Removal of mobile genetic elements from the genome of Clostridioides difficile and the implications for the organism's biology.

Clostridioides difficile is an emerging pathogen of One Health significance. Its highly variable genome contains mobile genetic elements (MGEs) such as transposons and prophages that influence its biology. Systematic deletion of each genetic element is required to determine their precise role in C. difficile biology and contribution to the wider mobilome. Here, Tn5397 (21 kb) and ϕ027 (56 kb) were deleted from C. difficile 630 and R20291, respectively, using allele replacement facilitated by CRISPR-Cas9. The 630 Tn5397 deletant transferred PaLoc at the same frequency (1 × 10-7) as 630 harboring Tn5397, indicating that Tn5397 alone did not mediate conjugative transfer of PaLoc. The R20291 ϕ027 deletant was sensitive to ϕ027 infection, and contained two unexpected features, a 2.7 kb remnant of the mutagenesis plasmid, and a putative catalase gene adjacent to the deleted prophage was also deleted. Growth kinetics of R20291 ϕ027 deletant was similar to wild type (WT) in rich medium but marginally reduced compared with WT in minimal medium. This work indicates the commonly used pMTL8000 plasmid series works well for CRISPR-Cas9-mediated gene deletion, resulting in the largest deleted locus (56.8 kb) described in C. difficile. Removal of MGEs was achieved by targeting conjugative/integrative regions to promote excision and permanent loss. The deletants created will be useful strains for investigating Tn5397 or ϕ027 prophage contribution to host virulence, fitness, and physiology, and a platform for other mutagenesis studies aimed at functional gene analysis without native transposon or phage interference in C. difficile 630 and R20291.

CRISPR-based electrochemical biosensors: an alternative for point-of-care diagnostics?

The combination of CRISPR technology and electrochemical sensors has sparked a paradigm shift in the landscape of point-of-care (POC) diagnostics. This review explores the dynamic convergence between CRISPR and electrochemical sensing, elucidating their roles in rapid and precise biosensing platforms. CRISPR, renowned for its remarkable precision in genome editing and programmability capability, has found a novel application in conjunction with electrochemical sensors, promising highly sensitive and specific detection of nucleic acids and biomarkers associated with diverse diseases. This article navigates through fundamental principles, research developments, and applications of CRISPR-based electrochemical sensors, highlighting their potential to revolutionize healthcare accessibility and patient outcomes. In addition, some key points and challenges regarding applying CRISPR-powered electrochemical sensors in real POC settings are presented. By discussing recent advancements and challenges in this interdisciplinary field, this review evaluates the potential of these innovative sensors as an alternative for decentralized, rapid, and accurate POC testing, offering some insights into their applications across clinical scenarios and their impact on the future of diagnostics.

Application of CRISPR/Cas12a in miRNA-155 detection: A novel homogeneous electrochemiluminescence biosensor.

BACKGROUND: MicroRNAs (miRNAs) are important non-coding RNA entities that affect gene expression and function by binding to target mRNAs, leading to degradation of the mRNAs or inhibiting their translation. MiRNAs are widely involved in a variety of biological processes, such as cell differentiation, development, metabolism, and apoptosis. In addition, miRNAs are associated with many diseases, including cancer. However, conventional detection techniques often suffer from shortcomings such as low sensitivity, so we need to develop a rapid and efficient detection strategy for accurate detection of miRNAs. RESULTS: We have developed an innovative homogeneous electrochemiluminescence (ECL) biosensor. This biosensor employs CRISPR/Cas12a gene editing technology for accurate and efficient detection of microRNA (miRNA). Compared to conventional technologies, this biosensor employs a unique homogeneous detection format that eliminates laborious probe fixation steps and greatly simplifies the detection process. By using two amplification techniques - isothermal amplification and T7 RNA polymerase amplification - the biosensor improves the sensitivity and specificity of the assay, providing excellent detection performance in the assay. This makes it possible to evaluate miRNA directly from a variety of biological samples such as cell lysates and diluted human serum. Experimental results convincingly demonstrate the extraordinary performance of this biosensor, including its extremely low detection limit of 1.27 aM, high sensitivity, reproducibility and stability. SIGNIFICANCE: The application of our constructed sensor in distinguishing between cancerous and non-cancerous cell lines highlights its potential for early cancer detection and monitoring. This innovative approach represents a major advancement in the field of miRNA detection, providing a user-friendly, cost-effective, and sensitive solution with broad implications for clinical diagnosis and patient care, especially in point-of-care settings.

Field-deployable viral diagnostic tools for dengue virus based on Cas13a and Cas12a.

The diagnosis of dengue virus (DENV) has been challenging particularly in areas far from clinical laboratories. Early diagnosis of pathogens is a prerequisite for the timely treatment and pathogen control. An ideal diagnostic for viral infections should possess high sensitivity, specificity, and flexibility. In this study, we implemented dual amplification involving Cas13a and Cas12a, enabling sensitive and visually aided diagnostics for the dengue virus. Cas13a recognized the target RNA by crRNA and formed the assembly of the Cas13a/crRNA/RNA ternary complex, engaged in collateral cleavage of nearby crRNA of Cas12a. The Cas12a/crRNA/dsDNA activator ternary complex could not be assembled due to the absence of crRNA of Cas12a. Moreover, the probe, with 5' and 3' termini labeled with FAM and biotin, could not be separated. The probes labeled with FAM and biotin, combined the Anti-FAM and the Anti-Biotin Ab-coated gold nanoparticle, and conformed sandwich structure on the T-line. The red line on the paper strip caused by clumping of AuNPs on the T-line indicated the detection of dengue virus. This technique, utilizing an activated Cas13a system cleaving the crRNA of Cas12a, triggered a cascade that amplifies the virus signal, achieving a low detection limit of 190 fM with fluorescence. Moreover, even at 1 pM, the red color on the T-line was easily visible by naked eyes. The developed strategy, incorporating cascade enzymatic amplification, exhibited good sensitivity and may serve as a field-deployable diagnostic tool for dengue virus.

Gene editing therapy for cardiovascular diseases.

The development of gene editing tools has been a significant area of research in the life sciences for nearly 30 years. These tools have been widely utilized in disease detection and mechanism research. In the new century, they have shown potential in addressing various scientific challenges and saving lives through gene editing therapies, particularly in combating cardiovascular disease (CVD). The rapid advancement of gene editing therapies has provided optimism for CVD patients. The progress of gene editing therapy for CVDs is a comprehensive reflection of the practical implementation of gene editing technology in both clinical and basic research settings, as well as the steady advancement of research and treatment of CVDs. This article provides an overview of the commonly utilized DNA-targeted gene editing tools developed thus far, with a specific focus on the application of these tools, particularly the clustered regularly interspaced short palindromic repeat/CRISPR-associated genes (Cas) (CRISPR/Cas) system, in CVD gene editing therapy. It also delves into the challenges and limitations of current gene editing therapies, while summarizing ongoing research and clinical trials related to CVD. The aim is to facilitate further exploration by relevant researchers by summarizing the successful applications of gene editing tools in the field of CVD.

Exploiting viral vectors to deliver genome editing reagents in plants.

Genome editing holds great promise for the molecular breeding of plants, yet its application is hindered by the shortage of simple and effective means of delivering genome editing reagents into plants. Conventional plant transformation-based methods for delivery of genome editing reagents into plants often involve prolonged tissue culture, a labor-intensive and technically challenging process for many elite crop cultivars. In this review, we describe various virus-based methods that have been employed to deliver genome editing reagents, including components of the CRISPR/Cas machinery and donor DNA for precision editing in plants. We update the progress in these methods with recent successful examples of genome editing achieved through virus-based delivery in different plant species, highlight the advantages and limitations of these delivery approaches, and discuss the remaining challenges.

Genome editing toward biofortified soybean with minimal trade-off between low phytic acid and yield.

Phytic acid (PA) in grain seeds reduces the bioavailability of nutrient elements in monogastric animals, and an important objective for crop seed biofortification is to decrease the seed PA content. Here, we employed CRISPR/Cas9 to generate a PA mutant population targeting PA biosynthesis and transport genes, including two multi-drug-resistant protein 5 (MRP5) and three inositol pentose-phosphate kinases (IPK1). We characterized a variety of lines containing mutations on multiple IPK and MRP5 genes. The seed PA was more significantly decreased in higher-order mutant lines with multiplex mutations. However, such mutants also exhibited poor agronomic performance. In the population, we identified  two lines carrying single mutations in ipk1b and ipk1c, respectively. These mutants exhibited moderately reduced PA content, and regular agronomic performance compared to the wild type. Our study indicates that moderately decreasing PA by targeting single GmIPK1 genes, rather than multiplex mutagenesis toward ultra-low PA, is an optimal strategy for low-PA soybean with a minimal trade-off in yield performance. Supplementary Information: The online version contains supplementary material available at 10.1007/s42994-024-00158-4.

Efficient genome editing in rice with miniature Cas12f variants.

Genome editing, particularly using the CRISPR/Cas system, has revolutionized biological research and crop improvement. Despite the widespread use of CRISPR/Cas9, it faces limitations such as PAM sequence requirements and challenges in delivering its large protein into plant cells. The hypercompact Cas12f, derived from Acidibacillus sulfuroxidans (AsCas12f), stands out due to its small size of only 422 amino acids and its preference for a T-rich motif, presenting advantageous features over SpCas9. However, its editing efficiency is extremely low in plants. Recent studies have generated two AsCas12f variants, AsCas12f-YHAM and AsCas12f-HKRA, demonstrating higher editing efficiencies in mammalian cells, yet their performance in plants remains unexplored. In this study, through a systematic investigation of genome cleavage activity in rice, we unveiled a substantial enhancement in editing efficiency for both AsCas12f variants, particularly for AsCas12f-HKRA, which achieved an editing efficiency of up to 53%. Furthermore, our analysis revealed that AsCas12f predominantly induces deletion in the target DNA, displaying a unique deletion pattern primarily concentrated at positions 12, 13, 23, and 24, resulting in deletion size mainly of 10 and 11 bp, suggesting significant potential for targeted DNA deletion using AsCas12f. These findings expand the toolbox for efficient genome editing in plants, offering promising prospects for precise genetic modifications in agriculture. Supplementary Information: The online version contains supplementary material available at 10.1007/s42994-024-00168-2.

Regulation of gene-edited plants in Europe: from the valley of tears into the shining sun?

Some 20 years ago, the EU introduced complex regulatory rules for the growth of transgenic crops, which resulted in a de facto ban to grow these plants in fields within most European countries. With the rise of novel genome editing technologies, it has become possible to improve crops genetically in a directed way without the need for incorporation of foreign genes. Unfortunately, in 2018, the European Court of Justice ruled that such gene-edited plants are to be regulated like transgenic plants. Since then, European scientists and breeders have challenged this decision and requested a revision of this outdated law. Finally, after 5 years, the European Commission has now published a proposal on how, in the future, to regulate crops produced by new breeding technologies. The proposal tries to find a balance between the different interest groups in Europe. On one side, genetically modified plants, which cannot be discerned from their natural counterparts, will exclusively be used for food and feed and are-besides a registration step-not to be regulated at all. On the other side, plants expressing herbicide resistance are to be excluded from this regulation, a concession to the strong environmental associations and NGOs in Europe. Moreover, edited crops are to be excluded from organic farming to protect the business interests of the strong organic sector in Europe. Nevertheless, if this law passes European parliament and council, unchanged, it will present a big step forward toward establishing a more sustainable European agricultural system. Thus, it might soon be possible to develop and grow crops that are more adapted to global warming and whose cultivation will require lower amounts of pesticides. However, there is still a long way to go until the law is passed. Too often, the storm of arguments raised by the opponents, based on irrational fears of mutations and a naive understanding of nature, has fallen on fruitful ground in Europe.

C-terminal frameshift mutations generate viable knockout mutants with developmental defects for three essential protein kinases.

Loss-of-function mutants are fundamental resources for gene function studies. However, it is difficult to generate viable and heritable knockout mutants for essential genes. Here, we show that targeted editing of the C-terminal sequence of the embryo lethal gene MITOGEN-ACTIVATED PROTEIN KINASES 1 (OsMPK1) results in weak mutants. This C-terminal-edited osmpk1 mutants displayed severe developmental defects and altered disease resistance but generated tens of viable seeds that inherited the mutations. Using the same C-terminal editing approach, we also obtained viable mutants for a wall-associated protein kinase (Os07g0493200) and a leucine-rich repeat receptor-like protein kinase (Os01g0239700), while the null mutations of these genes were lethal. These data suggest that protein kinase activity could be reduced by introducing frameshift mutations adjacent to the C-terminus, which could generate valuable resources for gene function studies and tune protein kinase activity for signaling pathway engineering. Supplementary Information: The online version contains supplementary material available at 10.1007/s42994-024-00165-5.

TaqMan-quantitative PCR assays applied in Neospora caninum knock-outs generated through CRISPR-Cas9 allow to determine the copy numbers of integrated dihydrofolate reductase-thymidylate synthase drug selectable markers.

As for many other organisms, CRISPR-Cas9 mediated genetic modification has gained increasing importance for the identification of vaccine candidates and drug targets in Neospora caninum, an apicomplexan parasite causing abortion in cattle and neuromuscular disease in dogs. A widely used approach for generating knock-out (KO) strains devoid of virulence factors is the integration of a drug selectable marker such as mutated dihydrofolate reductase-thymidylate synthase (mdhfr-ts) into the target gene, thus preventing the synthesis of respective protein and mediating resistance to pyrimethamine. However, CRISPR-Cas9 mutagenesis is not free of off-target effects, which can lead to integration of multiple mdhfr-ts copies into other sites of the genome. To determine the number of integrated mdhfr-ts in N. caninum, a duplex quantitative TaqMan PCR was developed. For this purpose, primers were designed that amplifies a 106 bp fragment from wild-type (WT) parasites corresponding to the single copy wtdhfrs-ts gene, as well as the mutated mdhfrs-ts present in KO parasites that confers resistance and were used simultaneously with primers amplifying the diagnostic NC5 gene. Thus, the dhfr-ts to NC5 ratio should be approximately 1 in WT parasites, while in KO parasites with a single integrated mdhrf-ts gene this ratio is doubled, and in case of multiple integration events even higher. This approach was applied to the Neospora KO strains NcΔGRA7 and NcΔROP40. For NcΔGRA7, the number of tachyzoites determined by dhfr-ts quantification was twice the number of tachyzoites determined by NC5 quantification, thus indicating that only one mdhfr-ts copy was integrated. The results obtained with the NcΔROP40 strain, however, showed that the number of dhfr-ts copies per genome was substantially higher, indicating that at least three copies of the selectable mdhfr-ts marker were integrated into the genomic DNA during gene editing by CRISPR-Cas9. This duplex TaqMan-qPCR provides a reliable and easy-to-use tool for assessing CRISPR-Cas9 mediated mutagenesis in WT N. caninum strains.

Genome Editing to Produce Knockout Mutations of Seed Dormancy Genes in Wheat.

Knockout mutants provide definitive information about the functions of genes related to agronomic traits, including seed dormancy. However, it takes many years to produce knockout mutants using conventional techniques in polyploid plants such as hexaploid wheat. Genome editing with sequence-specific nucleases is a promising approach for obtaining knockout mutations in all targeted homoeologs of wheat simultaneously. Here, we describe a procedure to produce a triple recessive mutant in wheat via genome editing. This protocol covers the evaluation of gRNA and Agrobacterium-mediated transformation to obtain edited wheat seedlings.

FRETting about CRISPR-Cas Assays: Dual-Channel Reporting Lowers Detection Limits and Times-to-Result.

Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR-Associated Protein (CRISPR-Cas) systems have evolved several mechanisms to specifically target foreign DNA. These properties have made them attractive as biosensors. The primary drawback associated with contemporary CRISPR-Cas biosensors is their weak signaling capacity, which is typically compensated for by coupling the CRISPR-Cas systems to nucleic acid amplification. An alternative strategy to improve signaling capacity is to engineer the reporter, i.e., design new signal-generating substrates for Cas proteins. Unfortunately, due to their reliance on custom synthesis, most of these engineered reporter substrates are inaccessible to many researchers. Herein, we investigate a substrate based on a fluorescein (FAM)-tetramethylrhodamine (TAMRA) Förster resonant energy-transfer (FRET) pair that functions as a seamless "drop-in" replacement for existing reporters, without the need to change any other aspect of a CRISPR-Cas12a-based assay. The reporter is readily available and employs FRET to produce two signals upon cleavage by Cas12a. The use of both signals in a ratiometric manner provides for improved assay performance and a decreased time-to-result for several CRISPR-Cas12a assays when compared to a traditional FAM-Black Hole Quencher (BHQ) quench-based reporter. We comprehensively characterize this reporter to better understand the reasons for the improved signaling capacity and benchmark it against the current standard CRISPR-Cas reporter. Finally, to showcase the real-world utility of the reporter, we employ it in a Recombinase Polymerase Amplification (RPA)-CRISPR-Cas12a DNA Endonuclease-Targeted CRISPR Trans Reporter (DETECTR) assay to detect Human papillomavirus in patient-derived samples.

Revisiting the impact of genomic hot spots: C12orf35 locus as a hot spot and engineering target.

Traditional Chinese hamster ovary (CHO) cell line development is based on random integration (RI) of transgene that causes clonal variation and subsequent large-scale clone screening. Therefore, site-specific integration (SSI) of transgenes into genomic hot spots has recently emerged as an alternative method for cell line development. However, the specific mechanisms underlying hot spot site formation remain unclear. In this study, we aimed to generate landing pad (LP) cell lines via the RI of transgenes encoding fluorescent reporter proteins flanked by recombination sites to facilitate recombinase-mediated cassette exchange. The RI-based LP cell line expressing high reporter levels with spontaneous C12orf35 locus deletion exhibited similar reporter fluorescent protein levels compared to targeted integrants with an identical reporter LP construct at the CHO genome hot spot, the C12orf35 locus. Additionally, Resf1, a C12orf35 locus gene, knockout (KO) in the RI-based LP cell line with conserved C12orf35 increased reporter expression levels, comparable to those in cell lines with C12orf35 locus disruption. These results indicate that the effect of SSI into the C12orf35 locus, a genomic hot spot, on high-level transgene expression was caused by C12orf35 disruption. In contrast to C12orf35 KO, KO at other well-known hot spot sites at specific loci of genes, including Fer1L4, Hprt1, Adgrl4, Clcc1, Dop1b, and Ddc, did not increase transgene expression. Overall, our findings suggest that C12orf35 is a promising engineering target and a hot spot for SSI-based cell line development.

Utilizing codon degeneracy in the design of donor DNA for CRISPR/Cas9-mediated gene editing to streamline the screening process for single amino acid mutations.

Chlamydomonas reinhardtii, a unicellular green alga, has been widely used as a model organism for studies of algal, plant and ciliary biology. The generation of targeted amino acid mutations is often necessary, and this can be achieved using CRISPR/Cas9 induced homology-directed repair to install genomic modifications from exogenous donor DNA. Due to the low gene editing efficiency, the technical challenge lies in identifying the mutant cells. Direct sequencing is not practical, and pre-screening is required. Here, we report a strategy for generating and screening for amino acid point mutations using the CRISPR/Cas9 gene editing system. The strategy is based on designing donor DNA using codon degeneracy, which enables the design of specific primers to facilitate mutant screening by PCR. An in vitro assembled RNP complex, along with a dsDNA donor and an antibiotic resistance marker, was electroporated into wild-type cells, followed by PCR screening. To demonstrate this principle, we have generated the E102K mutation in centrin and the K40R mutation in α-tubulin. The editing efficiencies at the target sites for Centrin, TUA1, TUA2 were 4, 24 and 8% respectively, based on PCR screening. More than 80% of the mutants with the expected size of PCR products were precisely edited, as revealed by DNA sequencing. Subsequently, the precision-edited mutants were biochemically verified. The introduction of codon degeneracy did not affect the gene expression of centrin and α-tubulins. Thus, this approach can be used to facilitate the identification of point mutations, especially in genes with low editing rates.

Methods for Functional Characterization of Genetic Polymorphisms of Non-Coding Regulatory Regions of the Human Genome.

Currently, numerous associations between genetic polymorphisms and various diseases have been characterized through the Genome-Wide Association Studies. Majority of the clinically significant polymorphisms are localized in non-coding regions of the genome. While modern bioinformatic resources make it possible to predict molecular mechanisms that explain influence of the non-coding polymorphisms on gene expression, such hypotheses require experimental verification. This review discusses the methods for elucidating molecular mechanisms underlying dependence of the disease pathogenesis on specific genetic variants within the non-coding sequences. A particular focus is on the methods for identification of transcription factors with binding efficiency dependent on polymorphic variations. Despite remarkable progress in bioinformatic resources enabling prediction of the impact of polymorphisms on the disease pathogenesis, there is still the need for experimental approaches to investigate this issue.

Engineered crRNA for CRISPR/Cas-assisted biosensing.

CRISPR/Cas-based diagnostics (CRISPR-Dx) face challenges, including difficulty in detecting ultrashort nucleotides, preamplification dependency, cross-contamination, insufficiency in on-pot detection paradigms, and inconvenience in detecting non-nucleic acid targets. This forum outlines the advances in engineered CRISPR RNA (crRNA) that address the aforementioned problems, highlighting challenges, opportunities, and future directions.

Enhancing the production of L-proline in recombinant Escherichia coli BL21 by metabolic engineering.

L-proline is widely used in the fields of food, medicine and agriculture, and is also an important raw material for the synthesis of trans-4-hydroxy-L-proline. In this study, enhancing the production of L-proline by metabolic engineering was investigated. Three genes, proB, proA and proC, were introduced into Escherichia coli BL21 by molecular biology technology to increase the metabolic flow of L-proline from glucose. The genes putP and proP related to the proline transfer were knocked out by CRISPR/Cas9 gene editing technology to weaken the feedback inhibition of proB to increase the production of L-proline. The fermentation curves of the engineered strain at different glucose concentrations were determined, and a glucose concentration of 10 g/L was chosen to expand the batch culture to 1 L shake flask. Ultimately, through these efforts, the titer of L-proline reached 832.19 mg/L in intermittent glucose addition fermentation in a 1 L shake flask.

Human Organoids for Rapid Validation of Gene Variants Linked to Cochlear Malformations.

Developmental anomalies of the hearing organ, the cochlea, are diagnosed in approximately one-fourth of individuals with congenital deafness. Most patients with cochlear malformations remain etiologically undiagnosed due to insufficient knowledge about underlying genes or the inability to make conclusive interpretations of identified genetic variants. We used exome sequencing for genetic evaluation of hearing loss associated with cochlear malformations in three probands from unrelated families. We subsequently generated monoclonal induced pluripotent stem cell (iPSC) lines, bearing patient-specific knockins and knockouts using CRISPR/Cas9 to assess pathogenicity of candidate variants. We detected FGF3 (p.Arg165Gly) and GREB1L (p.Cys186Arg), variants of uncertain significance in two recognized genes for deafness, and PBXIP1(p.Trp574*) in a candidate gene. Upon differentiation of iPSCs towards inner ear organoids, we observed significant developmental aberrations in knockout lines compared to their isogenic controls. Patient-specific single nucleotide variants (SNVs) showed similar abnormalities as the knockout lines, functionally supporting their causality in the observed phenotype. Therefore, we present human inner ear organoids as a tool to rapidly validate the pathogenicity of DNA variants associated with cochlear malformations.