RESUMO
Type V CRISPR-associated systems (Cas)12 family nucleases are considered to have evolved from transposon-associated TnpB, and several of these nucleases have been engineered as versatile genome editors. Despite the conserved RNA-guided DNA-cleaving functionality, these Cas12 nucleases differ markedly from the currently identified ancestor TnpB in aspects such as guide RNA origination, effector complex composition, and protospacer adjacent motif (PAM) specificity, suggesting the presence of earlier evolutionary intermediates that could be mined to develop advanced genome manipulation biotechnologies. Using evolutionary and biochemical analyses, we identify that the miniature type V-U4 nuclease (referred to as Cas12n, 400-700 amino acids) is likely the earliest evolutionary intermediate between TnpB and large type V CRISPR systems. We demonstrate that with the exception of CRISPR array emergence, CRISPR-Cas12n shares several similar characteristics with TnpB-ωRNA, including a miniature and likely monomeric nuclease for DNA targeting, origination of guide RNA from nuclease coding sequence, and generation of a small sticky end following DNA cleavage. Cas12n nucleases recognize a unique 5'-AAN PAM sequence, of which the A nucleotide at the -2 position is also required for TnpB. Moreover, we demonstrate the robust genome-editing capacity of Cas12n in bacteria and engineer a highly efficient CRISPR-Cas12n (termed Cas12Pro) with up to 80% indel efficiency in human cells. The engineered Cas12Pro enables base editing in human cells. Our results further expand the understanding regarding type V CRISPR evolutionary mechanisms and enrich the miniature CRISPR toolbox for therapeutic applications.
Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Humanos , Sistemas CRISPR-Cas/genética , Edição de Genes/métodos , Endonucleases/genética , DNA/genética , RNARESUMO
CRISPR-Cas12f nucleases are currently one of the smallest genome editors, exhibiting advantages for efficient delivery via cargo-size-limited adeno-associated virus delivery vehicles. Most characterized Cas12f nucleases recognize similar T-rich protospacer adjacent motifs (PAMs) for DNA targeting, substantially restricting their targeting scope. Here we report the cryogenic electron microscopy structure and engineering of a miniature Clostridium novyi Cas12f1 nuclease (CnCas12f1, 497 amino acids) with rare C-rich PAM specificity. Structural characterizations revealed detailed PAM recognition, asymmetric homodimer formation and single guide RNA (sgRNA) association mechanisms. sgRNA engineering transformed CRISPR-CnCas12f1, which initially was incapable of genome targeting in bacteria, into an effective genome editor in human cells. Our results facilitate further understanding of CRISPR-Cas12f1 working mechanism and expand the mini-CRISPR toolbox.
Assuntos
Sistemas CRISPR-Cas , RNA Guia de Sistemas CRISPR-Cas , Humanos , Sistemas CRISPR-Cas/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , DNA/química , Genoma , Endonucleases/genética , Endonucleases/metabolismo , Edição de GenesRESUMO
Pseudomonas aeruginosa harbors sophisticated transcription factor (TF) networks to coordinately regulate cellular metabolic states for rapidly adapting to changing environments. The extraordinary capacity in fine-tuning the metabolic states enables its success in tolerance to antibiotics and evading host immune defenses. However, the linkage among transcriptional regulation, metabolic states and antibiotic tolerance in P. aeruginosa remains largely unclear. By screening the P. aeruginosa TF mutant library constructed by CRISPR/Cas12k-guided transposase, we identify that rccR (PA5438) is a major genetic determinant in aminoglycoside antibiotic tolerance, the deletion of which substantially enhances bacterial tolerance. We further reveal the inhibitory roles of RccR in pyruvate metabolism (aceE/F) and glyoxylate shunt pathway (aceA and glcB), and overexpression of aceA or glcB enhances bacterial tolerance. Moreover, we identify that 2-keto-3-deoxy-6-phosphogluconate (KDPG) is a signal molecule that directly binds to RccR. Structural analysis of the RccR/KDPG complex reveals the detailed interactions. Substitution of the key residue R152, K270 or R277 with alanine abolishes KDPG sensing by RccR and impairs bacterial growth with glycerol or glucose as the sole carbon source. Collectively, our study unveils the connection between aminoglycoside antibiotic tolerance and RccR-mediated central carbon metabolism regulation in P. aeruginosa, and elucidates the KDPG-sensing mechanism by RccR.
Assuntos
Proteínas de Bactérias , Carbono , Pseudomonas aeruginosa , Aminoglicosídeos/farmacologia , Antibacterianos/farmacologia , Antibacterianos/metabolismo , Carbono/metabolismo , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , Proteínas de Bactérias/metabolismo , Redes Reguladoras de GenesRESUMO
The cell-wall recycling process is important for bacterial survival in nutrient-limited conditions and, in certain cases, is directly involved in antibiotic resistance. In the sophisticated cell-wall recycling process in Escherichia coli, the transcriptional repressor MurR controls the expression of murP and murQ, which are involved in transporting and metabolizing N-acetylmuramic acid (MurNAc), generating N-acetylmuramic acid-6-phosphate (MurNAc-6-P) and N-acetylglucosamine-6-phosphate (GlcNAc-6-P). Here, we report that both MurNAc-6-P and GlcNAc-6-P can bind to MurR and weaken the DNA binding ability of MurR. Structural characterizations of MurR in complex with MurNAc-6-P or GlcNAc-6-P as well as in the apo form revealed the detailed ligand recognition chemistries. Further studies showed that only MurNAc-6-P, but not GlcNAc-6-P, is capable of derepressing the expression of murQP controlled by MurR in cells and clarified the substrate specificity through the identification of key residues responsible for ligand binding in the complex structures. In summary, this study deciphered the molecular mechanism of the cell wall recycling process regulated by MurR in E. coli.
Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli , Proteínas Repressoras/metabolismo , Parede Celular/genética , Parede Celular/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Glicosídeo Hidrolases/genética , Ligantes , Fosfatos/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
The RNA-guided CRISPR-associated (Cas) nucleases are versatile tools for genome editing in various organisms. The large sizes of the commonly used Cas9 and Cas12a nucleases restrict their flexibility in therapeutic applications that use the cargo-size-limited adeno-associated virus delivery vehicle. More compact systems would thus offer more therapeutic options and functionality for this field. Here, we report a miniature class 2 type V-F CRISPR-Cas genome-editing system from Acidibacillus sulfuroxidans (AsCas12f1, 422 amino acids). AsCas12f1 is an RNA-guided endonuclease that recognizes 5' T-rich protospacer adjacent motifs and creates staggered double-stranded breaks to target DNA. We show that AsCas12f1 functions as an effective genome-editing tool in both bacteria and human cells using various delivery methods, including plasmid, ribonucleoprotein and adeno-associated virus. The small size of AsCas12f1 offers advantages for cellular delivery, and characterizations of AsCas12f1 may facilitate engineering more compact genome-manipulation technologies.
Assuntos
Bacillales/química , Proteínas de Bactérias/genética , Proteínas Associadas a CRISPR/genética , Proteínas de Bactérias/metabolismo , Proteínas Associadas a CRISPR/metabolismo , Edição de GenesRESUMO
The rise of antimicrobial resistance limits therapeutic options for infections by methicillin-resistant staphylococci. The staphylococcal cassette chromosome mec (SCCmec) is a mobile genetic element as the only carrier of the methicillin-resistance determinants, the mecA or mecC gene. The use of antibiotics increases the spread of antibiotic resistance, but the mechanism by which antibiotics promote horizontal dissemination of SCCmec is largely unknown. In this study, we demonstrate that many antibiotics, including ß-lactams, can induce the expression of ccrC1 and SCCmec excision from the bacterial chromosome. In particular, three widely used antibiotics targeting DNA replication and repair (sulfamethoxazole, ciprofloxacin and trimethoprim) induced higher levels of ccrC1 expression and higher rates of SCCmec excision even at low concentrations (1/8 × minimum inhibitory concentration). LexA was identified as a repressor of ccrC1 and ccrAB by binding to the promoter regions of ccrC1 and ccrAB. The activation of RecA after antibiotic induction alleviated the repression by LexA and increased the expression of ccrC1 or ccrAB, consequently increasing the excision frequency of the SCCmec for SCCmec transfer. These findings lead us to propose a mechanism by which antimicrobial agents can promote horizontal gene transfer of the mecA gene and facilitate the spread of methicillin resistance.
Assuntos
Elementos de DNA Transponíveis , Transferência Genética Horizontal , Resistência a Meticilina/genética , Recombinases/genética , Resposta SOS em Genética , Staphylococcus/genética , Antibacterianos/toxicidade , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Dano ao DNA , Regulação Bacteriana da Expressão Gênica , Regiões Promotoras Genéticas , Recombinases Rec A/metabolismo , Recombinases/biossíntese , Serina Endopeptidases/metabolismo , Staphylococcus/efeitos dos fármacos , Staphylococcus/metabolismoRESUMO
OBJECTIVES: The bla regulatory system is critical in the regulation of mecA expression particularly when staphylococci lack the mec regulator. We sought to evaluate the effect of bla regulators on the cryptic methicillin-resistant phenotype and resistance conversion under ß-lactam exposure in oxacillin-susceptible or oxacillin-resistant mecA-positive staphylococcal isolates. METHODS: Methicillin-resistant staphylococci isolates, NW19 and DY39, and their mutants, were used in this study. Both NW19 and DY39 carried intact mecA, a truncated mecR1 gene and a single copy of the bla regulatory system. Oxacillin MICs were determined using the agar dilution method. Increased and reduced expression of bla regulators was achieved by overexpression and antisense RNA, respectively. Expression of mecA, blaR1 and blaI was quantified in the presence or absence of oxacillin. RESULTS: NW19 had high expression of blaR1-blaI, low expression of mecA and was oxacillin susceptible, while DY39 expressed a low level of blaR1-blaI, expressed a high level of mecA and was oxacillin resistant. Increased expression of blaR1-blaI in DY39-RI led to an oxacillin-susceptible phenotype. Overexpressing blaR1 in DY39-R did not result in any phenotypic change. Under serial exposure of oxacillin, NW19, DY39-RI and DY39-R, with higher expression of blaR1, converted to be highly resistant at a faster speed compared with DY39 and NW19-KD, which had lower expression of blaR1. CONCLUSIONS: The expression level of BlaI was mainly responsible for the oxacillin-resistant phenotype in oxacillin-susceptible mecA-positive Staphylococcus without mec regulators. The initial amount of BlaR1 was determinative for the phenotypic conversion speed under ß-lactam exposure.
Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Genes Reguladores , Resistência a Meticilina , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Oxacilina/farmacologia , Proteínas de Ligação às Penicilinas/genética , beta-Lactamases/biossíntese , Perfilação da Expressão Gênica , Staphylococcus aureus Resistente à Meticilina/genética , Testes de Sensibilidade Microbiana , FenótipoRESUMO
Excision and integration of staphylococcal cassette chromosome mec (SCCmec) are mediated by cassette chromosome recombinases (Ccr), which play a crucial role in the worldwide spread of methicillin resistance in staphylococci. We report a novel ccr gene, ccrC2, in the SCCmec of a Staphylococcus aureus isolate, BA01611, which showed 62.6% to 69.4% sequence identities to all published ccrC1 sequences. A further survey found that the ccrC2 gene was mainly located among coagulase-negative staphylococci (CoNS) and could be found in staphylococcal isolates from China, the United States, France, and Germany. The ccr gene complex harboring the ccrC2 gene was designated a type 9 complex, and the SCCmec of BA01611 was considered a novel type and was designated type XII (9C2). This novel SCCmec element in BA01611 was flanked by a pseudo-SCC element (ΨSCCBA01611) carrying a truncated ccrA1 gene. Both individual SCC elements and a composite SCC were excised from the chromosome based on detection of extrachromosomal circular intermediates. We advocate inclusion of the ccrC2 gene and type 9 ccr gene complex during revision of the SCCmec typing method.
Assuntos
Proteínas de Bactérias/genética , DNA Bacteriano/genética , Resistência a Meticilina/genética , Staphylococcus aureus Resistente à Meticilina/genética , Leite/microbiologia , Recombinases/genética , Animais , Antibacterianos/farmacologia , Proteínas de Bactérias/metabolismo , Bovinos , Mapeamento Cromossômico , Cromossomos Bacterianos/química , Cromossomos Bacterianos/metabolismo , DNA Bacteriano/metabolismo , DNA Circular/genética , DNA Circular/metabolismo , Feminino , Expressão Gênica , Genótipo , Mastite/tratamento farmacológico , Mastite/microbiologia , Meticilina/farmacologia , Staphylococcus aureus Resistente à Meticilina/classificação , Staphylococcus aureus Resistente à Meticilina/enzimologia , Staphylococcus aureus Resistente à Meticilina/isolamento & purificação , Recombinases/metabolismo , Infecções Estafilocócicas/tratamento farmacológico , Infecções Estafilocócicas/microbiologiaRESUMO
The structure of a composite staphylococcal cassette chromosome (SCC) carried by a methicillin-resistant Staphylococcus haemolyticus (NW19A) isolated from a bovine milk sample was analyzed. The formation of the circular forms of both single SCC elements and composite SCC elements was detected in NW19A. Twenty heavy metal and antibiotic resistance-related genes coexisted in this composite SCC, suggesting that these genes might be coselected under environmental pressure. The mec gene complex in NW19A, designated type C3, is different from classic C1 or C2 gene complexes structurally and likely evolves differently. Furthermore, results from alignment of the SCC composite island of NW19A with 50 related sequences from different staphylococcal strains provided additional evidence to support the notion that coagulase-negative staphylococci (CoNS) are the original host of heavy metal resistance genes among staphylococci. Given that a SCC composite island could transfer freely among different staphylococcal species from different hosts, more attention should be paid to contamination with heavy metals and antibiotics in dairy farming environments, including wastewater, soil, feces, and feed.
Assuntos
Mastite Bovina/microbiologia , Metais Pesados/metabolismo , Leite/microbiologia , Staphylococcus haemolyticus/efeitos dos fármacos , Animais , Bovinos , Cromossomos Bacterianos/genética , Biologia Computacional , Resistência Microbiana a Medicamentos , Testes de Sensibilidade Microbiana , Staphylococcus haemolyticus/genéticaRESUMO
Cholangiocarcinoma is a highly aggressive cancer with a dismal prognosis and limited resectability. Chemotherapy has demonstrated tremendous benefits for patients with advanced and inoperable cancer, but drug resistance poses a significant obstacle. Despite recent progress in cancer therapy, the mechanisms driving drug resistance are multifaceted and not completely comprehended. Non-coding RNA refers to RNA molecules that are endogenous and do not code for proteins. Particularly microRNAs, long non-coding RNAs, circular RNAs, are widely acknowledged to be involved in cancer initiation, proliferation, and metastasis. Recently, evidences suggests that abnormal expression of non-coding RNAs contributes to resistance to different type of cancer therapies in cholangiocarcinoma. This occurs via the rewiring of signaling pathways including the reduction of anticancer drugs, apoptosis, interaction between cholangiocarcinoma and tumor-infiltrating immune cells, and cancer stemness. Thus, our review aims to demonstrate the potential of targeting non-coding RNA to override drug resistance and summarize the molecular mechanisms of how non-coding RNA contributes to drug resistance in cholangiocarcinoma.
RESUMO
CRISPR-Cas9 systems have been widely harnessed for diverse genome editing applications because of their ease of use and high efficiency. However, the large molecular sizes and strict PAM requirements of commonly used CRISPR-Cas9 systems restrict their broad applications in therapeutics. Here, we report the molecular basis and genome editing applications of a novel compact type II-A Eubacterium ventriosum CRISPR-Cas9 system (EvCas9) with 1107 residues and distinct 5'-NNGDGN-3' (where D represents A, T, or G) PAM specificity. We determine the cryo-EM structure of EvCas9 in a complex with an sgRNA and a target DNA, revealing the detailed PAM recognition and sgRNA and target DNA association mechanisms. Additionally, we demonstrate the robust genome editing capacity of EvCas9 in bacteria and human cells with superior fidelity compared to SaCas9 and SpCas9, and we engineer it to be efficient base editors by fusing a cytidine or adenosine deaminase. Collectively, our results facilitate further understanding of CRISPR-Cas9 working mechanisms and expand the compact CRISPR-Cas9 toolbox.
Assuntos
Sistemas CRISPR-Cas , Eubacterium , Edição de Genes , Humanos , Edição de Genes/métodos , Sistemas CRISPR-Cas/genética , RNA Guia de Sistemas CRISPR-Cas , DNA/genéticaRESUMO
Prime editing allows precise installation of any single base substitution and small insertions and deletions without requiring homologous recombination or double-strand DNA breaks in eukaryotic cells. However, the applications in bacteria are hindered and the underlying mechanisms that impede efficient prime editing remain enigmatic. Here, we report the determination of vital cellular factors that affect prime editing in bacteria. Genetic screening of 129 Escherichia coli transposon mutants identified sbcB, a 3'â5' DNA exonuclease, as a key genetic determinant in impeding prime editing in E. coli, combinational deletions of which with two additional 3'â5' DNA exonucleases, xseA and exoX, drastically enhanced the prime editing efficiency by up to 100-fold. Efficient prime editing in wild-type E. coli can be achieved by simultaneously inhibiting the DNA exonucleases via CRISPRi. Our results pave the way for versatile applications of prime editing for bacterial genome engineering.
Assuntos
Proteínas de Escherichia coli , Exodesoxirribonucleases , Exodesoxirribonucleases/genética , Escherichia coli/genética , Escherichia coli/metabolismo , DNA/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Quebras de DNA de Cadeia Dupla , Sistemas CRISPR-Cas/genéticaRESUMO
Previous studies have investigated the determinants of urban tourism development from the various attributes of neighborhood quality. However, traditional methods to assess neighborhood quality are often subjective, costly, and only on a small scale. To fill this research gap, this study applies the recent development in big data of street view images, deep learning algorithms, and image processing technology to assess quantitatively four attributes of neighborhood quality, namely street facilities, architectural landscape, green or ecological environment, and scene visibility. The paper collects more than 7.8 million Baidu SVPs of 232 prefecture-level cities in China and applies deep learning techniques to recognize these images. This paper then tries to examine the influence of neighborhood quality on regional tourism development. Empirical results show that both levels of street facilities and greenery environment promote tourism. However, the construction intensity of the landscape has an inhibitory influence on the development of tourism. The threshold test shows that the intensity of the influence varies with the city's overall economic level. These conclusions are of great significance for the development of China's urban construction and tourism economy, and also provide a useful reference for policymakers. The methodological procedure is reduplicative and can be applied to other challenging cases.
Assuntos
Aprendizado Profundo , Turismo , Características de Residência , Cidades , China , Desenvolvimento EconômicoRESUMO
Gene therapy is limited by inefficient delivery of large clustered regularly interspaced short palindromic repeat (CRISPR) effectors, such as Cas9 and Cas12a nucleases. Cas12f nucleases are currently one of the most compact CRISPR genome editors. However, the available toolkit of efficient Cas12f editors is limited. Here, we report the characterization and engineering of a miniature CRISPR-Cas12f system from Syntrophomonas palmitatica (SpaCas12f1, 497 amino acids). We show that CRISPR-SpaCas12f1 cleaves double-stranded DNA (dsDNA) with 5' T-rich PAM specificity and is naturally active for genome editing in bacteria. We identify that CRISPR-SpaCas12f1 trans-activating CRISPR RNA (tracrRNA) harbors a unique head-to-toe hairpin structure, and the natural hairpin structure is a key factor in restricting genome editing by SpaCas12f1 in human cells. Systematical engineering of SpaCas12f1 guide RNA transforms CRISPR-SpaCas12f1 into an efficient genome editor comparable to Francisella novicida CRISPR-Cas12a. Our findings expand the mini CRISPR toolbox, paving the way for therapeutic applications of CRISPR-SpaCas12f1 and engineering compact genome manipulation technologies.
Assuntos
Sistemas CRISPR-Cas , Aminoácidos/metabolismo , Sistemas CRISPR-Cas/genética , Clostridiales , Endonucleases/genética , Endonucleases/metabolismo , Edição de Genes , Humanos , RNA/metabolismo , RNA Guia de Sistemas CRISPR-CasRESUMO
Lactiplantibacillus plantarum are probiotic bacteria, maintaining the integrity of the gastrointestinal epithelial barrier, and preventing the infection of pathogenic bacteria. Exopolysaccharides (EPSs) are often involved in the probiotic property of L. plantarum. Here, we identified a new EPS-synthetic gene cluster, cpsWc, carrying 13 genes, laid on a large plasmid in a well-characterized probiotic L. plantarum strain LTC-113. The cpsWc gene cluster was horizontally acquired from Weissella cibaria, enhancing the biofilm formation ability of the host strain and its tolerance to harsh environmental stresses, including heat, acid, and bile. Transfer of cpsWc also conferred the probiotic properties to other L. plantarum strains. Moreover, cpsWc strengthened the adhesion of LTC-113 to intestinal epithelial cells. Both the cpsWc-carrying LTC-113 and its EPSs per se effectively attenuated the LPS-induced pro-inflammatory effect of intestinal epithelial cells, and inhibited the adhesion of pathogenic bacteria, such as S. typhimurium and E. coli by exclusion and competition. The newly identified cpsWc gene cluster emphasized the contribution of mobile EPS-synthetic element on the probiotic activity of L. plantarum, and shed a light on the engineering of probiotic bacteria.
RESUMO
Microbes employ sophisticated cellular networks encoded by complex genomes to rapidly adapt to changing environments. High-throughput genome engineering methods are valuable tools for functionally profiling genotype-phenotype relationships and understanding the complexity of cellular networks. However, current methods either rely on special homologous recombination systems and are thus applicable in only limited bacterial species or can generate only nonspecific mutations and thus require extensive subsequent screening. Here, we report a site-specific transposon-assisted genome engineering (STAGE) method that allows high-throughput Cas12k-guided mutagenesis in various microorganisms, such as Pseudomonas aeruginosa and Klebsiella pneumoniae. Exploiting the powerful STAGE technique, we construct a site-specific transposon mutant library that focuses on all possible transcription factors (TFs) in P. aeruginosa, enabling the comprehensive identification of essential genes and antibiotic-resistance-related factors. Given its broad host range activity and easy programmability, this method can be widely adapted to diverse microbial species for rapid genome engineering and strain evolution.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas Associadas a CRISPR/metabolismo , Farmacorresistência Bacteriana/genética , Edição de Genes , Klebsiella pneumoniae/genética , Pseudomonas aeruginosa/genética , Fatores de Transcrição/genética , Transposases/metabolismo , Proteínas de Bactérias/genética , Proteínas Associadas a CRISPR/genética , Regulação Bacteriana da Expressão Gênica , Biblioteca Gênica , Genoma Bacteriano , Sequenciamento de Nucleotídeos em Larga Escala , Klebsiella pneumoniae/enzimologia , Mutagênese , Mutação , Pseudomonas aeruginosa/enzimologia , Fatores de Transcrição/metabolismo , Transposases/genéticaRESUMO
Base editing is a promising technique, allowing precise single-base mutagenesis in genomes without double-strand DNA breaks or donor templates. Cytosine base editors (CBEs) convert cytosine to thymidine. In particular, CBEs can transform four codons, CAA, CAG, CGA, and TGG, into stop codons, providing a new means to rapidly inactivate a gene of interest and enabling loss-of-function study in recombination-deficient species and the construction of gene-inactivation libraries. However, designing single guide RNAs (sgRNAs) for gene inactivation is more complicated and more restricted in applicability than using the lustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (CRISPR/Cas9) system only, especially for researchers who do not specialize in the bioinformatics skills needed to design and evaluate sgRNAs. Here, we present a new user-friendly designing tool kit, namely, CRISPR-CBEI (cytosine base editor-mediated gene inactivation), including a Web tool and a command-line tool. The Web tool is dedicated to the design of sgRNAs for CBE-mediated gene inactivation and integrates various functions, including open reading frame (ORF) identification, CBE customization, sgRNA designing, summarizing, and front-end off-target searching against user-defined unlimited-file-size local genome files without the necessity of uploading to the server. The command-line version serves the same purpose but for a larger number of coding DNA sequences (CDSs), for instance, for designing a CBE-inactivation library in a target species which provides comprehensive evaluations of CBEs and target genomes. We envision that this tool would contribute to CBE-inactivation design.IMPORTANCE Life science has been in pursuit of precise and efficient genome editing in living cells since the very beginning of the first restriction cloning attempt. The introduction of RNA-guided CRISPR-associated (Cas) nucleases contributed to this ultimate goal through their ability to deliver a double-strand break (DSB) to a precise target location in various species, obsoleting the preceding editing tools, such as zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs). The derivative technology, base editing, combines the catalytically inactivated Cas nuclease and nucleotide deaminase and mediates the genetic modifications at single-nucleotide precision without introducing a DSB. Moreover, the cytosine base editors (CBEs) are able to transform multiple codons into stop codons, rapidly inactivating a gene of interest and enabling loss-of-function study in some recombination-deficient species. Here, we present the CRISPR-CBEI tool kit to assist the design of sgRNAs for CBE-mediated gene inactivation.
RESUMO
Staphylococcus aureus is a major opportunistic pathogen, infecting animals, and human beings. The bacterial cell wall plays a crucial role in antimicrobial resistance and its infection to host cells. Peptidoglycans (PGs) are a major component of the cell wall in S. aureus, which is heavily decorated with wall teichoic acids (WTAs) and capsular polysaccharides (CPs). The ligation of WTAs and CPs to PGs is catalyzed by LytR-CpsA-Psr (LCP) family proteins, including LcpA, LcpB, and LcpC. However, the involvement of LcpC in antimicrobial resistance of S. aureus and its infection to host cells remains unknown. By creating the LcpC-knockout strains, we showed that the deficiency in LcpC decreased the antimicrobial resistance to ß-lactams and glycopeptides and impeded the binding to various epithelial cells. These changes were accompanied by the morphological changes in bacterial cell wall. More importantly, the knockout of LcpC significantly reduced the pathogenicity of methicillin-resistant S. aureus (MRSA) in mice. Our results suggest that LcpC might be an appealing target for developing a therapeutic approach against MRSA infections.
RESUMO
Laccases are multicopper oxidases with important industrial value. In the study, a novel laccase gene (mco) in a Staphylococcus haemolyticus isolate is identified and heterologously expressed in Escherichia coli. Mco shares less than 40% of amino acid sequence identities with the other characterized laccases, exhibiting the maximal activity at pH 4.0 and 60°C with 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt (ABTS) as a substrate. Additionally, the Mco is tolerant to a wide range of pH, heavy metal ions and many organic solvents, and it has a high decolorization capability toward textile dyes in the absence of redox mediators. The characteristics of the Mco make this laccase potentially useful for industrial applications such as textile finishing. Based on BLASTN results, mco is found to be widely distributed in both the bacterial genome and bacterial plasmids. Its potential role in oxidative defense ability of staphylococci may contribute to the bacterial colonization and survival.
Assuntos
Proteínas de Bactérias/metabolismo , Corantes/metabolismo , Lacase/metabolismo , Staphylococcus haemolyticus/enzimologia , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Clonagem Molecular , Escherichia coli/genética , Temperatura Alta , Concentração de Íons de Hidrogênio , Lacase/química , Lacase/genética , Lacase/isolamento & purificação , Metais/metabolismo , Modelos Moleculares , Filogenia , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Solventes/metabolismo , Especificidade da Espécie , Staphylococcus haemolyticus/classificação , Staphylococcus haemolyticus/genética , Especificidade por SubstratoRESUMO
Precise genetic manipulation is vital to studying bacterial physiology, but is difficult to achieve in some bacterial species due to the weak intrinsic homologous recombination (HR) capacity and lack of a compatible exogenous HR system. Here we report the establishment of a rapid and efficient method for directly converting adenine to guanine in bacterial genomes using the fusion of an adenine deaminase and a Cas9 nickase. The method achieves the conversion of adenine to guanine via an enzymatic deamination reaction and a subsequent DNA replication process rather than HR, which is utilized in conventional bacterial genetic manipulation methods, thereby substantially simplifying the genome editing process. A systematic screening targeting the possibly editable adenine sites of cntBC, the importer of the staphylopine/metal complex in Staphylococcus aureus, pinpoints key residues for metal importation, demonstrating that application of the system would greatly facilitate the genomic engineering of bacteria.