Diseases.

Advances in experimental medicine and biology, 1396:19-33.

Metabolic and cardiovascular diseases are world-concerning pathologies that affect an important percentage of the population. Nowadays, advances in the genetic background of these diseases allow new approaches to models and therapies, as well as different gene edition trials. Furthermore, technological improvements in gene editing go along with the development of new online and biocomputational tools that provide us alternative ways to explore pathologies. In this chapter, historical gene editing methods are discussed but focusing on CRISPR-Cas system in detail and also online resources available to perform these types of experiments. Here, the different strategies for gene editing and their online tools are gathered, putting the light on its application in the study and treatment of cardiovascular and metabolic diseases.

RevDate: 2022-12-01

Sun YJ, Chen WD, Liu J, et al (2022)

A Conformational Restriction Strategy for the Control of CRISPR/Cas Gene Editing with Photoactivatable Guide RNAs.

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

The CRISPR/Cas system is one of the most powerful tools for gene editing. However, approaches for precise control of genome editing and regulatory events are still desirable. Here, we report a spatiotemporal and efficient control of CRISPR/Cas9- and Cas12a-mediated editing with conformational restricted gRNAs. This approach relied on only two or three pre-installed photo-labile substituents followed by an intramolecular cyclization, representing a robust synthetic method in comparison to the heavily modified linear gRNAs which often required extensive screening and time-consuming optimization. The tactic could direct precise cleavage of GFP and VEGFA within a predefined cutting region without notable editing leakage in live cells. We also achieved light-mediated MSTN gene editing in embryos, wherein a new bow-knot-type gRNA was constructed with excellent OFF/ON switch efficiency. Overall, our work provides a significantly new strategy in CRISPR/Cas editing with modified circular guide RNAs to precisely manipulate where and when genes are edited.

RevDate: 2022-12-04
CmpDate: 2022-12-02

Forte-Gomez HF, Gioia R, Tonelli F, et al (2022)

Structure, evolution and expression of zebrafish cartilage oligomeric matrix protein (COMP, TSP5). CRISPR-Cas mutants show a dominant phenotype in myosepta.

Frontiers in endocrinology, 13:1000662.

COMP (Cartilage Oligomeric Matrix Protein), also named thrombospondin-5, is a member of the thrombospondin family of extracellular matrix proteins. It is of clinical relevance, as in humans mutations in COMP lead to chondrodysplasias. The gene encoding zebrafish Comp is located on chromosome 11 in synteny with its mammalian orthologs. Zebrafish Comp has a domain structure identical to that of tetrapod COMP and shares 74% sequence similarity with murine COMP. Zebrafish comp is expressed from 5 hours post fertilization (hpf) on, while the protein is first detectable in somites of 11 hpf embryos. During development and in adults comp is strongly expressed in myosepta, craniofacial tendon and ligaments, around ribs and vertebra, but not in its name-giving tissue cartilage. As in mammals, zebrafish Comp forms pentamers. It is easily extracted from 5 days post fertilization (dpf) whole zebrafish. The lack of Comp expression in zebrafish cartilage implies that its cartilage function evolved recently in tetrapods. The expression in tendon and myosepta may indicate a more fundamental function, as in evolutionary distant Drosophila muscle-specific adhesion to tendon cells requires thrombospondin. A sequence encoding a calcium binding motif within the first TSP type-3 repeat of zebrafish Comp was targeted by CRISPR-Cas. The heterozygous and homozygous mutant Comp zebrafish displayed a patchy irregular Comp staining in 3 dpf myosepta, indicating a dominant phenotype. Electron microscopy revealed that the endoplasmic reticulum of myosepta fibroblasts is not affected in homozygous fish. The disorganized extracellular matrix may indicate that this mutation rather interferes with extracellular matrix assembly, similar to what is seen in a subgroup of chondrodysplasia patients. The early expression and easy detection of mutant Comp in zebrafish points to the potential of using the zebrafish model for large scale screening of small molecules that can improve secretion or function of disease-associated COMP mutants.

RevDate: 2022-11-30

Yao Y, Cao J, Wang W, et al (2022)

Highly Efficient One-Step Tagging of Endogenous Genes in Primary Cells Using CRISPR-Cas Ribonucleoproteins.

The CRISPR journal [Epub ahead of print].

Genome editing tools have simplified the generation of knock-in gene fusions, which are widely used to study proteins in their natural context. However, strategies for tagging endogenous genes in primary cells are few and inefficient. In this study, we developed a one-step endogenous gene-tagging strategy by co-delivery of clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 ribonucleoprotein complexes and chemically modified donor DNA into cells. Upon CRISPR-Cas9 blunt-end double-strand breaks, highly efficient site-specific insertion of genetic materials (3 × FLAG or eGFP) was achieved in both cell lines and primary cells. We further optimized the gene-tagging efficiency and precision by using CRISPR-Cas12a, which produces a staggered cut with a 5' overhang and thus enables precise ligation of DNA donors with a complementary 3' overhang. With high efficiency and flexibility, this platform would be extremely useful for multiplex endogenous genes tagging and further exploration of protein functions in various cell types.

RevDate: 2022-12-05
CmpDate: 2022-12-02

Simpson DM, EB Chuong (2023)

Genetic Knockout of TE Insertions by CRISPR-Cas9.

Methods in molecular biology (Clifton, N.J.), 2607:369-379.

Transposable elements (TEs) are abundant in the genome, and specific insertions may be co-opted to act as coding or noncoding functional elements. CRISPR-based genome editing technologies enable functional studies of TE insertions in cell lines. Here, we describe the use of CRISPR-Cas9 to create and validate genetic knockouts of TEs in mammalian cell lines.

RevDate: 2022-12-05
CmpDate: 2022-12-02

Weber VM, Doucet AJ, G Cristofari (2023)

Precise and Scarless Insertion of Transposable Elements by Cas9-Mediated Genome Engineering.

Methods in molecular biology (Clifton, N.J.), 2607:329-353.

Transposable element insertions can have broad effects on gene expression, ranging from new regulatory functions to pathogenic consequences by transplanting new cis-regulating elements or perturbing existing ones. Genetic manipulation of such DNA sequences can help decipher their mechanism of action. Here, we describe a CRISPR-Cas9-mediated two-step approach to precisely insert transposable elements into into the genome of cultured human cells, without scar or reporter gene. First, a double-selection cassette is inserted into the desired target locus. Once a clone containing a single copy of this cassette has been isolated, a second editing step is performed to exchange the double-selection cassette with a markerless transposable element sequence. More generally, this method can be used for knocking in any large insert without genetic markers.

RevDate: 2022-11-29

Yip CC, Sridhar S, Chan WM, et al (2022)

Development and Validation of a Novel COVID-19 nsp8 One-Tube RT-LAMP-CRISPR Assay for SARS-CoV-2 Diagnosis.

Microbiology spectrum [Epub ahead of print].

Accurate and simple diagnostic tests for coronavirus disease 2019 (COVID-19) are essential components of the pandemic response. In this study, we evaluated a one-tube reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay coupled with clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein-mediated endpoint detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in clinical samples. RT-LAMP-CRISPR is fast and affordable, does not require bulky thermocyclers, and minimizes carryover contamination risk. Results can be read either visually or with a fluorometer. RT-LAMP-CRISPR assays using primers targeting a highly expressed nsp8 gene and previously described nucleocapsid (N) gene primers were designed. The analytical characteristics and diagnostic performance of RT-LAMP-CRISPR assays were compared to those of a commercial real-time RT-PCR E gene assay. The limits of detection (LODs) of the nsp8 and N RT-LAMP-CRISPR assays were 750 and 2,000 copies/mL, which were higher than that of the commercial real-time RT-PCR assay (31.3 copies/mL). Despite the higher LOD, RT-LAMP-CRISPR assays showed diagnostic sensitivity and specificity of 98.6% and 100%, respectively, equivalent to those of the real-time RT-PCR assay (P = 0.5). The median fluorescence reading from the nsp8 assay (378.3 raw fluorescence unit [RFU] [range, 215.6 to 592.6]) was significantly higher than that of the N gene assay (342.0 RFU [range, 143.0 to 576.6]) (P < 0.0001). In conclusion, we demonstrate that RT-LAMP-CRISPR assays using primers rationally designed from highly expressed gene targets are highly sensitive, specific, and easy to perform. Such assays are a valuable asset in resource-limited settings. IMPORTANCE Accurate tests for the diagnosis of SARS-CoV-2, the virus causing coronavirus disease 2019 (COVID-19), are important for timely treatment and infection control decisions. Conventional tests such as real-time reverse transcription-PCR (RT-PCR) require specialized equipment and are expensive. On the other hand, rapid antigen tests suffer from a lack of sensitivity. In this study, we describe a novel assay format for the diagnosis of COVID-19 that is based on principles of loop-mediated isothermal amplification (LAMP) and clustered regularly interspaced short palindromic repeat (CRISPR)-Cas chemistry. A major advantage of this assay format is that it does not require expensive equipment to perform, and results can be read visually. This method proved to be fast, easy to perform, and inexpensive. The test compared well against an RT-PCR assay in terms of the ability to detect SARS-CoV-2 RNA in clinical samples. No false-positive test results were observed. The new assay format is ideal for SARS-CoV-2 diagnosis in resource-limited settings.

RevDate: 2022-12-02
CmpDate: 2022-11-30

McGrail M, Sakuma T, L Bleris (2022)

Genome editing.

Scientific reports, 12(1):20497.

Recent advances in genome editing technologies have redefined our ability to probe and precisely edit the human genome and epigenome in vitro and in vivo. More specifically, RNA-guided CRISPR/Cas systems have revolutionized the field due to their simplicity in design and adaptability across biological systems. This Collection highlights results in CRISPR/Cas technology that increase the efficiency of precision genome editing, and allow genetic manipulation in model systems traditionally intractable to site-directed gene modification.

RevDate: 2022-12-02
CmpDate: 2022-11-30

Huang Z, You X, Chen L, et al (2022)

mTORC1 links pathology in experimental models of Still's disease and macrophage activation syndrome.

Nature communications, 13(1):6915.

Still's disease is a severe inflammatory syndrome characterized by fever, skin rash and arthritis affecting children and adults. Patients with Still's disease may also develop macrophage activation syndrome, a potentially fatal complication of immune dysregulation resulting in cytokine storm. Here we show that mTORC1 (mechanistic target of rapamycin complex 1) underpins the pathology of Still's disease and macrophage activation syndrome. Single-cell RNA sequencing in a murine model of Still's disease shows preferential activation of mTORC1 in monocytes; both mTOR inhibition and monocyte depletion attenuate disease severity. Transcriptomic data from patients with Still's disease suggest decreased expression of the mTORC1 inhibitors TSC1/TSC2 and an mTORC1 gene signature that strongly correlates with disease activity and treatment response. Unrestricted activation of mTORC1 by Tsc2 deletion in mice is sufficient to trigger a Still's disease-like syndrome, including both inflammatory arthritis and macrophage activation syndrome with hemophagocytosis, a cellular manifestation that is reproduced in human monocytes by CRISPR/Cas-mediated deletion of TSC2. Consistent with this observation, hemophagocytic histiocytes from patients with macrophage activation syndrome display prominent mTORC1 activity. Our study suggests a mechanistic link of mTORC1 to inflammation that connects the pathogenesis of Still's disease and macrophage activation syndrome.

RevDate: 2022-11-28

Hu T, Ke X, Li W, et al (2022)

CRISPR/Cas12a-Enabled Multiplex Biosensing Strategy Via an Affordable and Visual Nylon Membrane Readout.

Advanced science (Weinheim, Baden-Wurttemberg, Germany) [Epub ahead of print].

Most multiplex nucleic acids detection methods require numerous reagents and high-priced instruments. The emerging clustered regularly interspaced short palindromic repeats (CRISPR)/Cas has been regarded as a promising point-of-care (POC) strategy for nucleic acids detection. However, how to achieve CRISPR/Cas multiplex biosensing remains a challenge. Here, an affordable means termed CRISPR-RDB (CRISPR-based reverse dot blot) for multiplex target detection in parallel, which possesses the advantages of high sensitivity and specificity, cost-effectiveness, instrument-free, ease to use, and visualization is reported. CRISPR-RDB integrates the trans-cleavage activity of CRISPR-Cas12a with a commercial RDB technique. It utilizes different Cas12a-crRNA complexes to separately identify multiple targets in one sample and converts targeted information into colorimetric signals on a piece of accessible nylon membrane that attaches corresponding specific-oligonucleotide probes. It has demonstrated that the versatility of CRISPR-RDB by constructing a four-channel system to simultaneously detect influenza A, influenza B, respiratory syncytial virus, and SARS-CoV-2. With a simple modification of crRNAs, the CRISPR-RDB can be modified to detect human papillomavirus, saving two-thirds of the time compared to a commercial PCR-RDB kit. Further, a user-friendly microchip system for convenient use, as well as a smartphone app for signal interpretation, is engineered. CRISPR-RDB represents a desirable option for multiplexed biosensing and on-site diagnosis.

RevDate: 2022-12-05

Patra P, B R D, Kundu P, et al (2022)

Recent advances in machine learning applications in metabolic engineering.

Biotechnology advances, 62:108069 pii:S0734-9750(22)00165-3 [Epub ahead of print].

Metabolic engineering encompasses several widely-used strategies, which currently hold a high seat in the field of biotechnology when its potential is manifesting through a plethora of research and commercial products with a strong societal impact. The genomic revolution that occurred almost three decades ago has initiated the generation of large omics-datasets which has helped in gaining a better understanding of cellular behavior. The itinerary of metabolic engineering that has occurred based on these large datasets has allowed researchers to gain detailed insights and a reasonable understanding of the intricacies of biosystems. However, the existing trail-and-error approaches for metabolic engineering are laborious and time-intensive when it comes to the production of target compounds with high yields through genetic manipulations in host organisms. Machine learning (ML) coupled with the available metabolic engineering test instances and omics data brings a comprehensive and multidisciplinary approach that enables scientists to evaluate various parameters for effective strain design. This vast amount of biological data should be standardized through knowledge engineering to train different ML models for providing accurate predictions in gene circuits designing, modification of proteins, optimization of bioprocess parameters for scaling up, and screening of hyper-producing robust cell factories. This review briefs on the premise of ML, followed by mentioning various ML methods and algorithms alongside the numerous omics datasets available to train ML models for predicting metabolic outcomes with high-accuracy. The combinative interplay between the ML algorithms and biological datasets through knowledge engineering have guided the recent advancements in applications such as CRISPR/Cas systems, gene circuits, protein engineering, metabolic pathway reconstruction, and bioprocess engineering. Finally, this review addresses the probable challenges of applying ML in metabolic engineering which will guide the researchers toward novel techniques to overcome the limitations.

RevDate: 2022-11-30
CmpDate: 2022-11-30

Takacs CN, Nakajima Y, Haber JE, et al (2022)

Cas9-mediated endogenous plasmid loss in Borrelia burgdorferi.

PloS one, 17(11):e0278151 pii:PONE-D-22-25882.

The spirochete Borrelia burgdorferi, which causes Lyme disease, has the most segmented genome among known bacteria. In addition to a linear chromosome, the B. burgdorferi genome contains over 20 linear and circular endogenous plasmids. While many of these plasmids are dispensable under in vitro culture conditions, they are maintained during the natural life cycle of the pathogen. Plasmid-encoded functions are required for colonization of the tick vector, transmission to the vertebrate host, and evasion of host immune defenses. Different Borrelia strains can vary substantially in the type of plasmids they carry. The gene composition within the same type of plasmid can also differ from strain to strain, impeding the inference of plasmid function from one strain to another. To facilitate the investigation of the role of specific B. burgdorferi plasmids, we developed a Cas9-based approach that targets a plasmid for removal. As a proof-of-principle, we showed that targeting wild-type Cas9 to several loci on the endogenous plasmids lp25 or lp28-1 of the B. burgdorferi type strain B31 results in sgRNA-specific plasmid loss even when homologous sequences (i.e., potential sequence donors for DNA recombination) are present nearby. Cas9 nickase versions, Cas9D10A or Cas9H840A, also cause plasmid loss, though not as robustly. Thus, sgRNA-directed Cas9 DNA cleavage provides a highly efficient way to eliminate B. burgdorferi endogenous plasmids that are non-essential in axenic culture.

RevDate: 2022-12-05
CmpDate: 2022-11-30

Xie Y, Wang M, Gu L, et al (2022)

CRISPR/Cas9-mediated knock-in strategy at the Rosa26 locus in cattle fetal fibroblasts.

PloS one, 17(11):e0276811 pii:PONE-D-22-09714.

The genetic modification of cattle has many agricultural and biomedical applications. However, random integration often leads to the unstable or differentially expression of the exogenous genes, which limit the application and development of transgenic technologies. Finding a safe locus suitable for site-specific insertion and efficient expression of exogenous genes is a good way to overcome these hurdles. In this study, we efficiently integrated three targeted vector into the cattle Rosa26 (cRosa26) by CRISPR/Cas9 technology in which EGFP was driven by CAG, EF1a, PGK and cRosa26 endogenous promoter respectively. The CRISPR/Cas9 knock-in system allows highly efficient gene insertion of different expression units at the cRosa26 locus. We also find that in the four cell lines, EGFP was stable expressed at different times, and the CAG promoter has the highest activity to activate the expression of EGFP, when compared with the cRosa26, EF1a and PGK promoter. Our results proved that cRosa26 was a locus that could integrate different expression units efficiently, and supported the friendly expression of different expression units. Our findings described here will be useful for a variety of studies using cattle.

RevDate: 2022-11-29

Bhoobalan-Chitty Y, Duan X, X Peng (2022)

High-MOI induces rapid CRISPR spacer acquisition in Sulfolobus from an acr deficient virus.

microPublication biology, 2022:.

Spacer acquisition, the first step in CRISPR-Cas adaptive immunity, plays a critical role in establishing and strengthening host defense against mobile genetic elements (MGEs). Here we present a host-virus system, where an increase in the multiplicity of infection (MOI), of a CRISPR-Cas susceptible virus, forces rapid spacer acquisition in the Sulfolobus islandicus LAL14/1 CRISPR arrays. Spacer acquisition was observed as early as 30 minutes post infection, with the newly acquired spacers uniformly distributed across the genome of the virus. Although the newly acquired spacers were predominantly effective only against the CRISPR-Cas susceptible mutant virus, we were able to isolate a host mutant with a novel spacer which provides immunity against the multiple Acr encoding wildtype virus, Sulfolobus islandicus rod-shaped virus 2 (SIRV2).

RevDate: 2022-11-30

Liu S, Xie T, Pei X, et al (2023)

CRISPR-Cas12a coupled with universal gold nanoparticle strand-displacement probe for rapid and sensitive visual SARS-CoV-2 detection.

Sensors and actuators. B, Chemical, 377:133009.

Point of care (POC) diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are particularly significant for preventing transmission of coronavirus disease 2019 (COVID-19) by any user at any given time and place. CRISPR/Cas-assisted SARS-CoV-2 assays are viewed as supplemental to RT-PCR due to simple operation, convenient use and low cost. However, most current CRISPR molecular diagnostics based on fluorescence measurement increased the difficulty of POC test with need of the additional light sources. Some instrument-free visual detection with the naked eye has limitations in probe universality. Herein, we developed a universal, rapid, sensitive and specific SARS-CoV-2 POC test that combines the outstanding DNase activity of Cas12a with universal AuNPs strand-displacement probe. The oligo trigger, which is the switch the AuNPs of the strand-displacement probe, is declined as a result of Cas12a recognition and digestion. The amount of released AuNPs produced color change which can be visual with the naked eye and assessed by UV-Vis spectrometer for quantitative detection. Furthermore, a low-cost hand warmer is used as an incubator for the visual assay, enabling an instrument-free, visual SARS-CoV-2 detection within 20 min. A real coronavirus GX/P2V instead of SARS-CoV-2 were chosen for practical application validation. After rapid virus RNA extraction and RT-PCR amplification, a minimum of 2.7 × 10[2] copies/mL was obtained successfully. The modular design can be applied to many nucleic acid detection applications, such as viruses, bacteria, species, etc., by simply modifying the crRNA, showing great potential in POC diagnosis.

RevDate: 2022-11-29

Park CH, Jeoung YH, Zhang L, et al (2022)

Establishment, characterization, and validation of novel porcine embryonic fibroblasts as a potential source for genetic modification.

Frontiers in cell and developmental biology, 10:1059710.

Fibroblasts are the common cell type in the connective tissue-the most abundant tissue type in the body. Fibroblasts are widely used for cell culture, for the generation of induced pluripotent stem cells (iPSCs), and as nuclear donors for somatic cell nuclear transfer (SCNT). We report for the first time, the derivation of embryonic fibroblasts (EFs) from porcine embryonic outgrowths, which share similarities in morphology, culture characteristics, molecular markers, and transcriptional profile to fetal fibroblasts (FFs). We demonstrated the efficient use of EFs as nuclear donors in SCNT, for enhanced post-blastocyst development, implantation, and pregnancy outcomes. We further validated EFs as a source for CRISPR/Cas genome editing with overall editing frequencies comparable to that of FFs. Taken together, we established an alternative and efficient pipeline for genome editing and for the generation of genetically engineered animals.

RevDate: 2022-11-29

Cobos M, Condori RC, Grandez MA, et al (2022)

Genomic analysis and biochemical profiling of an unaxenic strain of Synechococcus sp. isolated from the Peruvian Amazon Basin region.

Frontiers in genetics, 13:973324.

Cyanobacteria are diverse photosynthetic microorganisms able to produce a myriad of bioactive chemicals. To make possible the rational exploitation of these microorganisms, it is fundamental to know their metabolic capabilities and to have genomic resources. In this context, the main objective of this research was to determine the genome features and the biochemical profile of Synechococcus sp. UCP002. The cyanobacterium was isolated from the Peruvian Amazon Basin region and cultured in BG-11 medium. Growth parameters, genome features, and the biochemical profile of the cyanobacterium were determined using standardized methods. Synechococcus sp. UCP002 had a specific growth rate of 0.086 ± 0.008 μ and a doubling time of 8.08 ± 0.78 h. The complete genome of Synechococcus sp. UCP002 had a size of ∼3.53 Mb with a high coverage (∼200x), and its quality parameters were acceptable (completeness = 99.29%, complete and single-copy genes = 97.5%, and contamination = 0.35%). Additionally, the cyanobacterium had six plasmids ranging from 24 to 200 kbp. The annotated genome revealed ∼3,422 genes, ∼ 3,374 protein-coding genes (with ∼41.31% hypothetical protein-coding genes), two CRISPR Cas systems, and 61 non-coding RNAs. Both the genome and plasmids had the genes for prokaryotic defense systems. Additionally, the genome had genes coding the transcription factors of the metalloregulator ArsR/SmtB family, involved in sensing heavy metal pollution. The biochemical profile showed primary nutrients, essential amino acids, some essential fatty acids, pigments (e.g., all-trans-β-carotene, chlorophyll a, and phycocyanin), and phenolic compounds. In conclusion, Synechococcus sp. UCP002 shows biotechnological potential to produce human and animal nutrients and raw materials for biofuels and could be a new source of genes for synthetic biological applications.

RevDate: 2022-11-30
CmpDate: 2022-11-29

Yoshida M, Saito T, Takayanagi Y, et al (2022)

Necessity of integrated genomic analysis to establish a designed knock-in mouse from CRISPR-Cas9-induced mutants.

Scientific reports, 12(1):20390.

The CRISPR-Cas9 method for generation of knock-in mutations in rodent embryos yields many F0 generation candidates that may have the designed mutations. The first task for selection of promising F0 generations is to analyze genomic DNA which likely contains a mixture of designed and unexpected mutations. In our study, while generating Prlhr-Venus knock-in reporter mice, we found that genomic rearrangements near the targeted knock-in allele, tandem multicopies at a target allele locus, and mosaic genotypes for two different knock-in alleles occurred in addition to the designed knock-in mutation in the F0 generation. Conventional PCR and genomic sequencing were not able to detect mosaicism nor discriminate between the designed one-copy knock-in mutant and a multicopy-inserted mutant. However, by using a combination of Southern blotting and the next-generation sequencing-based RAISING method, these mutants were successfully detected in the F0 generation. In the F1 and F2 generations, droplet digital PCR assisted in establishing the strain, although a multicopy was falsely detected as one copy by analysis of the F0 generation. Thus, the combination of these methods allowed us to select promising F0 generations and facilitated establishment of the designed strain. We emphasize that focusing only on positive evidence of knock-in can lead to erroneous selection of undesirable strains.

RevDate: 2022-11-30
CmpDate: 2022-11-29

Ianevski A, Ahmad S, Anunnitipat K, et al (2022)

Seven classes of antiviral agents.

Cellular and molecular life sciences : CMLS, 79(12):605.

The viral epidemics and pandemics have stimulated the development of known and the discovery of novel antiviral agents. About a hundred mono- and combination antiviral drugs have been already approved, whereas thousands are in development. Here, we briefly reviewed 7 classes of antiviral agents: neutralizing antibodies, neutralizing recombinant soluble human receptors, antiviral CRISPR/Cas systems, interferons, antiviral peptides, antiviral nucleic acid polymers, and antiviral small molecules. Interferons and some small molecules alone or in combinations possess broad-spectrum antiviral activity, which could be beneficial for treatment of emerging and re-emerging viral infections.

RevDate: 2022-11-29
CmpDate: 2022-11-29

Liu Z, Chen S, Xie W, et al (2022)

Versatile and efficient genome editing with Neisseria cinerea Cas9.

Communications biology, 5(1):1296.

The CRISPR/Cas9 system is a versatile genome editing platform in biotechnology and therapeutics. However, the requirement of protospacer adjacent motifs (PAMs) limits the genome targeting scope. To expand this repertoire, we revisited and engineered a compact Cas9 orthologue derived from Neisseria cinerea (NcCas9) for efficient genome editing in mammal cells. We demonstrated that NcCas9 generates genome editing at target sites with N4GYAT (Y = T/C) PAM which cannot be recognized by existing Cas9s. By optimizing the NcCas9 architecture and its spacer length, editing efficacy of NcCas9 was further improved in human cells. In addition, the NcCas9-derived Base editors can efficiently generate base conversions. Six anti-CRISPR (Acr) proteins were identified as off-switches for NcCas9. Moreover, NcCas9 successfully generated efficient editing of mouse embryos by microinjection of NcCas9 mRNA and the corresponding sgRNA. Thus, the NcCas9 holds the potential to broaden the CRISPR/Cas9 toolsets for efficient gene modifications and therapeutic applications.

RevDate: 2022-12-03

Mingarro G, ML Del Olmo (2022)

Improvements in the genetic editing technologies: CRISPR-Cas and beyond.

Gene, 852:147064 pii:S0378-1119(22)00884-8 [Epub ahead of print].

Gene editing is a great hope not only for the scientific community, but also for society in general. This is due to its potential therapeutic applications that would allow curing diseases of genetic origin. The first realistic approach to achieve this goal was the development of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) tools. This review deals with some of the improvements that have been designed to obtain more efficient and safer genome editing. Initial CRISPR-Cas (CRISPR associated) editing systems yield low efficiency and undesired editing products. To solve these problems, new approaches emerged, such as the creation of base editors. Recent discoveries have led to the development of many interesting alternatives, such as the CRISPR-associated transposable systems, which open the range by generating guided insertions, or the discovery of other programmable nucleases like the IscB family, which greatly increase the range of proteins available for editing uses. Also, to address the limitations of base editors, prime editors were created; this novel system, despite having some disadvantages compared to base editor systems, has the potential to generate all the possible point mutations. On the other hand, dual prime editing systems (like twin and homologous 3' extension-mediated prime editors) have been developed to create targeted insertions and enhance the editing outcomes, respectively. Furthermore, advances in gene editing do not reside solely in CRISPR-dependent systems, as we will discuss when treating the Replication Interrupted Template-Driven DNA Modification technique.

RevDate: 2022-11-26

Schmitz M, Querques I, Oberli S, et al (2022)

Structural basis for the assembly of the type V CRISPR-associated transposon complex.

Cell pii:S0092-8674(22)01421-0 [Epub ahead of print].

CRISPR-Cas systems have been co-opted by Tn7-like transposable elements to direct RNA-guided transposition. Type V-K CRISPR-associated transposons rely on the concerted activities of the pseudonuclease Cas12k, the AAA+ ATPase TnsC, the Zn-finger protein TniQ, and the transposase TnsB. Here we present a cryo-electron microscopic structure of a target DNA-bound Cas12k-transposon recruitment complex comprised of RNA-guided Cas12k, TniQ, a polymeric TnsC filament and, unexpectedly, the ribosomal protein S15. Complex assembly, mediated by a network of interactions involving the guide RNA, TniQ, and S15, results in R-loop completion. TniQ contacts two TnsC protomers at the Cas12k-proximal filament end, likely nucleating its polymerization. Transposition activity assays corroborate our structural findings, implying that S15 is a bona fide component of the type V crRNA-guided transposon machinery. Altogether, our work uncovers key mechanistic aspects underpinning RNA-mediated assembly of CRISPR-associated transposons to guide their development as programmable tools for site-specific insertion of large DNA payloads.

RevDate: 2022-11-29
CmpDate: 2022-11-29

Zhuo C, Ruan Q, Zhao X, et al (2022)

CXCL1 promotes colon cancer progression through activation of NF-κB/P300 signaling pathway.

Biology direct, 17(1):34.

BACKGROUND: The upregulated expression of CXCL1 has been validated in colorectal cancer patients. As a potential biotherapeutic target for colorectal cancer, the mechanism by which CXCL1 affects the development of colorectal cancer is not clear.

METHODS: Expression data of CXCL1 in colorectal cancer were obtained from the GEO database and verified using the GEPIA database and the TIMER 2.0 database. Knockout and overexpression of CXCL1 in colorectal cancer cells by CRISPR/Cas and "Sleeping Beauty" transposon-mediated gene editing techniques. Cell biological function was demonstrated by CCK-8, transwell chamber and Colony formation assay. RT-qPCR and Western Blot assays measured RNA and protein expression. Protein localization and expression were measured by immunohistochemistry and immunofluorescence.

RESULTS: Bioinformatics analysis showed significant overexpression of CXCL1 in the colorectal cancer tissues compared to normal human tissues, and identified CXCL1 as a potential therapeutic target for colorectal cancer. We demonstrate that CXCL1 promotes the proliferation and migration of colon cancer cells and has a facilitative effect on tumor angiogenesis. Furthermore, CXCL1 elevation promoted the migration of M2-tumor associated macrophages (TAMs) while disrupting the aggregation of CD4+ and CD8+ T cells at tumor sites. Mechanistic studies suggested that CXCL1 activates the NF-κB pathway. In the in vivo colon cancer transplantation tumor model, treatment with the P300 inhibitor C646 significantly inhibited the growth of CXCL1-overexpressing colon cancer.

CONCLUSION: CXCL1 promotes colon cancer development through activation of NF-κB/P300, and that CXCL1-based therapy is a potential novel strategy to prevent colon cancer development.

RevDate: 2022-11-29
CmpDate: 2022-11-29

Pappadà M, Bonuccelli O, Buratto M, et al (2022)

Suppressing gain-of-function proteins via CRISPR/Cas9 system in SCA1 cells.

Scientific reports, 12(1):20285.

SCAs are autosomal dominant neurodegenerative disorders caused by a gain-of-function protein with toxic activities, containing an expanded polyQ tract in the coding region. There are no treatments available to delay the onset, stop or slow down the progression of these pathologies. In this work we focus our attention on SCA1 which is one of the most common genotypes circulating in Italy. Here, we develop a CRISPR/Cas9-based approach to reduce both forms of the ATXN1 protein, normal and mutated with expanded polyQ. We started with the screening of 10 different sgRNAs able to target Exon 8 of the ATXN1 gene. The two most promising sgRNAs were validated in fibroblasts isolated from SCA1 patients, following the identification of the best transfection method for this type of cell. Our silencing approach significantly downregulated the expression of ataxin1, due to large deletions and the introduction of small changes in the ATXN1 gene, evidenced by NGS analysis, without major effects on cell viability. Furthermore, very few significant guide RNA-dependent off-target effects were observed. These preliminary results not only allowed us to identify the best transfection method for SCA1 fibroblasts, but strongly support CRISPR/Cas9 as a promising approach for the treatment of expanded polyQ diseases. Further investigations will be needed to verify the efficacy of our silencing system in SCA1 neurons and animal models.

RevDate: 2022-11-29
CmpDate: 2022-11-29

He X, Zhang Z, Xue J, et al (2022)

Low-dose AAV-CRISPR-mediated liver-specific knock-in restored hemostasis in neonatal hemophilia B mice with subtle antibody response.

Nature communications, 13(1):7275.

AAV-delivered CRISPR/Cas9 (AAV-CRISPR) has shown promising potentials in preclinical models to efficiently insert therapeutic gene sequences in somatic tissues. However, the AAV input doses required were prohibitively high and posed serious risk of toxicity. Here, we performed AAV-CRISPR mediated homology-independent knock-in at a new target site in mAlb 3'UTR and demonstrated that single dose of AAVs enabled long-term integration and expression of hF9 transgene in both adult and neonatal hemophilia B mice (mF9 -/-), yielding high levels of circulating human Factor IX (hFIX) and stable hemostasis restoration during entire 48-week observation period. Furthermore, we achieved hemostasis correction with a significantly lower AAV dose (2 × 10[9] vg/neonate and 1 × 10[10] vg/adult mouse) through liver-specific gene knock-in using hyperactive hF9[R338L] variant. The plasma antibodies against Cas9 and AAV in the neonatal mice receiving low-dose AAV-CRISPR were negligible, which lent support to the development of AAV-CRISPR mediated somatic knock-in for treating inherited diseases.

RevDate: 2022-11-28

Fu Q, Hu L, Shen T, et al (2022)

Recent Advances in Gene Therapy for Familial Hypercholesterolemia: An Update Review.

Journal of clinical medicine, 11(22): pii:jcm11226773.

(1) Background: Existing lipid-lowering therapies have difficulty in achieving lipid target levels in patients with familial hypercholesterolemia (FH), especially in the treatment of patients with homozygous familial hypercholesterolemia. (2) Method: All of the literature data containing "Familial hypercholesterolemia" and "Gene Therapy" in PubMed and Clinical Trials from 2018 to 2022 were selected. (3) Results: The rapid development of gene therapy technology in recent years is expected to change the treatment status of FH patients. As emerging gene therapy vectors, the optimized adeno-associated viruses, exosomes, and lipid nanoparticles have demonstrated an improved safety and higher transfection efficiency. Various RNA-targeted therapies are in phase 1-3 clinical trials, such as small interfering RNA-based drugs inclisiran, ARO-ANG3, ARO-APOC3, olpasiran, SLN360, and antisense oligonucleotide-based drugs AZD8233, vupanorsen, volanesorsen, IONIS-APO(a)Rx, etc., all of which have demonstrated excellent lipid-lowering effects. With gene editing technologies, such as CRISPR-Cas 9 and meganuclease, completing animal experiments in mice or cynomolgus monkeys and demonstrating lasting lipid-lowering effects, patients with FH are expected to reach a permanent cure in the future. (4) Conclusion: Gene therapy is being widely used for the lipid-lowering treatment of FH patients and has shown excellent therapeutic promise, but the current delivery efficiency, economic burden, immunogenicity and the precision of gene therapy can be further optimized.

RevDate: 2022-11-29
CmpDate: 2022-11-29

Kandasamy S, Yoo J, Yun J, et al (2022)

Probiogenomic In-Silico Analysis and Safety Assessment of Lactiplantibacillus plantarum DJF10 Strain Isolated from Korean Raw Milk.

International journal of molecular sciences, 23(22): pii:ijms232214494.

The whole genome sequence of Lactiplantibacillus plantarum DJF10, isolated from Korean raw milk, is reported, along with its genomic analysis of probiotics and safety features. The genome consists of 29 contigs with a total length of 3,385,113 bp and a GC content of 44.3%. The average nucleotide identity and whole genome phylogenetic analysis showed the strain belongs to Lactiplantibacillus plantarum with 99% identity. Genome annotation using Prokka predicted a total of 3235 genes, including 3168 protein-coding sequences (CDS), 59 tRNAs, 7 rRNAs and 1 tmRNA. The functional annotation results by EggNOG and KEGG showed a high number of genes associated with genetic information and processing, transport and metabolism, suggesting the strain's ability to adapt to several environments. Various genes conferring probiotic characteristics, including genes related to stress adaptation to the gastrointestinal tract, biosynthesis of vitamins, cell adhesion and production of bacteriocins, were identified. The CAZyme analysis detected 98 genes distributed under five CAZymes classes. In addition, several genes encoding carbohydrate transport and metabolism were identified. The genome also revealed the presence of insertion sequences, genomic islands, phage regions, CRISPR-cas regions, and the absence of virulence and toxin genes. However, the presence of hemolysin and antibiotic-resistance-related genes detected in the KEGG search needs further experimental validation to confirm the safety of the strain. The presence of two bacteriocin clusters, sactipeptide and plantaricin J, as detected by the BAGEL 4 webserver, confer the higher antimicrobial potential of DJF10. Altogether, the analyses in this study performed highlight this strain's functional characteristics. However, further in vitro and in vivo studies are required on the safety assurance and potential application of L. plantarum DJF10 as a probiotic agent.

RevDate: 2022-11-29
CmpDate: 2022-11-29

Yu H, Wang Y, Fu F, et al (2022)

Transgenic Improvement for Biotic Resistance of Crops.

International journal of molecular sciences, 23(22): pii:ijms232214370.

Biotic constraints, including pathogenic fungi, viruses and bacteria, herbivory insects, as well as parasitic nematodes, cause significant yield loss and quality deterioration of crops. The effect of conventional management of these biotic constraints is limited. The advances in transgenic technologies provide a direct and directional approach to improve crops for biotic resistance. More than a hundred transgenic events and hundreds of cultivars resistant to herbivory insects, pathogenic viruses, and fungi have been developed by the heterologous expression of exogenous genes and RNAi, authorized for cultivation and market, and resulted in a significant reduction in yield loss and quality deterioration. However, the exploration of transgenic improvement for resistance to bacteria and nematodes by overexpression of endogenous genes and RNAi remains at the testing stage. Recent advances in RNAi and CRISPR/Cas technologies open up possibilities to improve the resistance of crops to pathogenic bacteria and plant parasitic nematodes, as well as other biotic constraints.

RevDate: 2022-11-29
CmpDate: 2022-11-29

Wang Q, Bai H, Zada A, et al (2022)

DORN1 Is Involved in Drought Stress Tolerance through a Ca[2+]-Dependent Pathway.

International journal of molecular sciences, 23(22): pii:ijms232214213.

Water shortages caused by climate change seriously threaten the survival and production of plants and are also one of the major environmental pressures faced by plants. DORN1 was the first identified purinoceptor for the plant response to extracellular ATP. It has been established that DORN1 could play key roles in a series of biological activities in plants. However, the biological roles of DORN1 and the mechanism remain unclear under drought stress conditions in plants. Here, DORN1 was targeted for knockout by using the CRISPR/Cas 9 system. It was found that the loss function of DORN1 resulted in a significant decrease in the effective quantum yield of PSII [Y(II)], the photochemical quenching coefficient (qP), and the rate of photosynthetic electron transport through PSII (ETR), which reflected plants' photochemical efficiency. Whereas Y(NO) values showed obvious enhancement under drought stress conditions. Further experimental results showed that the Y(II), qP, and ETR, which reflect plants' photochemical efficiency, increased significantly with CaCl2 treatment. These results indicated that the drought tolerance of the mutant was decreased, and the exogenous application of calcium ions could effectively promote the drought tolerance of the dorn1 mutant. Transpiration loss controlled by stomata is closely related to drought tolerance, further, we examined the transpirational water loss in dorn1 and found that it was greater than wild-type (WT). Besides, the dorn1 mutant's stomatal aperture significantly increased compared with the WT and the stomata of dorn1 mutant plants tend to close after CaCl2 treatment. Taken together, our results show that DORN1 plays a key role in drought stress tolerance in plants, which may depend on calcium and calcium-related signaling pathways.

RevDate: 2022-11-29
CmpDate: 2022-11-29

Li C, Gong C, Wu J, et al (2022)

Improvement of Rice Agronomic Traits by Editing Type-B Response Regulators.

International journal of molecular sciences, 23(22): pii:ijms232214165.

Type-B response regulator proteins in rice contain a conserved receiver domain, followed by a GARP DNA binding domain and a longer C-terminus. Some type-B response regulators such as RR21, RR22 and RR23 are involved in the development of rice leaf, root, flower and trichome. In this study, to evaluate the application potential of type-B response regulators in rice genetic improvement, thirteen type-B response regulator genes in rice were respectively knocked out by using CRISPR/Cas9 genome editing technology. Two guide RNAs (gRNAs) were simultaneously expressed on a knockout vector to mutate one gene. T0 transformed plants were used to screen the plants with deletion of large DNA fragments through PCR with specific primers. The mutants of CRISPR/Cas9 gene editing were detected by Cas9 specific primer in the T1 generation, and homozygous mutants without Cas9 were screened, whose target regions were confirmed by sequencing. Mutant materials of 12 OsRRs were obtained, except for RR24. Preliminary phenotypic observation revealed variations of various important traits in different mutant materials, including plant height, tiller number, tillering angle, heading date, panicle length and yield. The osrr30 mutant in the T2 generation was then further examined. As a result, the heading date of the osrr30 mutant was delayed by about 18 d, while the yield was increased by about 30%, and the chalkiness was significantly reduced compared with those of the wild-type under field high temperature stress. These results indicated that osrr30 has great application value in rice breeding. Our findings suggest that it is feasible to perform genetic improvement of rice by editing the type-B response regulators.

RevDate: 2022-11-29
CmpDate: 2022-11-29

Pechenov PY, Garagulya DA, Stanovov DS, et al (2022)

New Effective Method of Lactococcus Genome Editing Using Guide RNA-Directed Transposition.

International journal of molecular sciences, 23(22): pii:ijms232213978.

Lactococcus lactis is an important industrial microorganism and a widely used model object for research in the field of lactic acid bacteria (LAB) biology. The development of new L. lactis and related LAB strains with improved properties, including phage-resistant strains for dairy fermentation, LAB-based vaccines or strains with altered genotypes for research purposes, are hindered by the lack of genome-editing tools that allow for the easy and straightforward incorporation of a significant amount of the novel genetic material, such as large genes or operons, into the chromosomes of these bacteria. We recently employed a suggested system based on the CRISPR-Cas-associated transposon for the editing of the L. lactis genome. After the in-depth redesign of the system, we were able to achieve the stable incorporation of the fragments that were sized up to 10 kbp into the L. lactis beta-galactosidase gene. The efficiency of editing under the optimized conditions were 2 × 10[-4] and 4 × 10[-5] for 1 kbp and 10 kbp, respectively, which are sufficient for fast and easy modifications if a positive selection marker can be used.

RevDate: 2022-11-29
CmpDate: 2022-11-29

Baranova SV, Zhdanova PV, Lomzov AA, et al (2022)

Structure- and Content-Dependent Efficiency of Cas9-Assisted DNA Cleavage in Genome-Editing Systems.

International journal of molecular sciences, 23(22): pii:ijms232213889.

Genome-editing systems, being some of the key tools of molecular biologists, represent a reasonable hope for progress in the field of personalized medicine. A major problem with such systems is their nonideal accuracy and insufficient selectivity. The selectivity of CRISPR-Cas9 systems can be improved in several ways. One efficient way is the proper selection of the consensus sequence of the DNA to be cleaved. In the present work, we attempted to evaluate the effect of formed non-Watson-Crick pairs in a DNA duplex on the efficiency of DNA cleavage in terms of the influence of the structure of the formed partially complementary pairs. We also studied the effect of the location of such pairs in DNA relative to the PAM (protospacer-adjacent motif) on the cleavage efficiency. We believe that the stabilization of the Cas9-sgRNA complex with a DNA substrate containing noncomplementary pairs is due to loop reorganization in the RuvC domain of the enzyme. In addition, PAM-proximal mismatches in the DNA substrate lower enzyme efficiency because the "seed" region is involved in binding and cleavage, whereas PAM-distal mismatches have no significant impact on target DNA cleavage. Our data suggest that in the case of short duplexes with mismatches, the stages of recognition and binding of dsDNA substrates by the enzyme determine the reaction rate and time rather than the thermodynamic parameters affected by the "unwinding" of DNA. The results will provide a theoretical basis for predicting the efficiency and accuracy of CRISPR-Cas9 systems at cleaving target DNA.

RevDate: 2022-11-29
CmpDate: 2022-11-29

Nasrallah A, Sulpice E, Kobaisi F, et al (2022)

CRISPR-Cas9 Technology for the Creation of Biological Avatars Capable of Modeling and Treating Pathologies: From Discovery to the Latest Improvements.

Cells, 11(22): pii:cells11223615.

This is a spectacular moment for genetics to evolve in genome editing, which encompasses the precise alteration of the cellular DNA sequences within various species. One of the most fascinating genome-editing technologies currently available is Clustered Regularly Interspaced Palindromic Repeats (CRISPR) and its associated protein 9 (CRISPR-Cas9), which have integrated deeply into the research field within a short period due to its effectiveness. It became a standard tool utilized in a broad spectrum of biological and therapeutic applications. Furthermore, reliable disease models are required to improve the quality of healthcare. CRISPR-Cas9 has the potential to diversify our knowledge in genetics by generating cellular models, which can mimic various human diseases to better understand the disease consequences and develop new treatments. Precision in genome editing offered by CRISPR-Cas9 is now paving the way for gene therapy to expand in clinical trials to treat several genetic diseases in a wide range of species. This review article will discuss genome-editing tools: CRISPR-Cas9, Zinc Finger Nucleases (ZFNs), and Transcription Activator-Like Effector Nucleases (TALENs). It will also encompass the importance of CRISPR-Cas9 technology in generating cellular disease models for novel therapeutics, its applications in gene therapy, and challenges with novel strategies to enhance its specificity.

RevDate: 2022-11-29
CmpDate: 2022-11-29

Yang Y, Li D, Wan F, et al (2022)

Identification and Analysis of Small Molecule Inhibitors of CRISPR-Cas9 in Human Cells.

Cells, 11(22): pii:cells11223574.

Genome editing tools based on CRISPR-Cas systems can repair genetic mutations in situ; however, off-target effects and DNA damage lesions that result from genome editing remain major roadblocks to its full clinical implementation. Protein and chemical inhibitors of CRISPR-Cas systems may reduce off-target effects and DNA damage. Here we describe the identification of several lead chemical inhibitors that could specifically inhibit the activity of Streptococcus pyogenes Cas9 (SpCas9). In addition, we obtained derivatives of lead inhibitors that could penetrate the cell membrane and inhibit SpCas9 in cellulo. Two of these compounds, SP2 and SP24, were able to improve the specificity of SpCas9 in cellulo at low-micromolar concentration. Furthermore, microscale thermophoresis (MST) assays showed that SP24 might inhibit SpCas9 activity by interacting with both the SpCas9 protein and the SpCas9-gRNA ribonucleoprotein complex. Taken together, SP24 is a novel chemical inhibitor of SpCas9 which has the potential to enhance therapies that utilize SpCas9.

RevDate: 2022-11-29
CmpDate: 2022-11-29

Gu J, Wang J, Bi H, et al (2022)

CRISPR/Cas9-Mediated Mutagenesis of Sex-Specific Doublesex Splicing Variants Leads to Sterility in Spodoptera frugiperda, a Global Invasive Pest.

Cells, 11(22): pii:cells11223557.

Spodoptera frugiperda (J. E. Smith), an emerging invasive pest worldwide, has posed a serious agricultural threat to the newly invaded areas. Although somatic sex differentiation is fundamentally conserved among insects, the sex determination cascade in S. frugiperda is largely unknown. In this study, we cloned and functionally characterized Doublesex (dsx), a "molecular switch" modulating sexual dimorphism in S. frugiperda using male- and female-specific isoforms. Given that Lepidoptera is recalcitrant to RNAi, CRISPR/Cas9-mediated mutagenesis was employed to construct S. frugiperda mutants. Specifically, we designed target sites on exons 2, 4, and 5 to eliminate the common, female-specific, and male-specific regions of S. frugiperda dsx (Sfdsx), respectively. As expected, abnormal development of both the external and internal genitalia was observed during the pupal and adult stages. Interestingly, knocking out sex-specific dsx variants in S. frugiperda led to significantly reduced fecundity and fertility in adults of corresponding sex. Our combined results not only confirm the conserved function of dsx in S. frugiperda sex differentiation but also provide empirical evidence for dsx as a potential target for the Sterile Insect Technique (SIT) to combat this globally invasive pest in a sustainable and environmentally friendly way.

RevDate: 2022-11-29
CmpDate: 2022-11-29

Yagi Y, Teramoto T, Kaieda S, et al (2022)

Construction of a Versatile, Programmable RNA-Binding Protein Using Designer PPR Proteins and Its Application for Splicing Control in Mammalian Cells.

Cells, 11(22): pii:cells11223529.

RNAs play many essential roles in gene expression and are involved in various human diseases. Although genome editing technologies have been established, the engineering of sequence-specific RNA-binding proteins that manipulate particular cellular RNA molecules is immature, in contrast to nucleotide-based RNA manipulation technology, such as siRNA- and RNA-targeting CRISPR/Cas. Here, we demonstrate a versatile RNA manipulation technology using pentatricopeptide-repeat (PPR)-motif-containing proteins. First, we developed a rapid construction and evaluation method for PPR-based designer sequence-specific RNA-binding proteins. This system has enabled the steady construction of dozens of functional designer PPR proteins targeting long 18 nt RNA, which targets a single specific RNA in the mammalian transcriptome. Furthermore, the cellular functionality of the designer PPR proteins was first demonstrated by the control of alternative splicing of either a reporter gene or an endogenous CHK1 mRNA. Our results present a versatile protein-based RNA manipulation technology using PPR proteins that facilitates the understanding of unknown RNA functions and the creation of gene circuits and has potential for use in future therapeutics.

RevDate: 2022-12-07
CmpDate: 2022-12-07

Wu WY, Mohanraju P, Liao C, et al (2022)

The miniature CRISPR-Cas12m effector binds DNA to block transcription.

Molecular cell, 82(23):4487-4502.e7.

CRISPR-Cas are prokaryotic adaptive immune systems. Cas nucleases generally use CRISPR-derived RNA guides to specifically bind and cleave DNA or RNA targets. Here, we describe the experimental characterization of a bacterial CRISPR effector protein Cas12m representing subtype V-M. Despite being less than half the size of Cas12a, Cas12m catalyzes auto-processing of a crRNA guide, recognizes a 5'-TTN' protospacer-adjacent motif (PAM), and stably binds a guide-complementary double-stranded DNA (dsDNA). Cas12m has a RuvC domain with a non-canonical catalytic site and accordingly is incapable of guide-dependent cleavage of target nucleic acids. Despite lacking target cleavage activity, the high binding affinity of Cas12m to dsDNA targets allows for interference as demonstrated by its ability to protect bacteria against invading plasmids through silencing invader transcription and/or replication. Based on these molecular features, we repurposed Cas12m by fusing it to a cytidine deaminase that resulted in base editing within a distinct window.

RevDate: 2022-12-06

Shiriaeva AA, Kuznedelov K, Fedorov I, et al (2022)

Host nucleases generate prespacers for primed adaptation in the E. coli type I-E CRISPR-Cas system.

Science advances, 8(47):eabn8650.

CRISPR-Cas systems provide prokaryotes with adaptive immunity against foreign nucleic acids. In Escherichia coli, immunity is acquired upon integration of 33-bp spacers into CRISPR arrays. DNA targets complementary to spacers get degraded and serve as a source of new spacers during a process called primed adaptation. Precursors of such spacers, prespacers, are ~33-bp double-stranded DNA fragments with a ~4-nt 3' overhang. The mechanism of prespacer generation is not clear. Here, we use FragSeq and biochemical approaches to determine enzymes involved in generation of defined prespacer ends. We demonstrate that RecJ is the main exonuclease trimming 5' ends of prespacer precursors, although its activity can be partially substituted by ExoVII. The RecBCD complex allows single strand-specific RecJ to process double-stranded regions flanking prespacers. Our results reveal intricate functional interactions of genome maintenance proteins with CRISPR interference and adaptation machineries during generation of prespacers capable of integration into CRISPR arrays.

RevDate: 2022-12-02
CmpDate: 2022-11-29

Maguire JA, Gadue P, DL French (2022)

Highly Efficient CRISPR/Cas9-Mediated Genome Editing in Human Pluripotent Stem Cells.

Current protocols, 2(11):e590.

Human pluripotent stem cells hold tremendous potential for both basic biology and cell-based therapies for a wide variety of diseases. The ability to manipulate the genome of these cells using the CRISPR/Cas9 technology has expanded this potential by providing a valuable tool to engineer or correct disease-associated mutations. Because of the high efficiency with which CRISPR/Cas9 creates targeted double-strand breaks, a major challenge has been the introduction of precise genetic modifications on one allele without indel formation on the non-targeted allele. To overcome this obstacle, we describe use of two oligonucleotide repair templates: one expressing the sequence change and the other maintaining the normal sequence. In addition, we have streamlined both the transfection and screening methodologies to make the protocols efficient, with small numbers of cells and a limited amount of labor-intensive clone passaging. This article provides a technically simple approach for generating valuable tools to model human disease in stem cells. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Application and optimization of CRISPR-based genome editing in human pluripotent stem cells Basic Protocol 2: Genetic modification of human pluripotent stem cells using a double-oligonucleotide CRISPR/Cas9 recombination system.

RevDate: 2022-11-26

de Oliveira HL, Dias GM, BC Neves (2022)

Genome sequence of Pseudomonas aeruginosa PA1-Petro-A role model of environmental adaptation and a potential biotechnological tool.

Heliyon, 8(11):e11566.

Pseudomonas aeruginosa is a ubiquitous microorganism, capable of colonizing a wide range of habitats due to its metabolic versatility and wide adaptability to different conditions. Industrial and environmental research involving petroleum microbiology play a pivotal role in controlling many technical, operational, and environmental issues. P. aeruginosa PA1-Petro strain was isolated from oil production water in Northeastern Brazil. Herein we report the genomic sequencing and annotation of PA1-Petro, and a comparative genomics study against two widely used reference P. aeruginosa strains (PAO1 and PA14). PA1-Petro has a genome of 6,893,650 bp, the largest among the three analyzed in this study, with a 65.87% GC content. The analyzes resulted in a wide repertoire of 544 unique genes in PA1-Petro, and the highest copy numbers of common genes among the three strains (PA1-Petro, PAO1 and PA14). Unique sequences are hypothetical proteins, prophage sequences, mobile genetic elements, transcriptional regulators, metal resistance genes to copper, tellurium and arsenic, type IE CRISPR-Cas, Type VI Secretion System (T6SS)-associated proteins, and a toxin-antitoxin system. Taken together, these results provide intriguing insights on adaptive evolution within PA1-Petro genome, adding unprecedented information to the species' plasticity and ubiquitous characteristics.

RevDate: 2022-12-07
CmpDate: 2022-11-28

Nasef M, Khweis SA, JA Dunkle (2022)

The effect of crRNA-target mismatches on cOA-mediated interference by a type III-A CRISPR-Cas system.

RNA biology, 19(1):1293-1304.

CRISPR systems elicit interference when a foreign nucleic acid is detected by its ability to base-pair to crRNA. Understanding what degree of complementarity between a foreign nucleic acid and crRNA is required for interference is a central question in the study of CRISPR systems. A clear description of which target-crRNA mismatches abrogate interference in type III, Cas10-containing, CRISPR systems has proved elusive due to the complexity of the system which utilizes three distinct interference activities. We characterized the effect of target-crRNA mismatches on in vitro cyclic oligoadenylate (cOA) synthesis and in vivo in an interference assay that depends on cOA synthesis. We found that sequence context affected whether a mismatched target was recognized by crRNA both in vitro and in vivo. We also investigated how the position of a mismatch within the target-crRNA duplex affected recognition by crRNA. Our data provide support for the hypothesis that a Cas10-activating region exists in the crRNA-target duplex, that the Cas10-proximal region of the duplex is the most critical in regulating cOA synthesis. Understanding the rules governing target recognition by type III CRISPR systems is critical: as one of the most prevalent CRISPR systems in nature, it plays an important role in the survival of many genera of bacteria. Recently, type III systems were re-purposed as a sensitive and accurate molecular diagnostic tool. Understanding the rules of target recognition in this system will be critical as it is engineered for biotechnology purposes.

RevDate: 2022-12-06

Prasad D, Mani NK, DM Pandey (2022)

CRISPR/Cas technology: Opportunities for phytopathogenic viruses detection.

Journal of biotechnology, 360:211-217.

Detection and monitoring of viruses are essential for healthy plants and prosperity. Recent development in CRISPR/Cas system in diagnosis has open an avenue well suited for pathogen detection. Variety of CRISPR associated proteins are being discovered, suggesting array of application and detection strategies in diagnosis. Phytopathogenic viruses are diverse with respect to their nucleic acid compositions, which presents a challenge in developing a single device applicable for almost all viruses. The review describes about the efficient use of CRISPR/Cas Technology in diagnosis, such as SHERLOCK, DETECTR and SATORI. These methods are different in their characteristic to identify specific nucleic acids and processing the detectable signals. These technologies are in their infancy and lot of scope is there to develop commercial kits. Plant tissue culture-based industries, climate control green houses, indoor cultivation facilities etc. has been considered as few examples. This review will be beneficial for researchers seeking to develop detection mechanism based on CRISPR/Cas technology. The outcome in the form of cost-effective detection of viruses will be boon for agro-based industries, which are facing challenges through virus contamination.

RevDate: 2022-12-02
CmpDate: 2022-11-28

Al-Shayeb B, Skopintsev P, Soczek KM, et al (2022)

Diverse virus-encoded CRISPR-Cas systems include streamlined genome editors.

Cell, 185(24):4574-4586.e16.

CRISPR-Cas systems are host-encoded pathways that protect microbes from viral infection using an adaptive RNA-guided mechanism. Using genome-resolved metagenomics, we find that CRISPR systems are also encoded in diverse bacteriophages, where they occur as divergent and hypercompact anti-viral systems. Bacteriophage-encoded CRISPR systems belong to all six known CRISPR-Cas types, though some lack crucial components, suggesting alternate functional roles or host complementation. We describe multiple new Cas9-like proteins and 44 families related to type V CRISPR-Cas systems, including the Casλ RNA-guided nuclease family. Among the most divergent of the new enzymes identified, Casλ recognizes double-stranded DNA using a uniquely structured CRISPR RNA (crRNA). The Casλ-RNA-DNA structure determined by cryoelectron microscopy reveals a compact bilobed architecture capable of inducing genome editing in mammalian, Arabidopsis, and hexaploid wheat cells. These findings reveal a new source of CRISPR-Cas enzymes in phages and highlight their value as genome editors in plant and human cells.

RevDate: 2022-12-07
CmpDate: 2022-12-07

Yang H, Li F, Xue T, et al (2022)

Csm6-DNAzyme Tandem Assay for One-Pot and Sensitive Analysis of Lead Pollution and Bioaccumulation in Mice.

Analytical chemistry, 94(48):16953-16959.

Lead contamination in the environment tends to enter the food chain and further into the human body, causing serious health issues. Herein, we proposed a Csm6-DNAzyme tandem assay (termed cDNAzyme) using CRISPR/Cas III-A Csm6 and GR-5 DNAzyme, enabling one-pot and sensitive detection of lead contamination. We found that Pb[2+]-activated GR-5 DNAzyme produced cleaved substrates that can serve as the activator of Csm6, and the Csm6-DNAzyme tandem improved the sensitivity for detecting Pb[2+] by 6.1 times compared to the original GR-5 DNAzyme. Due to the high specificity of DNAzyme, the cDNAzyme assay can discriminate Pb[2+] from other bivalent and trivalent interfering ions and allowed precise detection of Pb[2+] in water and food samples. Particularly, the assay can achieve one-step, mix-and-read detection of Pb[2+] at room temperature. We used the cDNAzyme assay to investigate the accumulation of lead in mice, and found that lead accumulated at higher levels in the colon and kidney compared to the liver, and most of the lead was excreted. The cDNAzyme assay is promising to serve as analytical tools for lead-associated environmental and biosafety issues.

RevDate: 2022-11-30
CmpDate: 2022-11-30

Kato K, Okazaki S, Schmitt-Ulms C, et al (2022)

RNA-triggered protein cleavage and cell growth arrest by the type III-E CRISPR nuclease-protease.

Science (New York, N.Y.), 378(6622):882-889.

The type III-E CRISPR-Cas7-11 effector binds a CRISPR RNA (crRNA) and the putative protease Csx29 and catalyzes crRNA-guided RNA cleavage. We report cryo-electron microscopy structures of the Cas7-11-crRNA-Csx29 complex with and without target RNA (tgRNA), and demonstrate that tgRNA binding induces conformational changes in Csx29. Biochemical experiments revealed tgRNA-dependent cleavage of the accessory protein Csx30 by Csx29. Reconstitution of the system in bacteria showed that Csx30 cleavage yields toxic protein fragments that cause growth arrest, which is regulated by Csx31. Csx30 binds Csx31 and the associated sigma factor RpoE (RNA polymerase, extracytoplasmic E), suggesting that Csx30-mediated RpoE inhibition modulates the cellular response to infection. We engineered the Cas7-11-Csx29-Csx30 system for programmable RNA sensing in mammalian cells. Overall, the Cas7-11-Csx29 effector is an RNA-dependent nuclease-protease.

RevDate: 2022-11-30
CmpDate: 2022-11-30

Strecker J, Demircioglu FE, Li D, et al (2022)

RNA-activated protein cleavage with a CRISPR-associated endopeptidase.

Science (New York, N.Y.), 378(6622):874-881.

In prokaryotes, CRISPR-Cas systems provide adaptive immune responses against foreign genetic elements through RNA-guided nuclease activity. Recently, additional genes with non-nuclease functions have been found in genetic association with CRISPR systems, suggesting that there may be other RNA-guided non-nucleolytic enzymes. One such gene from Desulfonema ishimotonii encodes the TPR-CHAT protease Csx29, which is associated with the CRISPR effector Cas7-11. Here, we demonstrate that this CRISPR-associated protease (CASP) exhibits programmable RNA-activated endopeptidase activity against a sigma factor inhibitor to regulate a transcriptional response. Cryo-electron microscopy of an active and substrate-bound CASP complex reveals an allosteric activation mechanism that reorganizes Csx29 catalytic residues upon target RNA binding. This work reveals an RNA-guided function in nature that can be leveraged for RNA-sensing applications in vitro and in human cells.

RevDate: 2022-11-28
CmpDate: 2022-11-28

Atçeken N, Yigci D, Ozdalgic B, et al (2022)

CRISPR-Cas-Integrated LAMP.

Biosensors, 12(11):.

Pathogen-specific point-of-care (PoC) diagnostic tests have become an important need in the fight against infectious diseases and epidemics in recent years. PoC diagnostic tests are designed with the following parameters in mind: rapidity, accuracy, sensitivity, specificity, and ease of use. Molecular techniques are the gold standard for pathogen detection due to their accuracy and specificity. There are various limitations in adapting molecular diagnostic methods to PoC diagnostic tests. Efforts to overcome limitations are focused on the development of integrated molecular diagnostics by utilizing the latest technologies available to create the most successful PoC diagnostic platforms. With this point of view, a new generation technology was developed by combining loop-mediated isothermal amplification (LAMP) technology with clustered regularly interspaced short palindromic repeat (CRISPR)-associated (CRISPR-Cas) technology. This integrated approach benefits from the properties of LAMP technology, namely its high efficiency, short turnaround time, and the lack of need for a complex device. It also makes use of the programmable function of CRISPR-Cas technology and the collateral cleavage activity of certain Cas proteins that allow for convenient reporter detection. Thus, this combined technology enables the development of PoC diagnostic tests with high sensitivity, specificity, and ease of use without the need for complicated devices. In this review, we discuss the advantages and limitations of the CRISPR/Cas combined LAMP technology. We review current limitations to convert CRISPR combined LAMP into pathogen-specific PoC platforms. Furthermore, we point out the need to design more useful PoC platforms using microfabrication technologies by developing strategies that overcome the limitations of this new technology, reduce its complexity, and reduce the risk of contamination.

RevDate: 2022-12-06
CmpDate: 2022-12-06

Zhang Y, Lei J, Liu W, et al (2022)

Chemiluminescence resonance energy transfer as a simple and sensitive readout mode for a CRISPR/Cas12a-based biosensing platform.

The Analyst, 147(24):5687-5693.

Various CRISPR/Cas12a-based biosensing systems have been developed in the past few years, and most of these systems used Taqman probes to report signals through fluorescence resonance energy transfer (FRET). In this study, we explored chemiluminescence resonance energy transfer (CRET) as the readout mode for CRISPR/Cas12a-based biosensing. The chemiluminescence (CL) reaction of bis(2,4,6-trichlorophenyl) oxalate (TCPO) and H2O2 was used to excite the fluorophore dye of the Taqman probe. Different from FRET, CRET does not need external excitation light, which can effectively avoid autofluorescence and photobleaching. The detection limit of this CRET readout mode was estimated to be 10 pM for target DNA, which was about 8 times lower than that of the widely used FRET readout mode. These results suggest that CRET can serve as a rapid, sensitive and simple readout mode of CRISPR/Cas12-based biosensing, and can further enrich the toolbox of CRISPR/Cas12-based biosensing.

RevDate: 2022-11-25

Ding J, Schuergers N, Baehre H, et al (2022)

Enzymatic properties of CARF-domain proteins in Synechocystis sp. PCC 6803.

Frontiers in microbiology, 13:1046388.

Prokaryotic CRISPR-Cas (clustered regularly interspaced short palindromic repeats and CRISPR-associated genes) systems provide immunity against invading genetic elements such as bacteriophages and plasmids. In type III CRISPR systems, the recognition of target RNA leads to the synthesis of cyclic oligoadenylate (cOA) second messengers that activate ancillary effector proteins via their CRISPR-associated Rossmann fold (CARF) domains. Commonly, these are ribonucleases (RNases) that unspecifically degrade both invader and host RNA. To mitigate adverse effects on cell growth, ring nucleases can degrade extant cOAs to switch off ancillary nucleases. Here we show that the model organism Synechocystis sp. PCC 6803 harbors functional CARF-domain effector and ring nuclease proteins. We purified and characterized the two ancillary CARF-domain proteins from the III-D type CRISPR system of this cyanobacterium. The Csx1 homolog, SyCsx1, is a cyclic tetraadenylate(cA4)-dependent RNase with a strict specificity for cytosine nucleotides. The second CARF-domain protein with similarity to Csm6 effectors, SyCsm6, did not show RNase activity in vitro but was able to break down cOAs and attenuate SyCsx1 RNase activity. Our data suggest that the CRISPR systems in Synechocystis confer a multilayered cA4-mediated defense mechanism.

RevDate: 2022-12-02
CmpDate: 2022-12-02

Ledford H (2022)

CRISPR tools found in thousands of viruses could boost gene editing.

Nature, 612(7938):21.

RevDate: 2022-11-26
CmpDate: 2022-11-25

Huang J, Rowe D, Subedi P, et al (2022)

CRISPR-Cas12a induced DNA double-strand breaks are repaired by multiple pathways with different mutation profiles in Magnaporthe oryzae.

Nature communications, 13(1):7168.

CRISPR-Cas mediated genome engineering has revolutionized functional genomics. However, understanding of DNA repair following Cas-mediated DNA cleavage remains incomplete. Using Cas12a ribonucleoprotein genome editing in the fungal pathogen, Magnaporthe oryzae, we detail non-canonical DNA repair outcomes from hundreds of transformants. Sanger and nanopore sequencing analysis reveals significant variation in DNA repair profiles, ranging from small INDELs to kilobase size deletions and insertions. Furthermore, we find the frequency of DNA repair outcomes varies between loci. The results are not specific to the Cas-nuclease or selection procedure. Through Ku80 deletion analysis, a key protein required for canonical non-homologous end joining, we demonstrate activity of an alternative end joining mechanism that creates larger DNA deletions, and uses longer microhomology compared to C-NHEJ. Together, our results suggest preferential DNA repair pathway activity in the genome that can create different mutation profiles following repair, which could create biased genome variation and impact genome engineering and genome evolution.

RevDate: 2022-11-29
CmpDate: 2022-11-25

Niklander SE, KD Hunter (2023)

A Protocol to Produce Genetically Edited Primary Oral Keratinocytes Using the CRISPR-Cas9 System.

Methods in molecular biology (Clifton, N.J.), 2588:217-229.

The Nobel Prize awarded gene editing system, CRISPR-Cas9, is probably one of the greatest achievements of the last decades. CRISPR-Cas9 can introduce irreversible genomic changes in its target DNA by simple specifying a 20-nucleotide sequence within its RNA guide. Due to its simplicity, efficacy, and relative low cost in comparison with other genome editing systems, it has become the most common gene editing system used in research laboratories. Here we describe a step-by-step protocol to produce genetically edited primary oral keratinocytes using the CRISPR-Cas9 system.

RevDate: 2022-11-26
CmpDate: 2022-11-25

Willis JCW, Silva-Pinheiro P, Widdup L, et al (2022)

Compact zinc finger base editors that edit mitochondrial or nuclear DNA in vitro and in vivo.

Nature communications, 13(1):7204.

DddA-derived cytosine base editors (DdCBEs) use programmable DNA-binding TALE repeat arrays, rather than CRISPR proteins, a split double-stranded DNA cytidine deaminase (DddA), and a uracil glycosylase inhibitor to mediate C•G-to-T•A editing in nuclear and organelle DNA. Here we report the development of zinc finger DdCBEs (ZF-DdCBEs) and the improvement of their editing performance through engineering their architectures, defining improved ZF scaffolds, and installing DddA activity-enhancing mutations. We engineer variants with improved DNA specificity by integrating four strategies to reduce off-target editing. We use optimized ZF-DdCBEs to install or correct disease-associated mutations in mitochondria and in the nucleus. Leveraging their small size, we use a single AAV9 to deliver into heart, liver, and skeletal muscle in post-natal mice ZF-DdCBEs that efficiently install disease-associated mutations. While off-target editing of ZF-DdCBEs is likely too high for therapeutic applications, these findings demonstrate a compact, all-protein base editing research tool for precise editing of organelle or nuclear DNA without double-strand DNA breaks.

RevDate: 2022-11-30
CmpDate: 2022-11-25

Yan MY, Zheng D, Li SS, et al (2022)

Application of combined CRISPR screening for genetic and chemical-genetic interaction profiling in Mycobacterium tuberculosis.

Science advances, 8(47):eadd5907.

CRISPR screening, including CRISPR interference (CRISPRi) and CRISPR-knockout (CRISPR-KO) screening, has become a powerful technology in the genetic screening of eukaryotes. In contrast with eukaryotes, CRISPR-KO screening has not yet been applied to functional genomics studies in bacteria. Here, we constructed genome-scale CRISPR-KO and also CRISPRi libraries in Mycobacterium tuberculosis (Mtb). We first examined these libraries to identify genes essential for Mtb viability. Subsequent screening identified dozens of genes associated with resistance/susceptibility to the antitubercular drug bedaquiline (BDQ). Genetic and chemical validation of the screening results suggested that it provided a valuable resource to investigate mechanisms of action underlying the effects of BDQ and to identify chemical-genetic synergies that can be used to optimize tuberculosis therapy. In summary, our results demonstrate the potential for efficient genome-wide CRISPR-KO screening in bacteria and establish a combined CRISPR screening approach for high-throughput investigation of genetic and chemical-genetic interactions in Mtb.

RevDate: 2022-11-25
CmpDate: 2022-11-25

Verkuijl SAN, Gonzalez E, Li M, et al (2022)

A CRISPR endonuclease gene drive reveals distinct mechanisms of inheritance bias.

Nature communications, 13(1):7145.

CRISPR/Cas gene drives can bias transgene inheritance through different mechanisms. Homing drives are designed to replace a wild-type allele with a copy of a drive element on the homologous chromosome. In Aedes aegypti, the sex-determining locus is closely linked to the white gene, which was previously used as a target for a homing drive element (w[GDe]). Here, through an analysis using this linkage we show that in males inheritance bias of w[GDe] did not occur by homing, rather through increased propagation of the donor drive element. We test the same w[GDe] drive element with transgenes expressing Cas9 with germline regulatory elements sds3, bgcn, and nup50. We only find inheritance bias through homing, even with the identical nup50-Cas9 transgene. We propose that DNA repair outcomes may be more context dependent than anticipated and that other previously reported homing drives may, in fact, bias their inheritance through other mechanisms.

RevDate: 2022-11-22

Gu S, Zhang J, Li L, et al (2022)

Repurposing the Endogenous CRISPR-Cas9 System for High-Efficiency Genome Editing in Lacticaseibacillus paracasei.

ACS synthetic biology [Epub ahead of print].

Lactobacilli such as Lacticaseibacillus (Lcb) paracasei are generally regarded as safe and health-promoting microbes, and have been widely applied in food and pharmaceutical industries. However, the genetic bases of their beneficial properties were mostly uncertain because of the lack of effective genetic manipulation tools. The type II CRISPR-Cas9 system is the largest family present in lactobacilli, but none of them yet have been developed for genetic modifications. Here, we establish the first endogenous CRISPR-Cas9 genome-editing system in lactobacilli. With a validated protospacer adjacent motif (PAM) and customized single guide RNA (sgRNA) expression cassette, the native CRISPR-Cas9 system was reprogrammed to achieve gene deletion and chromosomal insertion at over 90% efficiency, as well as nucleotide substitution at ≥50% efficiency. We also effectively accomplished deletions of large genomic fragments (5-10 kb) and simultaneous deletion of multiple genes at distal loci, both of which are the first cases in lactobacilli when either endogenous or exogenous CRISPR-Cas systems were employed. In addition, we designed a controllable plasmid-targeting sgRNA expression module and integrated it into the editing plasmid. The all-in-one vector realized gene deletion and plasmid curing at high efficiency (>90%). Collectively, the present study develops a convenient and precise genetic tool in Lcb. paracasei and contributes to the genetics and engineering of lactobacilli.

RevDate: 2022-11-22

Yang B, Zheng J, Y Yin (2022)

AcaFinder: Genome Mining for Anti-CRISPR-Associated Genes.

mSystems [Epub ahead of print].

Anti-CRISPR (Acr) proteins are encoded by (pro)viruses to inhibit their host's CRISPR-Cas systems. Genes encoding Acr and Aca (Acr associated) proteins often colocalize to form acr-aca operons. Here, we present AcaFinder as the first Aca genome mining tool. AcaFinder can (i) predict Acas and their associated acr-aca operons using guilt-by-association (GBA); (ii) identify homologs of known Acas using an HMM (Hidden Markov model) database; (iii) take input genomes for potential prophages, CRISPR-Cas systems, and self-targeting spacers (STSs); and (iv) provide a standalone program (https://github.com/boweny920/AcaFinder) and a web server (http://aca.unl.edu/Aca). AcaFinder was applied to mining over 16,000 prokaryotic and 142,000 gut phage genomes. After a multistep filtering, 36 high-confident new Aca families were identified, which is three times that of the 12 known Aca families. Seven new Aca families were from major human gut bacteria (Bacteroidota, Actinobacteria, and Fusobacteria) and their phages, while most known Aca families were from Proteobacteria and Firmicutes. A complex association network between Acrs and Acas was revealed by analyzing their operonic colocalizations. It appears very common in evolution that the same aca genes can recombine with different acr genes and vice versa to form diverse acr-aca operon combinations. IMPORTANCE At least four bioinformatics programs have been published for genome mining of Acrs since 2020. In contrast, no bioinformatics tools are available for automated Aca discovery. As the self-transcriptional repressor of acr-aca operons, Aca can be viewed as anti-anti-CRISPRs, with great potential in the improvement of CRISPR-Cas technology. Although all the 12 known Aca proteins contain a conserved helix-turn-helix (HTH) domain, not all HTH-containing proteins are Acas. However, HTH-containing proteins with adjacent Acr homologs encoded in the same genetic operon are likely Aca proteins. AcaFinder implements this guilt-by-association idea and the idea of using HMMs of known Acas for homologs into one software package. Applying AcaFinder in screening prokaryotic and gut phage genomes reveals a complex acr-aca operonic colocalization network between different families of Acrs and Acas.

RevDate: 2022-11-23
CmpDate: 2022-11-23

Hazrati A, Malekpour K, Soudi S, et al (2022)

CRISPR/Cas9-engineered mesenchymal stromal/stem cells and their extracellular vesicles: A new approach to overcoming cell therapy limitations.

Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie, 156:113943.

Cell therapy is one of the newest therapeutic approaches for treating tissue destruction diseases and replacing damaged parts in defective tissues. Among different cells, mesenchymal stem cells (MSCs) have received a lot of attention due to their advantages and desirable properties. Also, MSCs-derived secretome, which includes various growth factors, cytokines, and extracellular vesicles (EVs), is used in the treatment of different types of diseases. However, the application of MSCs in an intact form brings their functionality with limitations. For this reason, different methods are recommended to increase their efficiency and the extracellular vesicles derived from them. One of these methods is gene editing of these cells. Among the different techniques for MSCs gene editing, CRISPR/Cas9 can increase the therapeutic potential of MSCs in a targeted manner due to its advantages. In order to achieve the desired result, various genes have been manipulated in MSCs, including genes involved in stemness, aging, migration, proliferation, survival, and inflammatory responses. Engineering MSCs with this method affects the cells' characteristics, changes their cytokine and different growth factors secretions, and increases their therapeutic efficiency.

RevDate: 2022-11-22

Saad FA, Saad JF, Siciliano G, et al (2022)

Duchenne Muscular Dystrophy Gene therapy.

Current gene therapy pii:CGT-EPUB-127694 [Epub ahead of print].

Duchenne and Becker muscular dystrophies are allelic X-linked recessive neuromuscular diseases affecting both skeletal and cardiac muscles. Therefore, owing to their single X chromosome, the affected boys receive the pathogenic gene mutations from their unknowing carrier mothers. Current pharmacological drugs are palliative that address the symptoms of the disease rather than the genetic cause imbedded in the Dystrophin gene DNA sequence. Therefore, alternative therapies like gene drugs that can address the genetic cause of the disease at its root is crucial, which include gene transfer/implantation, exon skipping, and gene editing. Presently, it is possible through genetic reprogramming to engineer AAV vectors to deliver certain therapeutic cargos specifically to muscle or other organs regardless of their serotype. Similarly, it is possible to direct the biogenesis of exosomes to carry gene editing constituents or certain therapeutic cargos to specific tissue or cell type like brain and muscle. While autologous exosomes are immunologically inert, it is possible to camouflage AAV capsids, gold and lipid nanoparticles to evade the immune system recognition. In this review, we highlight current opportunities for Duchenne muscular dystrophy gene therapy, which has been known thus far as an incurable genetic disease. This article is a part of Gene Therapy of Rare Genetic Diseases thematic issue.

RevDate: 2022-12-06
CmpDate: 2022-12-06

Xing G, Shang Y, Wang X, et al (2023)

Multiplexed detection of foodborne pathogens using one-pot CRISPR/Cas12a combined with recombinase aided amplification on a finger-actuated microfluidic biosensor.

Biosensors & bioelectronics, 220:114885.

Foodborne pathogens have raised significant concerns in human public health. Rapid, high-sensitive, low-cost, and easy-to-use testing methods for food safety are needed. In this study, we developed a finger-actuated microfluidic biosensor (FA-MB) for multiplexed detection of Bacillus cereus and other six common foodborne pathogens based on one-pot CRISPR/Cas12a combined with recombinase aided amplification (RAA). Wells for RAA and CRISPR/Cas12a were isolated to avoid interference, while finger-actuated one-way control valves were incorporated to fulfill the unidirectional flow of RAA products to the CRISPR/Cas12a reaction wells, realizing one-pot RAA-CRISPR/Cas12a assay. The final fluorescent signal was acquired and processed by a smartphone. Under selected experimental conditions, seven pathogenic bacteria could be tested in about 1 h with the limits of detection (LODs) below 500 CFU/mL. Recoveries ranged from 90% to 116% of the spiked samples were readily achieved. The proposed FA-MB is highly integrated and easy-to-use, and could be used for rapid, high-sensitive point of care (POC) testing without the external driving device, suitable for resource-constrained settings.

RevDate: 2022-11-23
CmpDate: 2022-11-23

Zhao H, Yang M, Bishop J, et al (2022)

Identification and functional validation of super-enhancers in Arabidopsis thaliana.

Proceedings of the National Academy of Sciences of the United States of America, 119(48):e2215328119.

Super-enhancers (SEs) are exceptionally large enhancers and are recognized to play prominent roles in cell identity in mammalian species. We surveyed the genomic regions containing large clusters of accessible chromatin regions (ACRs) marked by deoxyribonuclease (DNase) I hypersensitivity in Arabidopsis thaliana. We identified a set of 749 putative SEs, which have a minimum length of 1.5 kilobases and represent the top 2.5% of the largest ACR clusters. We demonstrate that the genomic regions associating with these SEs were more sensitive to DNase I than other nonpromoter ACRs. The SEs were preferentially associated with topologically associating domains. Furthermore, the SEs and their predicted cognate genes were frequently associated with organ development and tissue identity in A. thaliana. Therefore, the A. thaliana SEs and their cognate genes mirror the functional characteristics of those reported in mammalian species. We developed CRISPR/Cas-mediated deletion lines of a 3,578-bp SE associated with the thalianol biosynthetic gene cluster (BGC). Small deletions (131-157 bp) within the SE resulted in distinct phenotypic changes and transcriptional repression of all five thalianol genes. In addition, T-DNA insertions in the SE region resulted in transcriptional alteration of all five thalianol genes. Thus, this SE appears to play a central role in coordinating the operon-like expression pattern of the thalianol BGC.

RevDate: 2022-11-21

Maslać N, Sidhu C, Teeling H, et al (2022)

Comparative Transcriptomics Sheds Light on Remodeling of Gene Expression during Diazotrophy in the Thermophilic Methanogen Methanothermococcus thermolithotrophicus.

mBio [Epub ahead of print].

Some marine thermophilic methanogens are able to perform energy-consuming nitrogen fixation despite deriving only little energy from hydrogenotrophic methanogenesis. We studied this process in Methanothermococcus thermolithotrophicus DSM 2095, a methanogenic archaeon of the order Methanococcales that contributes to the nitrogen pool in some marine environments. We successfully grew this archaeon under diazotrophic conditions in both batch and fermenter cultures, reaching the highest cell density reported so far. Diazotrophic growth depended strictly on molybdenum and, in contrast to other diazotrophs, was not inhibited by tungstate or vanadium. This suggests an elaborate control of metal uptake and a specific metal recognition system for the insertion into the nitrogenase cofactor. Differential transcriptomics of M. thermolithotrophicus grown under diazotrophic conditions with ammonium-fed cultures as controls revealed upregulation of the nitrogenase machinery, including chaperones, regulators, and molybdate importers, as well as simultaneous upregulation of an ammonium transporter and a putative pathway for nitrate and nitrite utilization. The organism thus employs multiple synergistic strategies for uptake of nitrogen nutrients during the early exponential growth phase without altering transcription levels for genes involved in methanogenesis. As a counterpart, genes coding for transcription and translation processes were downregulated, highlighting the maintenance of an intricate metabolic balance to deal with energy constraints and nutrient limitations imposed by diazotrophy. This switch in the metabolic balance included unexpected processes, such as upregulation of the CRISPR-Cas system, probably caused by drastic changes in transcription levels of putative mobile and virus-like elements. IMPORTANCE The thermophilic anaerobic archaeon M. thermolithotrophicus is a particularly suitable model organism to study the coupling of methanogenesis to diazotrophy. Likewise, its capability of simultaneously reducing N2 and CO2 into NH3 and CH4 with H2 makes it a viable target for biofuel production. We optimized M. thermolithotrophicus cultivation, resulting in considerably higher cell yields and enabling the successful establishment of N2-fixing bioreactors. Improved understanding of the N2 fixation process would provide novel insights into metabolic adaptations that allow this energy-limited extremophile to thrive under diazotrophy, for instance, by investigating its physiology and uncharacterized nitrogenase. We demonstrated that diazotrophic growth of M. thermolithotrophicus is exclusively dependent on molybdenum, and complementary transcriptomics corroborated the expression of the molybdenum nitrogenase system. Further analyses of differentially expressed genes during diazotrophy across three cultivation time points revealed insights into the response to nitrogen limitation and the coordination of core metabolic processes.

RevDate: 2022-11-22

An Y, Wang Y, Wang X, et al (2022)

Development of chloroplast transformation and gene expression regulation technology in land plants.

Frontiers in plant science, 13:1037038.

Chloroplasts in land plants have their own small circular DNA that is presumed to have originated from cyanobacteria-related endosymbionts, and the chloroplast genome is an attractive target to improve photosynthetic ability and crop yield. However, to date, most transgenic or genetic engineering technologies for plants are restricted to manipulations of the nuclear genome. In this review, we provide a comprehensive overview of chloroplast genetic engineering and regulation of gene expression from the perspective of history and biology, focusing on current and latest methods. In addition, we suggest techniques that may regulate the chloroplast gene expression at the transcriptional or post-transcriptional level.

RevDate: 2022-11-22

Ngamsom B, Iles A, Kamita M, et al (2022)

A sample-to-answer COVID-19 diagnostic device based on immiscible filtration and CRISPR-Cas12a-assisted detection.

Talanta open, 6:100166.

In response to the ongoing coronavirus disease 2019 (COVID-19) pandemic and disparities of vaccination coverage in low-and middle-income countries, it is vital to adopt a widespread testing and screening programme, combined with contact tracing, to monitor and effectively control the infection dispersion in areas where medical resources are limited. This work presents a lab-on-a-chip device, namely 'IFAST-LAMP-CRISPR', as an affordable, rapid and high-precision molecular diagnostic means for detection of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). The herein proposed 'sample-to-answer' platform integrates RNA extraction, amplification and molecular detection with lateral flow readout in one device. The microscale dimensions of the device containing immiscible liquids, coupled with the use of silica paramagnetic beads and guanidine hydrochloride, streamline sample preparation (including RNA extraction, concentration and purification) in 15 min with minimal hands-on steps. The pre-amplification in combination with CRISPR-Cas12a detection assays targeting the nucleoprotein (N) gene achieved visual identification of ≥ 470 copies mL[-1] genomic SARS-CoV-2 samples in 45 min. On-chip assays showed the ability to isolate and detect SARS-CoV-2 RNA from 100 genome copies mL[-1] of replication-deficient viral particles in 1 h. This simple, affordable and integrated platform demonstrated a visual, faster, and yet specificity- and sensitivity-comparable alternative to the costly gold-standard reverse transcription-polymerase chain reaction (RT-PCR) assay, requiring only a simple heating source. Initial testing illustrates the platform viability both on nasopharyngeal swab and saliva samples collected using the easily accessible Swan-brand cigarette filter, providing a complete workflow for COVID-19 diagnostics in low-resource settings.

RevDate: 2022-11-22

Zhu Y, Xing C, Yang L, et al (2022)

Dual-gene detection in a single-tube system based on CRISPR-Cas12a/Cas13a for severe fever thrombocytopenia syndrome virus.

Frontiers in microbiology, 13:977382.

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging infectious disease, which is caused by severe fever with thrombocytopenia syndrome virus (SFTSV). The disease results in high mortality and increased morbidity and threatens global public health. Rapid detection of SFTSV is crucial for epidemic prevention in low-resource settings. Here we developed deployable, sensitive and rapid detection methods based on CRISPR/Cas12a or Cas13a technologies. The CRISPR/Cas12a-based detection assay could stably detect the SFTSV L or M genes at 10 cp/μl. The Cas13a-based method could detect the L gene as low as 0.75 cp/μl. For point-of-care testing, we combined fluorescence visualization and lateral flow detection with CRISPR/Cas-based assays. Furthermore, using the orthogonal DNA/RNA collateral activity of the Cas12a/Cas13a system, we present the dual-gene detection platform for SFTSV, which can simultaneously detect the L and M genes in a single tube. Based on the dual-gene detection, we designed multiplexed test strips to detect SFTSV. All our methods were initially validated using 52 clinical samples, showing 100% sensitivity and specificity. These new CRISPR/Cas-based detection methods are promising candidates for on-site detection of SFTSV.

RevDate: 2022-11-22

Abriouel H, Manetsberger J, Caballero Gómez N, et al (2022)

In silico genomic analysis of the potential probiotic Lactiplantibacillus pentosus CF2-10N reveals promising beneficial effects with health promoting properties.

Frontiers in microbiology, 13:989824.

Lactiplantibacillus pentosus CF2-10 N, isolated from brines of naturally fermented Aloreña green table olives, exhibited high probiotic potential. High throughput sequencing and annotation of genome sequences underline the potential of L. pentosus CF2-10 N as excellent probiotic candidate of vegetable origin. In a previous study we could show the probiotic potential of CF2-10 N in vitro, while in this study in silico analysis of its genome revealed new insights into its safety and functionality. Our findings highlight the microorganism's ecological flexibility and adaptability to a broad range of environmental niches, food matrices and the gastrointestinal tract. These features are shared by both phylogenetically very close L. pentosus strains (CF2-10 N and MP-10) isolated from the same ecological niche with respect to their genome size (≅ 3.6 Mbp), the presence of plasmids (4-5) and several other properties. Nonetheless, additional and unique features are reported in the present study for L. pentosus CF2-10 N. Notably, the safety of L. pentosus CF2-10 N was shown by the absence of virulence determinants and the determination of acquired antibiotic resistance genes, i.e., resistome, which is mostly represented by efflux-pump resistance genes responsible for the intrinsic resistance. On the other hand, defense mechanisms of L. pentosus CF2-10 N include eight prophage regions and a CRISPR/cas system (CRISPR-I and CRISPR-II) as acquired immune system against mobile elements. Finally, the probiotic potential of this strain was further demonstrated by the presence of genes coding for proteins involved in adhesion, exopolysaccharide biosynthesis, tolerance to low pH and bile salts, immunomodulation, and vitamin and enzyme production. Taken together these results, we propose the use of L. pentosus CF2-10 N as a potential and promising probiotic candidate able to colonize several niches and adapt to different lifestyles. The strain can provide attractive functional and probiotic features necessary for its application as starter culture and probiotic.

RevDate: 2022-11-22

Álvarez-Rodríguez A, Jin BK, Radwanska M, et al (2022)

Recent progress in diagnosis and treatment of Human African Trypanosomiasis has made the elimination of this disease a realistic target by 2030.

Frontiers in medicine, 9:1037094.

Human African Trypanosomiasis (HAT) is caused by unicellular flagellated protozoan parasites of the genus Trypanosoma brucei. The subspecies T. b. gambiense is mainly responsible for mostly chronic anthroponotic infections in West- and Central Africa, accounting for roughly 95% of all HAT cases. Trypanosoma b. rhodesiense results in more acute zoonotic infections in East-Africa. Because HAT has a two-stage pathogenesis, treatment depends on clinical assessment of patients and the determination whether or not parasites have crossed the blood brain barrier. Today, ultimate confirmation of parasitemia is still done by microscopy analysis. However, the introduction of diagnostic lateral flow devices has been a major contributor to the recent dramatic drop in T. b. gambiense HAT. Other techniques such as loop mediated isothermal amplification (LAMP) and recombinant polymerase amplification (RPA)-based tests have been published but are still not widely used in the field. Most recently, CRISPR-Cas technology has been proposed to improve the intrinsic diagnostic characteristics of molecular approaches. This will become crucial in the near future, as preventing the resurgence of HAT will be a priority and will require tools with extreme high positive and negative predicted values, as well as excellent sensitivity and specificity. As for treatment, pentamidine and suramin have historically been the drugs of choice for the treatment of blood-stage gambiense-HAT and rhodesiense-HAT, respectively. For treatment of second-stage infections, drugs that pass the blood brain barrier are needed, and melarsoprol has been effectively used for both forms of HAT in the past. However, due to the high occurrence of post-treatment encephalopathy, the drug is not recommended for use in T. b. gambiense HAT. Here, a combination therapy of eflornithine and nifurtimox (NECT) has been the choice of treatment since 2009. As this treatment requires IV perfusion of eflornithine, efforts were launched in 2003 by the drugs for neglected disease initiative (DNDi) to find an oral-only therapy solution, suitable for rural sub-Saharan Africa treatment conditions. In 2019 this resulted in the introduction of fexinidazole, with a treatment regimen suitable for both the blood-stage and non-severe second-stage T. b. gambiense infections. Experimental treatment of T. b. rhodesiense HAT has now been initiated as well.

RevDate: 2022-11-30

Johnston M, Ceren Ates H, Glatz RT, et al (2022)

Multiplexed biosensor for point-of-care COVID-19 monitoring: CRISPR-powered unamplified RNA diagnostics and protein-based therapeutic drug management.

Materials today (Kidlington, England) [Epub ahead of print].

In late 2019 SARS-CoV-2 rapidly spread to become a global pandemic, therefore, measures to attenuate chains of infection, such as high-throughput screenings and isolation of carriers were taken. Prerequisite for a reasonable and democratic implementation of such measures, however, is the availability of sufficient testing opportunities (beyond reverse transcription PCR, the current gold standard). We, therefore, propose an electrochemical, microfluidic multiplexed polymer-based biosensor in combination with CRISPR/Cas-powered assays for low-cost and accessible point-of-care nucleic acid testing. In this study, we simultaneously screen for and identify SARS-CoV-2 infections (Omicron-variant) in clinical specimens (Sample-to-result time: ∼30 min), employing LbuCas13a, whilst bypassing reverse transcription as well as target amplification of the viral RNA (LODs of 2,000 and 7,520 copies/µl for the E and RdRP genes, respectively, and 50 copies/ml for combined targets), both of which are necessary for detection via PCR and other isothermal methods. In addition, we demonstrate the feasibility of combining synthetic biology-driven assays based on different classes of biomolecules, in this case protein-based ß-lactam antibiotic detection, on the same device. The programmability of the effector and multiplexing capacity (up to six analytes) of our platform, in combination with a miniaturized measurement setup, including a credit card sized near field communication (NFC) potentiostat and a microperistaltic pump, provide a promising on-site tool for identifying individuals infected with variants of concern and monitoring their disease progression alongside other potential biomarkers or medication clearance.

RevDate: 2022-11-26

Wang SY, Li X, Wang SG, et al (2022)

Base editing for reprogramming cyanobacterium Synechococcus elongatus.

Metabolic engineering, 75:91-99 pii:S1096-7176(22)00148-3 [Epub ahead of print].

Cyanobacteria can directly convert carbon dioxide (CO2) at the atmospheric level to biofuels, value-added chemicals and food products, making them ideal candidates to alleviate global climate change. Despite decades-long pioneering successes, the development of genome-editing tools, especially the CRISPR-Cas-based approaches, seems to lag behind other microbial chassis, slowing down the innovations of cyanobacteria. Here, we adapted and tailored base editing for cyanobacteria based on the CRISPR-Cas system and deamination. We achieved precise and efficient genome editing at a single-nucleotide resolution and demonstrated multiplex base editing in the model cyanobacterium Synechococcus elongatus. By using the base-editing tool, we successfully manipulated the glycogen metabolic pathway via the introduction of premature STOP codons in the relevant genes, building engineered strains with elevated potentials to produce chemicals and food from CO2. We present here the first report of base editing in the phylum of cyanobacteria, and a paradigm for applying CRISPR-Cas systems in bacteria. We believe that our work will accelerate the metabolic engineering and synthetic biology of cyanobacteria and drive more innovations to alleviate global climate change.

RevDate: 2022-11-30

Perveen S, Negi A, Gopalakrishnan V, et al (2022)

COVID-19 diagnostics: Molecular biology to nanomaterials.

Clinica chimica acta; international journal of clinical chemistry, 538:139-156 pii:S0009-8981(22)01384-5 [Epub ahead of print].

The SARS-CoV-2 pandemic has claimed around 6.4 million lives worldwide. The disease symptoms range from mild flu-like infection to life-threatening complications. The widespread infection demands rapid, simple, and accurate diagnosis. Currently used methods include molecular biology-based approaches that consist of conventional amplification by RT-PCR, isothermal amplification-based techniques such as RT-LAMP, and gene editing tools like CRISPR-Cas. Other methods include immunological detection including ELISA, lateral flow immunoassay, chemiluminescence, etc. Radiological-based approaches are also being used. Despite good analytical performance of these current methods, there is an unmet need for less costly and simpler tests that may be performed at point of care. Accordingly, nanomaterial-based testing has been extensively pursued. In this review, we discuss the currently used diagnostic techniques for SARS-CoV-2, their usefulness, and limitations. In addition, nanoparticle-based approaches have been highlighted as another potential means of detection. The review provides a deep insight into the current diagnostic methods and future trends to combat this deadly menace.

RevDate: 2022-12-06

Çerçi B, Uzay IA, Kara MK, et al (2022)

Clinical trials and promising preclinical applications of CRISPR/Cas gene editing.

Life sciences, 312:121204 pii:S0024-3205(22)00904-3 [Epub ahead of print].

Treatment of genetic disorders by genomic manipulation has been the unreachable goal of researchers for many decades. Although our understanding of the genetic basis of genetic diseases has advanced tremendously in the last few decades, the tools developed for genomic editing were not efficient and practical for their use in the clinical setting until now. The recent advancements in the research of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated protein (Cas) systems offered an easy and efficient way to edit the genome and accelerated the research on their potential use in the treatment of genetic disorders. In this review, we summarize the clinical trials that evaluate the CRISPR/Cas systems for treating different genetic diseases and highlight promising preclinical research on CRISPR/Cas mediated treatment of a great diversity of genetic disorders. Ultimately, we discuss the future of CRISPR/Cas mediated genome editing in genetic diseases.

RevDate: 2022-11-22
CmpDate: 2022-11-22

Leng K, M Kampmann (2022)

Towards elucidating disease-relevant states of neurons and glia by CRISPR-based functional genomics.

Genome medicine, 14(1):130.

Our understanding of neurological diseases has been tremendously enhanced over the past decade by the application of new technologies. Genome-wide association studies have highlighted glial cells as important players in diseases. Single-cell profiling technologies are providing descriptions of disease states of neurons and glia at unprecedented molecular resolution. However, significant gaps remain in our understanding of the mechanisms driving disease-associated cell states, and how these states contribute to disease. These gaps in our understanding can be bridged by CRISPR-based functional genomics, a powerful approach to systematically interrogate gene function. In this review, we will briefly review the current literature on neurological disease-associated cell states and introduce CRISPR-based functional genomics. We discuss how advances in CRISPR-based screens, especially when implemented in the relevant brain cell types or cellular environments, have paved the way towards uncovering mechanisms underlying neurological disease-associated cell states. Finally, we will delineate current challenges and future directions for CRISPR-based functional genomics to further our understanding of neurological diseases and potential therapeutic strategies.

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ESP Quick Facts

ESP Origins

In the early 1990's, Robert Robbins was a faculty member at Johns Hopkins, where he directed the informatics core of GDB — the human gene-mapping database of the international human genome project. To share papers with colleagues around the world, he set up a small paper-sharing section on his personal web page. This small project evolved into The Electronic Scholarly Publishing Project.

ESP Support

In 1995, Robbins became the VP/IT of the Fred Hutchinson Cancer Research Center in Seattle, WA. Soon after arriving in Seattle, Robbins secured funding, through the ELSI component of the US Human Genome Project, to create the original ESP.ORG web site, with the formal goal of providing free, world-wide access to the literature of classical genetics.

ESP Rationale

Although the methods of molecular biology can seem almost magical to the uninitiated, the original techniques of classical genetics are readily appreciated by one and all: cross individuals that differ in some inherited trait, collect all of the progeny, score their attributes, and propose mechanisms to explain the patterns of inheritance observed.

ESP Goal

In reading the early works of classical genetics, one is drawn, almost inexorably, into ever more complex models, until molecular explanations begin to seem both necessary and natural. At that point, the tools for understanding genome research are at hand. Assisting readers reach this point was the original goal of The Electronic Scholarly Publishing Project.

ESP Usage

Usage of the site grew rapidly and has remained high. Faculty began to use the site for their assigned readings. Other on-line publishers, ranging from The New York Times to Nature referenced ESP materials in their own publications. Nobel laureates (e.g., Joshua Lederberg) regularly used the site and even wrote to suggest changes and improvements.

ESP Content

When the site began, no journals were making their early content available in digital format. As a result, ESP was obliged to digitize classic literature before it could be made available. For many important papers — such as Mendel's original paper or the first genetic map — ESP had to produce entirely new typeset versions of the works, if they were to be available in a high-quality format.

ESP Help

Early support from the DOE component of the Human Genome Project was critically important for getting the ESP project on a firm foundation. Since that funding ended (nearly 20 years ago), the project has been operated as a purely volunteer effort. Anyone wishing to assist in these efforts should send an email to Robbins.

ESP Plans

With the development of methods for adding typeset side notes to PDF files, the ESP project now plans to add annotated versions of some classical papers to its holdings. We also plan to add new reference and pedagogical material. We have already started providing regularly updated, comprehensive bibliographies to the ESP.ORG site.

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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

Electronic Scholarly Publishing
961 Red Tail Lane
Bellingham, WA 98226

E-mail: RJR8222 @ gmail.com

Papers in Classical Genetics

The ESP began as an effort to share a handful of key papers from the early days of classical genetics. Now the collection has grown to include hundreds of papers, in full-text format.

Digital Books

Along with papers on classical genetics, ESP offers a collection of full-text digital books, including many works by Darwin (and even a collection of poetry — Chicago Poems by Carl Sandburg).

Timelines

ESP now offers a much improved and expanded collection of timelines, designed to give the user choice over subject matter and dates.

Biographies

Biographical information about many key scientists.

Selected Bibliographies

Bibliographies on several topics of potential interest to the ESP community are now being automatically maintained and generated on the ESP site.

ESP Picks from Around the Web (updated 07 JUL 2018 )