CRISPR-Cas13a cascade-based viral RNA assay for detecting SARS-CoV-2 and its mutations in clinical samples.

SARS-CoV-2 is one of the greatest threats to global human health. Point-of-care diagnostic tools for SARS-CoV-2 could facilitate rapid therapeutic intervention and mitigate transmission. In this work, we report CRISPR-Cas13a cascade-based viral RNA (Cas13C) assay for label-free and isothermal determination of SARS-CoV-2 and its mutations in clinical samples. Cas13a/crRNA was utilized to directly recognize the target of SARS-CoV-2 RNA, and the recognition events sequentially initiate the transcription amplification to produce light-up RNA aptamers for output fluorescence signal. The recognition of viral RNA via Cas13a-guide RNA ensures a high specificity to distinguish SARS-CoV-2 from MERS-CoV and SARS-CoV, as well as viral mutations. A post transcription amplification strategy was triggered after CRISPR-Cas13a recognition contributes to an amplification cascade that achieves high sensitivity for detecting SARS-CoV-2 RNA, with a limit of detection of 0.216 fM. In addition, the Cas13C assay could be able to discriminate single-nucleotide mutation, which was proven with N501Y in SARS-Cov-2 variant. This method was validated by a 100% agreement with RT-qPCR results from 12 clinical throat swab specimens. The Cas13C assay has the potential to be used as a routine nucleic acid test of SARS-CoV-2 virus in resource-limited regions.

Optimization of loop-mediated isothermal amplification (LAMP) assay for robust visualization in SARS-CoV-2 and emerging variants diagnosis.

Loop-mediated isothermal amplification (LAMP) is widely used in detection of pathogenic microorganisms including SARS-CoV-2. However, the performance of LAMP assay needs further exploration in the emerging SARS-CoV-2 variants test. Here, we design serials of primers and select an optimal set for LAMP-based on SARS-CoV-2 N gene for a robust and visual assay in SARS-CoV-2 diagnosis. The limit of detectable template reaches 10 copies of N gene per 25 µL reaction at isothermal 58℃ within 40 min. Importantly, the primers for LAMP assay locate at 12 to 213 nt of N gene, a highly conservative region, which serves as a compatible test in emerging SARS-CoV-2 variants. Comparison to a commercial qPCR assay, this LAMP assay exerts the high viability in diagnosis of 41 clinical samples. Our study optimizes an advantageous LAMP assay for colorimetric detection of SARS-CoV-2 and emerging variants, which is hopeful to be a promising test in COVID-19 surveillance.

Sensitive and rapid on-site detection of SARS-CoV-2 using a gold nanoparticle-based high-throughput platform coupled with CRISPR/Cas12-assisted RT-LAMP.

The outbreak of corona virus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to a global pandemic. The high infectivity of SARS-CoV-2 highlights the need for sensitive, rapid and on-site diagnostic assays of SARS-CoV-2 with high-throughput testing capability for large-scale population screening. The current detection methods in clinical application need to operate in centralized labs. Though some on-site detection methods have been developed, few tests could be performed for high-throughput analysis. We here developed a gold nanoparticle-based visual assay that combines with CRISPR/Cas12a-assisted RT-LAMP, which is called Cas12a-assisted RT-LAMP/AuNP (CLAP) assay for rapid and sensitive detection of SARS-CoV-2. In optimal condition, we could detect down to 4 copies/µL of SARS-CoV-2 RNA in 40 min. by naked eye. The sequence-specific recognition character of CRISPR/Cas12a enables CLAP a superior specificity. More importantly, the CLAP is easy for operation that can be extended to high-throughput test by using a common microplate reader. The CLAP assay holds a great potential to be applied in airports, railway stations, or low-resource settings for screening of suspected people. To the best of our knowledge, this is the first AuNP-based colorimetric assay coupled with Cas12 and RT-LAMP for on-site diagnosis of COVID-19. We expect CLAP assay will improve the current COVID-19 screening efforts, and make contribution for control and mitigation of the pandemic.

Current state of diagnostic, screening and surveillance testing methods for COVID-19 from an analytical chemistry point of view.

Since December 2019, we have been in the battlefield with a new threat to the humanity known as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In this review, we describe the four main methods used for diagnosis, screening and/or surveillance of SARS-CoV-2: Real-time reverse transcription polymerase chain reaction (RT-PCR); chest computed tomography (CT); and different complementary alternatives developed in order to obtain rapid results, antigen and antibody detection. All of them compare the highlighting advantages and disadvantages from an analytical point of view. The gold standard method in terms of sensitivity and specificity is the RT-PCR. The different modifications propose to make it more rapid and applicable at point of care (POC) are also presented and discussed. CT images are limited to central hospitals. However, being combined with RT-PCR is the most robust and accurate way to confirm COVID-19 infection. Antibody tests, although unable to provide reliable results on the status of the infection, are suitable for carrying out maximum screening of the population in order to know the immune capacity. More recently, antigen tests, less sensitive than RT-PCR, have been authorized to determine in a quicker way whether the patient is infected at the time of analysis and without the need of specific instruments.

Bacterial diversity and functional metagenomics expounding the diversity of xenobiotics, stress, defense and CRISPR gene ontology providing eco-efficiency to Himalayan Hot Springs.

Sikkim is one of the bio-diverse states of India, which harbors diverse alkaline and sulfur rich hot springs in its vicinity. However, there is a dearth of data present in terms of microbial and its functional diversity as only a few hot springs have been studied in this area. Thus, in this regard, microbial and functional diversity of two hot springs by NGS, PLFA, and culture-independent approaches were carried out. PLFA and culture-dependent analysis was complementary as the Gram-positive bacteria were abundant in both the hot springs with the dominance of phylum Firmicutes with Geobacillus. Metagenomic analysis revealed the abundance of Proteobacteria, Actinobacteria, and Firmicutes in both hot springs. Functional metagenomics suggested that both Yumthang and Reshi hot spring possess a diverse set of genes analogous to stress such as genes allied to osmotic, heat shock, and acid stresses; defense analogies such as multidrug resistance efflux pump, multidrug transport system, and ß-lactamase; and CRISPR analogues such as related to Cas1, Cas2, Cas3, cmr1-5 proteins, CT1972, and CT1133 gene families. The xenobiotic analogues were found against benzoate, nitrotolune, xylene, DDT, and chlorocyclohexane/chlorobenzene degradation. Thus, these defensive mechanisms against environmental and anthropogenic hiccups and hindrances provide the eco-efficiency to such thermal habitats. The higher enzymatic, degradation, defense, stress potential and the lower percentage identity (< 95%) of isolates encourage the further exploration and exploitation of these habitats for industrial and biotechnological purposes.

Carboxypeptidase D is the only enzyme responsible for antibody C-terminal lysine cleavage in Chinese hamster ovary (CHO) cells.

Heterogeneity of C-terminal lysine levels often observed in therapeutic monoclonal antibodies is believed to result from the proteolysis by endogenous carboxypeptidase(s) during cell culture production. Identifying the responsible carboxypeptidase(s) for C-terminal lysine cleavage in CHO cells would provide valuable insights for antibody production cell culture processes development and optimization. In this study, five carboxypeptidases, CpD, CpM, CpN, CpB, and CpE, were studied for message RNA (mRNA) expression by qRT-PCR analysis in two most commonly used blank hosts (DUXB-11 derived DHFR-deficient DP12 host and DHFR-positive CHOK1 host), used for therapeutic antibody production, as well an antibody-expressing cell line derived from each host. Our results showed that CpD had the highest mRNA expression. When CpD mRNA levels were reduced by RNAi (RNA interference) technology, C-terminal lysine levels increased, whereas there was no obvious change in C-terminal lysine levels when a different carboxypeptidase mRNA level was knocked down suggesting that carboxypeptidase D is the main contributor for C-terminal lysine processing. Most importantly, when CpD expression was knocked out by CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) technology, C-terminal lysine cleavage was completely abolished in CpD knockout cells based on mass spectrometry analysis, demonstrating that CpD is the only endogenous carboxypeptidase that cleaves antibody heavy chain C-terminal lysine in CHO cells. Hence, our work showed for the first time that the cleavage of antibody heavy chain C-terminal lysine is solely mediated by the carboxypeptidase D in CHO cells and our finding provides one solution to eliminating C-terminal lysine heterogeneity for therapeutic antibody production by knocking out CpD gene expression. Biotechnol. Bioeng. 2016;113: 2100-2106. © 2016 Wiley Periodicals, Inc.

Incomplete splicing of neutrophil-specific genes affects neutrophil development in a zebrafish model of poikiloderma with neutropenia.

Poikiloderma with neutropenia (PN) is a rare inherited disorder characterized by poikiloderma, facial dysmorphism, pachyonychia, short stature and neutropenia. The molecular testing of PN patients has identified mutations in the C16orf57 gene, which encodes a protein referred to as USB1 (U Six Biogenesis 1). In this study, we developed a zebrafish model of PN by the microinjection of morpholino antisense oligos to suppress usb1 gene function. Severe morphological defects, including a bent tail, thin yolk extension and reduced body length, were predominant in the Usb1-suppressed embryos (morphants). We also observed significantly decreased number of neutrophils in the morphants by Sudan Black staining. Interestingly, the splicing of genes involved in neutrophil differentiation and development, such as mpx, ncf1, ela3l and npsn, was aberrant in the morphants. However, the splicing of haematopoietic precursors and erythroid-specific genes was unaltered. Importantly, the neutrophil defects were almost completely rescued by co-injection of ela3l mRNA, the most markedly affected gene in the morphants. Our study demonstrated a possible role of USB1 in modulating the tissue-specific gene splicing that eventually leads to the impaired development of neutrophils. This zebrafish model could serve as a valuable tool to investigate the causative role of USB1 in PN pathogenesis.

Small indels induced by CRISPR/Cas9 in the 5' region of microRNA lead to its depletion and Drosha processing retardance.

MicroRNA knockout by genome editing technologies is promising. In order to extend the application of the technology and to investigate the function of a specific miRNA, we used CRISPR/Cas9 to deplete human miR-93 from a cluster by targeting its 5' region in HeLa cells. Various small indels were induced in the targeted region containing the Drosha processing site and seed sequences. Interestingly, we found that even a single nucleotide deletion led to complete knockout of the target miRNA with high specificity. Functional knockout was confirmed by phenotype analysis. Furthermore, de novo microRNAs were not found by RNA-seq. Nevertheless, expression of the pri-microRNAs was increased. When combined with structural analysis, the data indicated that biogenesis was impaired. Altogether, we showed that small indels in the 5' region of a microRNA result in sequence depletion as well as Drosha processing retard.

FORGEdb: a tool for identifying candidate functional variants and uncovering target genes and mechanisms for complex diseases.

The majority of disease-associated variants identified through genome-wide association studies are located outside of protein-coding regions. Prioritizing candidate regulatory variants and gene targets to identify potential biological mechanisms for further functional experiments can be challenging. To address this challenge, we developed FORGEdb ( https://forgedb.cancer.gov/ ; https://forge2.altiusinstitute.org/files/forgedb.html ; and https://doi.org/10.5281/zenodo.10067458 ), a standalone and web-based tool that integrates multiple datasets, delivering information on associated regulatory elements, transcription factor binding sites, and target genes for over 37 million variants. FORGEdb scores provide researchers with a quantitative assessment of the relative importance of each variant for targeted functional experiments.

A Localized CRISPR Assay that Detects Short Nucleic Acid Fragments in Unamplified Genetically Modified Samples.

Detecting short genetically modified (GM) nucleic acid fragments in GM crops and associated products is critically important for the global agriculture industry. Although nucleic acid amplification-based technologies have been widely used for genetically modified organism (GMO) detection, they still struggle to amplify and detect these ultra-short nucleic acid fragments in highly processed products. Here, we used a multiple-CRISPR-derived RNA (crRNA) strategy to detect ultra-short nucleic acid fragments. By combining confinement effects on local concentrations, an amplification-free CRISPR-based short nucleic acid (CRISPRsna) system was established to detect the cauliflower mosaic virus 35S promoter in GM samples. Moreover, we demonstrated assay sensitivity, specificity, and reliability by directly detecting nucleic acid samples from GM crops with a wide genomic range. The CRISPRsna assay avoided possible aerosol contamination from nucleic acid amplification and saved time due to an amplification-free approach. Given that our assay displayed distinct advantages over other technologies in detecting ultra-short nucleic acid fragments, it may have wide applications for detecting GM in highly processed products.

Sensitive and selective DNA detecting electrochemical sensor via double cleaving CRISPR Cas12a and dual polymerization on hyperbranched rolling circle amplification.

Electrochemical sensors are widely used for nucleic acid detection. However, they exhibit low sensitivity and specificity. To overcome these limitations, DNA amplification method is necessary. In this study, we introduced CRISPR (Clustered regularly interspaced short palindromic repeats) Cas12a-dependent hyperbranched rolling circle amplification (HRCA) into an electrochemical sensor platform. By resolving the existing false-positive issue of HRCA, CRISPR Cas12a determines the real positive amplification that able to enhance its sensitivity for extremely low concentrations of nucleic acids and specificity for single-point mutations. In detail, CRISPR Cas12a, which activates the nucleic acid amplification reaction, was used for both trans and cis cleavage for the first time. Finally, selectively amplified DNA was detected using a screen-printed electrode. Using the change in surface coverage by DNA, the electrochemical sensor detected a decrease in the redox signal. In summary, combining a novel DNA amplification method and electrochemical sensor platform, our proposed method compensates for the shortcomings of existing RCA and hyperbranched RCA, secures a high sensitivity of 10 aM, and overcomes false-positivity problems. Moreover, such creative applications of CRISPR Cas12a may lead to the expansion of its applications to other nucleic acid amplification methods.

Applications of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) as a Genetic Scalpel for the Treatment of Cancer: A Translational Narrative Review.

Cancer remains a global health challenge with high prevalence and mortality rates, imposing significant financial and emotional burdens on affected families. However, hope lies in genetic manipulation, with a focus on innovative approaches to combat genetically linked cancers. Clustered regularly interspaced short palindromic repeats (CRISPR), an adaptive immune system found in various bacteria and archaea, hold immense potential. We searched articles on PubMed Central, Medline, and PubMed databases indexed journals. The keywords from the research topic, i.e., "CRISPR AND genetic therapy," were searched, and we found 3397 articles. Following this, we explored the medical subject headings (MeSH) glossary and created a search strategy "Clustered Regularly Interspaced Short Palindromic Repeats"[Mesh] AND "Genetic Therapy"[Majr] and after applying a variety of filters we included 30 studies in our review. CRISPR consists of unique spacers and CRISPR-associated (Cas) genes, operating through acquisition, CRISPR ribonucleic acid (crRNA) biogenesis, and target interference phases. The type II CRISPR-Cas9 system is a well-researched avenue for gene editing, with Cas9 cleaving specific genomic regions and initiating deoxyribonucleic acid (DNA) repair mechanisms. Cancer results from genetic alterations, leading to tumor development with properties like metastasis. CRISPR/Cas9 offers precise genome editing to inhibit tumor formation by removing specific genomic sequences and promoting DNA repair. Challenges in CRISPR's use for cancer therapy, including delivery methods, cell adaptation, and ethical concerns, are recognized. Beyond cancer, CRISPR finds diverse applications in infectious diseases and non-cancerous conditions, signifying its transformative potential in modern medicine. CRISPR technology represents a groundbreaking frontier in cancer therapy and beyond, offering insights into genetic editing, disease mechanisms, and potential cures. Despite challenges and ethical considerations, precise genome editing promises improved cancer treatments and innovative medical interventions in the future.

Starting the conversation on gene therapy for phenylketonuria: Current perspectives of patients, caregivers, and advocates.

Phenylketonuria (PKU) is a rare genetic condition caused by inborn error(s) in the gene for the enzyme phenylalanine hydroxylase. Resulting loss of phenylalanine (Phe) metabolism requires strict dietary therapy and/or medication to prevent toxic accumulation of Phe. Novel investigational therapies, including gene therapies that aim to address underlying causes of PKU, are now entering clinical trials. However, perceptions of this technology in the PKU community have not been assessed. We conducted a qualitative survey recruiting adult patients, caregivers, and patient advocates from the US and 3 EU countries to assess the impact of living with PKU and the perceptions of gene therapy. Telephone interviews were conducted for up to 60 min following a standardized discussion guide. Interviewers classified each participant by their level of knowledge regarding gene therapy as either: low (little or no prior awareness); moderate (awareness of gene therapy as a concept in PKU); or high (working knowledge of gene therapy, e.g., vectors). In total, 33 participants were recruited (patients, n = 24; caregivers, n = 5; advocates, n = 4). The patient sample was well balanced among age groups, sex, and US/EU geographies. The participants' experiences and burden of living with PKU were largely negative, characterized by frustrations with current management consistent with prior reports. Most participants (n = 18/33) were identified as displaying moderate gene-therapy knowledge, 10/33 as displaying high knowledge, and 5/33 as displaying low knowledge. Both positive and negative perceptions were observed; positive perceptions were often linked to "hope" that gene therapy may represent a cure, whereas negative perceptions were linked to the "uncertainty" of outcomes. High knowledge of gene therapy appeared to trend with negative perceptions; 7/10 participants from this group reported high levels of concern over gene therapy. In contrast, participants who displayed low knowledge reported low (n = 3/5) or moderate (n = 2/5) concern, with predominantly positive perceptions. These data highlight the need for education around the theoretical risk:benefit profile of gene therapy. Despite current unknowns around gene therapy, our study demonstrates the important role of healthcare providers as educators who can use available data to provide balanced information to patients and caregivers.

BEtarget: A versatile web-based tool to design guide RNAs for base editing in plants.

CRISPR-dependent base editors enable direct nucleotide conversion without the introduction of double-strand DNA break or donor DNA template, thus expanding the CRISPR toolbox for genetic manipulation. However, designing guide RNAs (gRNAs) for base editors to enable gene correction or inactivation is more complicated than using the CRISPR system for gene disruption. Here, we present a user-friendly web tool named BEtarget dedicated to the design of gRNA for base editing. It is currently supported by 46 plant reference genomes and 5 genomes of non-plant model organisms. BEtarget supports the design of gRNAs with different types of protospacer adjacent motifs (PAM) and integrates various functions, including automatic identification of open reading frame, prediction of potential off-target sites, annotation of codon change, and assessment of gRNA quality. Moreover, the program provides an interactive interface for users to selectively display information about the desired target sites. In brief, we have developed a flexible and versatile web-based tool to simplify complications associated with the design of base editing technology. BEtarget is freely accessible at https://skl.scau.edu.cn/betarget/.

A one-step platform for screening high-efficient and minimal off-target CRISPR/Cas13 crRNAs to eradicate SARS-CoV-2 virus for treatment of COVID-19 patients.

Coronavirus disease 2019 (COVID-19) is a new respiratory illness caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and now spreads globally. Currently, therapeutics and effective treatment options remain scarce and there is no proven drug to treat COVID-19. Targeting the positive-sense RNA genome and viral mRNAs of SARS-CoV-2 to simultaneously degrade viral genome templates for replication and viral mRNAs for essential gene expression would be a strategy to completely realize virus elimination. Type VI CRISPR enzymes Cas13 have recently been identified as programmable RNA-guided, RNA-targeting Cas proteins with nuclease activity that allows for RNA cleavage and degradation. The precise viral RNA detection and antiviral application of the CRISPR/Cas13 system depend on high-efficient and minimal off-target crRNAs. Although a computer-based algorithm has been applied for the design of crRNAs targeting SRAS-CoV-2, the experimental screening system to identify optimal crRNA is not available. We develop a one-step experimental screening system to identify high-efficient crRNAs with minimal off-target effects for CRISPR/Cas13-based SARS-CoV-2 elimination. This platform provides the foundation for CRISPR/Cas13-based diagnostics and therapeutics for COVID-19. This platform is versatile and could also be applied for crRNAs screening for other RNA viruses.

Utilization and Potential of RNA-Based Therapies in Cardiovascular Disease.

Cardiovascular disease (CVD) remains the largest cause of mortality worldwide. The development of new effective therapeutics is a major unmet need. The current review focuses broadly on the concept of nucleic acid (NA)-based therapies, considering the use of various forms of NAs, including mRNAs, miRNAs, siRNA, and guide RNAs, the latter specifically for the purpose of CRISPR-Cas directed gene editing. We describe the current state-of-the-art of RNA target discovery and development, the status of RNA therapeutics in the context of CVD, and some of the challenges and hurdles to be overcome.

CRISPR-Cas9-mediated knockout of CYP79D1 and CYP79D2 in cassava attenuates toxic cyanogen production.

Cassava (Manihot esculenta) is a starchy root crop that supports over a billion people in tropical and subtropical regions of the world. This staple, however, produces the neurotoxin cyanide and requires processing for safe consumption. Excessive consumption of insufficiently processed cassava, in combination with protein-poor diets, can have neurodegenerative impacts. This problem is further exacerbated by drought conditions which increase this toxin in the plant. To reduce cyanide levels in cassava, we used CRISPR-mediated mutagenesis to disrupt the cytochrome P450 genes CYP79D1 and CYP79D2 whose protein products catalyze the first step in cyanogenic glucoside biosynthesis. Knockout of both genes eliminated cyanide in leaves and storage roots of cassava accession 60444; the West African, farmer-preferred cultivar TME 419; and the improved variety TMS 91/02324. Although knockout of CYP79D2 alone resulted in significant reduction of cyanide, mutagenesis of CYP79D1 did not, indicating these paralogs have diverged in their function. The congruence of results across accessions indicates that our approach could readily be extended to other preferred or improved cultivars. This work demonstrates cassava genome editing for enhanced food safety and reduced processing burden, against the backdrop of a changing climate.

A single allele of the hsa-miR-302/367 cluster maintains human pluripotent stem cells.

Introduction: In a diploid organism, two alleles from a single genetic locus are expressed to generate a normal phenotype. Heterozygous deleterious mutation causes a reduction of functional proteins to a half dose and insufficient amounts of functional proteins can occur to generate an in-normal phenotype, namely haploinsufficiency. Heterozygous deleterious mutation of microRNAs (miRs), non-coding RNAs that regulate the expression level of target transcripts, is still not well understood. The hsa-miR-302/367 cluster is the most abundant and specifically up-regulated miR cluster in human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) and plays an important role in the maintenance of pluripotency. Methods: We targeted the hsa-miR-302/367 region via a Cas9 nuclease complex with guide RNA and replaced that region with green fluorescent protein (GFP). Using a homologous donor, consisting of left and right arms and GFP, we confirmed deletion of the hsa-miR-302/367 cluster by homologous recombination without cellular destruction by microscopy. We sub-cloned GFP-positive colonies and checked the genotype of each sub-clone by genomic PCR. We then analyzed the pluripotency of heterozygous knockout cells with a hsa-miR-302/367 cluster by assessing cell proliferation ratio, morphology, and undifferentiated marker gene expression. We also used an embryoid body formation assay and transplanted wild-type and heterozygous knockout cells into immune-deficient mice. Furthermore, to analyze the lineage-specific differentiation potential of heterozygous knockout cells, we differentiated both wild-type and heterozygous knockout cells into neural stem cells. Results: Here, we show that the half dose of mature miRs from the hsa-miR-302/367 cluster loci was sufficient for the continued self-renewal of hiPSCs. All GFP-positive clones were revealed to be heterozygous knockout cells, suggesting hsa-miR-302/367 cluster homozygous knockout cells were not maintained. The cell proliferation ratio, morphology, and expression of undifferentiated marker genes were comparable between wild-type and heterozygous knockout of undifferentiated human iPSCs. In addition, we found that heterozygous knockout human iPSCs have the capacity to differentiate into three germ layers, including neural stem cells. Conclusions: Taken together, a single allele of the hsa-miR-302/367 cluster expresses a sufficient amount of miRs to maintain the pluripotent properties of human stem cells.

Multiplexed site-specific genome engineering in Mycolicibacterium neoaurum by Att/Int system.

Genomic integration of genes and pathway-sized DNA cassettes is often an indispensable way to construct robust and productive microbial cell factories. For some uncommon microbial hosts, such as Mycolicibacterium and Mycobacterium species, however, it is a challenge. Here, we present a multiplexed integrase-assisted site-specific recombination (miSSR) method to precisely and iteratively integrate genes/pathways with controllable copies in the chromosomes of Mycolicibacteria for the purpose of developing cell factories. First, a single-step multi-copy integration method was established in M. neoaurum by a combination application of mycobacteriophage L5 integrase and two-step allelic exchange strategy, the efficiencies of which were ∼100% for no more than three-copy integration events and decreased sharply to ∼20% for five-copy integration events. Second, the R4, Bxb1 and ΦC31 bacteriophage Att/Int systems were selected to extend the available integration toolbox for multiplexed gene integration events. Third, a reconstructed mycolicibacterial Xer recombinases (Xer-cise) system was employed to recycle the selection marker of gene recombination to facilitate the iterative gene manipulation. As a proof of concept, the biosynthetic pathway of ergothioneine (EGT) in Mycolicibacterium neoaurum ATCC 25795 was achieved by remodeling its metabolic pathway with a miSSR system. With six copies of the biosynthetic gene clusters (BGCs) of EGT and pentose phosphate isomerase (PRT), the titer of EGT in the resulting strain in a 30 mL shake flask within 5 days was enhanced to 66 mg/L, which was 3.77 times of that in the wild strain. The improvements indicated that the miSSR system was an effective, flexible, and convenient tool to engineer the genomes of Mycolicibacteria as well as other strains in the Mycobacteriaceae due to their proximate evolutionary relationships.

Plasticity and therapeutic potential of cAMP and cGMP-specific phosphodiesterases in Toxoplasma gondii.

Toxoplasma gondii is a common zoonotic protozoan pathogen adapted to intracellular parasitism in many host cells of diverse organisms. Our previous work has identified 18 cyclic nucleotide phosphodiesterase (PDE) proteins encoded by the parasite genome, of which 11 are expressed during the lytic cycle of its acutely-infectious tachyzoite stage in human cells. Here, we show that ten of these enzymes are promiscuous dual-specific phosphodiesterases, hydrolyzing cAMP and cGMP. TgPDE1 and TgPDE9, with a Km of 18 µM and 31 µM, respectively, are primed to hydrolyze cGMP, whereas TgPDE2 is highly specific to cAMP (Km, 14 µM). Immuno-electron microscopy revealed various subcellular distributions of TgPDE1, 2, and 9, including in the inner membrane complex, apical pole, plasma membrane, cytosol, dense granule, and rhoptry, indicating spatial control of signaling within tachyzoites. Notably, despite shared apical location and dual-catalysis, TgPDE8 and TgPDE9 are fully dispensable for the lytic cycle and show no functional redundancy. In contrast, TgPDE1 and TgPDE2 are individually required for optimal growth, and their collective loss is lethal to the parasite. In vitro phenotyping of these mutants revealed the roles of TgPDE1 and TgPDE2 in proliferation, gliding motility, invasion and egress of tachyzoites. Moreover, our enzyme inhibition assays in conjunction with chemogenetic phenotyping underpin TgPDE1 as a target of commonly-used PDE inhibitors, BIPPO and zaprinast. Finally, we identified a retinue of TgPDE1 and TgPDE2-interacting kinases and phosphatases, possibly regulating the enzymatic activity. In conclusion, our datasets on the catalytic function, physiological relevance, subcellular localization and drug inhibition of key phosphodiesterases highlight the previously-unanticipated plasticity and therapeutic potential of cyclic nucleotide signaling in T. gondii.