A Cas12a-based CRISPR interference system for multigene regulation in mycobacteria.
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ABSTRACT:
SUBMITTER: Fleck N
PROVIDER: S-EPMC8363830 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The CRISPR-Cas12a is a class II, type V clustered regularly interspaced short palindromic repeat (CRISPR) system with both RNase and DNase activity. Compared to the CRISPR-Cas9 system, it recognizes T-rich PAM sequences and has the advantage of multiplex genomic editing. Here, in fission yeast Schizosaccharomyces pombe, we successfully implemented the CRISPR-Cas12a system for versatile genomic editing and manipulation. In addition to the rrk1 promoter, we used new pol II promoters from endogenous coding genes to express crRNA for Cas12a and obtained a much higher editing efficiency. This new design expands the promoter choices for potential applications in fission yeast and other organisms. In addition, we expressed a gRNA array using a strong constitutive pol II promoter. The array transcript is processed by Cas12a itself to release multiple mature crRNAs. With this construct, multiplex genomic editing of up to three loci was achieved from a single yeast transformation. We also built a CRISPR interference system using a DNase-dead Cas12a to significantly repress endogenous gene expression. Our study provides the first CRISPR-Cas12a toolkit for efficient and rapid genomic gene editing and regulation in fission yeast.
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Project description:The Gram-positive, spore-forming, obligate anaerobic firmicute species that make up the Clostridium genus have broad feedstock consumption capabilities and produce value-added metabolic products, but genetic manipulation is difficult, limiting their broad appeal. CRISPR-Cas systems have recently been applied to Clostridium species, primarily using Cas9 as a counterselection marker in conjunction with plasmid-based homologous recombination. CRISPR interference is a method that reduces gene expression of specific genes via precision targeting of a nuclease deficient Cas effector protein. Here, we develop a dCas12a-based CRISPR interference system for transcriptional gene repression in multiple mesophilic Clostridium species. We show the Francisella novicida Cas12a-based system has a broader applicability due to the low GC content in Clostridium species compared to CRISPR Cas systems derived from other bacteria. We demonstrate >99% reduction in transcript levels of targeted genes in Clostridium acetobutylicum and >75% reduction in Clostridium pasteurianum. We also demonstrate multiplexed repression via use of a single synthetic CRISPR array, achieving 99% reduction in targeted gene expression and elucidating a unique metabolic profile for their reduced expression. Overall, this work builds a foundation for high throughput genetic screens without genetic editing, a key limitation in current screening methods used in the Clostridium community.
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Project description:Chemical modifications of CRISPR-Cas nucleases help decrease off-target editing and expand the biomedical applications of CRISPR-based gene manipulation tools. Here, we found that epigenetic modifications of guide RNA, such as m6A and m1A methylation, can effectively inhibit both the cis- and trans-DNA cleavage activities of CRISPR-Cas12a. The underlying mechanism is that methylations destabilize the secondary and tertiary structure of gRNA which prevents the assembly of the Cas12a-gRNA nuclease complex, leading to decreased DNA targeting ability. A minimum of three adenine methylated nucleotides are required to completely inhibit the nuclease activity. We also demonstrate that these effects are reversible through the demethylation of gRNA by demethylases. This strategy has been used in the regulation of gene expression, demethylase imaging in living cells and controllable gene editing. The results demonstrate that the methylation-deactivated and demethylase-activated strategy is a promising tool for regulation of the CRISPR-Cas12a system.
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Project description:As CRISPR technology is promoted to more fine-divided molecular biology applications, its inherent performance finds it increasingly difficult to cope with diverse needs in these different fields, and how to more accurately control the performance has become a key issue to develop CRISPR technology to a new stage. Herein, we propose a CRISPR/Cas12a regulation strategy based on the powerful programmability of nucleic acid nanotechnology. Unlike previous difficult and rigid regulation of core components Cas nuclease and crRNA, only a simple switch of different external RNA accessories is required to change the reaction kinetics or thermodynamics, thereby finely and almost steplessly regulating multi-performance of CRISPR/Cas12a including activity, speed, specificity, compatibility, programmability and sensitivity. In particular, the significantly improved specificity is expected to mark advance the accuracy of molecular detection and the safety of gene editing. In addition, this strategy was applied to regulate the delayed activation of Cas12a, overcoming the compatibility problem of the one-pot assay without any physical separation or external stimulation, and demonstrating great potential for fine-grained control of CRISPR. This simple but powerful CRISPR regulation strategy without any component modification has pioneering flexibility and versatility, and will unlock the potential for deeper applications of CRISPR technology in many finely divided fields.
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Project description:The development of new drug regimens that allow rapid, sterilizing treatment of tuberculosis has been limited by the complexity and time required for genetic manipulations in Mycobacterium tuberculosis. CRISPR interference (CRISPRi) promises to be a robust, easily engineered and scalable platform for regulated gene silencing. However, in M. tuberculosis, the existing Streptococcus pyogenes Cas9-based CRISPRi system is of limited utility because of relatively poor knockdown efficiency and proteotoxicity. To address these limitations, we screened eleven diverse Cas9 orthologues and identified four that are broadly functional for targeted gene knockdown in mycobacteria. The most efficacious of these proteins, the CRISPR1 Cas9 from Streptococcus thermophilus (dCas9Sth1), typically achieves 20- to 100-fold knockdown of endogenous gene expression with minimal proteotoxicity. In contrast to other CRISPRi systems, dCas9Sth1-mediated gene knockdown is robust when targeted far from the transcriptional start site, thereby allowing high-resolution dissection of gene function in the context of bacterial operons. We demonstrate the utility of this system by addressing persistent controversies regarding drug synergies in the mycobacterial folate biosynthesis pathway. We anticipate that the dCas9Sth1 CRISPRi system will have broad utility for functional genomics, genetic interaction mapping and drug-target profiling in M. tuberculosis.
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Project description:Toxoplasmosis, caused by the intracellular protozoon Toxoplasma gondii, is a significant parasitic zoonosis with a world-wide distribution. As a main transmission route, human infection can be acquired by the ingestion of T. gondii oocysts from the environment (e.g., soil, water, fruits and vegetables). Regarding the detection of T. gondii oocysts in environmental samples, the development of a time-saving, cost-effective and highly sensitive technique is crucial for the surveillance, prevention and control of toxoplasmosis. In this study, we developed a new method by combining recombinase-aided amplification (RAA) with CRISPR-Cas12a, designated as the RAA-Cas12a-Tg system. Here, we compared this system targeting the 529 bp repeat element (529 bp-RE) with the routine PCR targeting both 529 bp-RE and ITS-1 gene, respectively, to assess its ability to detect T. gondii oocysts in soil samples. Our results indicated that the 529 bp RE-based RAA-Cas12a-Tg system was able to detect T. gondii successfully in nearly an hour at body temperature and was more sensitive than the routine PCR assay. The sensitivity of this system reached as low as 1 fM with high specificity. Thus, RAA-Cas12a-Tg system provided a rapid, sensitive and easily operable method for point-of-care detection of T. gondii oocysts in soil, which will facilitate the control of T. gondii infection in humans and animals.
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Project description:In recent years, meat adulteration safety incidents have occurred frequently, triggering widespread attention and discussion. Although there are a variety of meat quality identification methods, conventional assays require high standards for personnel and experimental conditions and are not suitable for on-site testing. Therefore, there is an urgent need for a rapid, sensitive, high specificity and high sensitivity on-site meat detection method. This study is the first to apply RPA combined with CRISPR/Cas12a technology to the field of multiple meat identification. The system developed by parameter optimization can achieve specific detection of chicken, duck, beef, pork and lamb with a minimum target sequence copy number as low as 1 × 100 copies/μL for 60 min at a constant temperature. LFD test results can be directly observed with the naked eye, with the characteristics of fast, portable and simple operation, which is extremely in line with current needs. In conclusion, the meat identification RPA-CRISPR/Cas12a-LFD system established in this study has shown promising applications in the field of meat detection, with a profound impact on meat quality, and provides a model for other food safety control programs.
| S-EPMC10582345 | biostudies-literature
Project description:The type V-A Cas12a protein can process its CRISPR array, a feature useful for multiplexed gene editing and regulation. However, CRISPR arrays often exhibit unpredictable performance due to interference between multiple guide RNA (gRNAs). Here, we report that Cas12a array performance is hypersensitive to the GC content of gRNA spacers, as high-GC spacers can impair activity of the downstream gRNA. We analyze naturally occurring CRISPR arrays and observe that natural repeats always contain an AT-rich fragment that separates gRNAs, which we term a CRISPR separator. Inspired by this observation, we design short, AT-rich synthetic separators (synSeparators) that successfully remove the disruptive effects between gRNAs. We further demonstrate enhanced simultaneous activation of seven endogenous genes in human cells using an array containing the synSeparator. These results elucidate a previously underexplored feature of natural CRISPR arrays and demonstrate how nature-inspired engineering solutions can improve multi-gene control in mammalian cells.
| S-EPMC8478413 | biostudies-literature
Project description:BackgroundCampylobacter spp. are the leading cause of bacterial food-borne illness in humans worldwide, with Campylobacter jejuni responsible for 80% of these infections. There is an urgent need to understand fundamental C. jejuni biology for the development of new strategies to prevent and treat infections. The range of molecular tools available to regulate gene expression in C. jejuni is limited, which in turn constrains our ability to interrogate the function of essential and conditionally essential genes. We have addressed this by developing and utilising a CRISPR-based interference system known as CRISPRi in C. jejuni to control gene expression. To achieve this, a catalytically inactive ("dead") cas9 and sgRNA backbone from the Streptococcus pyogenes CRISPRi system was combined with C. jejuni-derived promoters of predetermined expression activities to develop a CRISPRi-based repression tool in C. jejuni strains M1Cam and 81-176.ResultsThe CRISPRi tool was validated through successful repression of the arylsulphatase-encoding gene astA using a range of sgRNA target sequences spanning the astA gene. The tool was also applied to target astA in an M1Cam CRISPR-Cas9 deletion strain, which showed that the presence of an endogenous CRISPR-Cas9 system did not affect the activity of the CRISPRi-based repression tool. The tool was further validated against the hippicurase-encoding gene hipO. Following this, the flagella genes flgR, flaA, flaB and both flaA and flaB were targeted for CRISPRi-based repression, which resulted in varying levels of motility reduction and flagella phenotypes as determined by phenotypical assays and transmission electron microscopy (TEM).ConclusionsThis is the first report of a CRISPRi-based tool in C. jejuni, which will provide a valuable resource to the Campylobacter community.
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Project description:(1) Background: Gene editing technology, as represented by CRISPR is a powerful tool used in biomedical science. However, the editing efficiency of such technologies, especially in induced pluripotent stem cells (iPSCs) and other types of stem cells, is low which hinders its application in regenerative medicine; (2) Methods: A gene-editing system, COE, was designed and constructed based on CRISPR/Cas12a and Orip/EBNA1, and its editing efficiency was evaluated in human embryonic kidney 293T (HEK-293T) cells with flow cytometry and restriction fragment length polymorphism (RFLP) analysis. The COE was nucleofected into iPSCs, then, the editing efficiency was verified by a polymerase chain reaction and Sanger sequencing; (3) Results: With the extension of time, COE enables the generation of up to 90% insertion or deletion rates in HEK-293T cells. Furthermore, the deletion of a 2.5 kb fragment containing Exon 51 of the dystrophin gene (DMD) in iPSCs was achieved with high efficiency; out of 14 clones analyzed, 3 were positive. Additionally, the Exon 51-deleted iPSCs derived from cardiomyocytes had similar expression profiles to those of Duchenne muscular dystrophy (DMD) patient-specific iPSCs. Moreover, there was no residue of each component of the plasmid in the editing cells; (4) Conclusions: In this study, a novel, efficient, and safe gene-editing system, COE, was developed, providing a powerful tool for gene editing.
| S-EPMC8620414 | biostudies-literature