RESUMO
The CRISPR/Cas9 system is a versatile genome editing platform in biotechnology and therapeutics. However, the requirement of protospacer adjacent motifs (PAMs) limits the genome targeting scope. To expand this repertoire, we revisited and engineered a compact Cas9 orthologue derived from Neisseria cinerea (NcCas9) for efficient genome editing in mammal cells. We demonstrated that NcCas9 generates genome editing at target sites with N4GYAT (Y = T/C) PAM which cannot be recognized by existing Cas9s. By optimizing the NcCas9 architecture and its spacer length, editing efficacy of NcCas9 was further improved in human cells. In addition, the NcCas9-derived Base editors can efficiently generate base conversions. Six anti-CRISPR (Acr) proteins were identified as off-switches for NcCas9. Moreover, NcCas9 successfully generated efficient editing of mouse embryos by microinjection of NcCas9 mRNA and the corresponding sgRNA. Thus, the NcCas9 holds the potential to broaden the CRISPR/Cas9 toolsets for efficient gene modifications and therapeutic applications.
Assuntos
Edição de Genes , Neisseria cinerea , Camundongos , Humanos , Animais , Sistemas CRISPR-Cas , Proteína 9 Associada à CRISPR/genética , Proteína 9 Associada à CRISPR/metabolismo , Neisseria cinerea/genética , Neisseria cinerea/metabolismo , Genoma , Mamíferos/genéticaRESUMO
Mosaic penA alleles have caused most of the cephalosporin resistance in Neisseria gonorrhoeae, but their evolution is mostly unknown. The penA gene from Neisseria cinerea strain AM1601 (ceftriaxone MIC, 1.0 µg/ml) caused ceftriaxone resistance (MIC, 1 µg/ml) in a ceftriaxone-susceptible gonococcal strain. The 3'-terminal half of AM1601 penA was almost identical to that of the ceftriaxone-resistant gonococcal GU140106 and FC428 strains. N. cinerea can serve as a reservoir of ceftriaxone resistance-mediating penA sequences that can be transferred to gonococci.
Assuntos
Bacteriemia/microbiologia , Proteínas de Transporte/genética , Resistência às Cefalosporinas/genética , Transferência Genética Horizontal , Gonorreia/microbiologia , Neisseria cinerea/genética , Neisseria gonorrhoeae/genética , Alelos , Bacteriemia/diagnóstico , Bacteriemia/tratamento farmacológico , Sequência de Bases , Proteínas de Transporte/metabolismo , Expressão Gênica , Gonorreia/diagnóstico , Gonorreia/tratamento farmacológico , Humanos , Testes de Sensibilidade Microbiana , Mutação , Neisseria cinerea/efeitos dos fármacos , Neisseria cinerea/metabolismo , Neisseria gonorrhoeae/efeitos dos fármacos , Neisseria gonorrhoeae/metabolismo , Alinhamento de Sequência , Homologia de Sequência do Ácido Nucleico , D-Ala-D-Ala Carboxipeptidase Tipo SerinaRESUMO
Neisseria cinerea is a human commensal. The first known case of meningitis and bacteremia due to Neisseria cinerea following percutaneous glycerol instillation of the trigeminal ganglion is reported. Conventional phenotypic methods and complete 16S RNA gene sequencing accurately identified the pathogen. Difficulties in differentiation from pathogenic neisseriae are discussed.
Assuntos
Bacteriemia/diagnóstico , Meningites Bacterianas/diagnóstico , Neisseria cinerea/isolamento & purificação , Infecções por Neisseriaceae/diagnóstico , Antibacterianos/farmacologia , Bacteriemia/complicações , Bacteriemia/microbiologia , Análise por Conglomerados , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Ribossômico/química , DNA Ribossômico/genética , Cabeça/diagnóstico por imagem , Humanos , Masculino , Meningites Bacterianas/complicações , Meningites Bacterianas/microbiologia , Testes de Sensibilidade Microbiana , Pessoa de Meia-Idade , Dados de Sequência Molecular , Neisseria cinerea/classificação , Neisseria cinerea/efeitos dos fármacos , Neisseria cinerea/genética , Infecções por Neisseriaceae/microbiologia , Infecções por Neisseriaceae/patologia , Filogenia , RNA Ribossômico 16S/genética , Rizotomia/efeitos adversos , Análise de Sequência de DNA , Tomografia Computadorizada por Raios X , Neuralgia do Trigêmeo/terapiaRESUMO
To better understand Neisseria meningitidis genomes and virulence, microarray comparative genome hybridization (mCGH) data were collected from one Neisseria cinerea, two Neisseria lactamica, two Neisseria gonorrhoeae and 48 Neisseria meningitidis isolates. For N. meningitidis, these isolates are from diverse clonal complexes, invasive and carriage strains, and all major serogroups. The microarray platform represented N. meningitidis strains MC58, Z2491 and FAM18, and N. gonorrhoeae FA1090. By comparing hybridization data to genome sequences, the core N. meningitidis genome and insertions/deletions (e.g. capsule locus, type I secretion system) related to pathogenicity were identified, including further characterization of the capsule locus, bioinformatics analysis of a type I secretion system, and identification of some metabolic pathways associated with intracellular survival in pathogens. Hybridization data clustered meningococcal isolates from similar clonal complexes that were distinguished by the differential presence of six distinct islands of horizontal transfer. Several of these islands contained prophage or other mobile elements, including a novel prophage and a transposon carrying portions of a type I secretion system. Acquisition of some genetic islands appears to have occurred in multiple lineages, including transfer between N. lactamica and N. meningitidis. However, island acquisition occurs infrequently, such that the genomic-level relationship is not obscured within clonal complexes. The N. meningitidis genome is characterized by the horizontal acquisition of multiple genetic islands; the study of these islands reveals important sets of genes varying between isolates and likely to be related to pathogenicity.
Assuntos
Transferência Genética Horizontal , Genoma Bacteriano , Genômica , Neisseria meningitidis/genética , Cápsulas Bacterianas/genética , Transporte Biológico/genética , Análise por Conglomerados , Elementos de DNA Transponíveis , Ilhas Genômicas , Sequências Repetitivas Dispersas , Neisseria cinerea/genética , Neisseria gonorrhoeae/genética , Neisseria lactamica/genética , Neisseria meningitidis/patogenicidade , Hibridização de Ácido Nucleico , Análise de Sequência com Séries de Oligonucleotídeos , Prófagos/genética , Sintenia , Virulência/genéticaRESUMO
The genome of Neisseria meningitidis serogroup B strain MC58 contains three genes - nmb0278, nmb0294 and nmb0407 - encoding putative homologues of DsbA, a periplasmic thiol disulphide oxidoreductase protein-folding catalyst of the Dsb protein family. DsbA assists the folding of periplasmic and membrane proteins in diverse organisms. While all three cloned genes complemented the DTT sensitivity of dsbA-null Escherichia coli, they showed different activities in folding specific target proteins in this background. NMB0278 protein was the most active in complementing defects in motility and alkaline phosphatase activity, while NMB0294 was the most active in folding periplasmic MalF. NMB0407 showed the weakest activity in all assays. It is extremely unusual for organisms to contain more than one chromosomal dsbA. Among the members of the genus Neisseria, only the meningococcus carries all three of these genes. Strains of Neisseria gonorrhoeae, Neisseria lactamica, Neisseria cinerea and Neisseria polysaccharea contained only homologues of nmb0278 and nmb0407, while Neisseria flava, Neisseria subflava and Neisseria flavescens carried only nmb0294. It is speculated that the versatility of the meningococcus in surviving in different colonizing and invasive disease settings may be derived in part from an enhanced potential to deploy outer-membrane proteins, a consequence of carrying an extended repertoire of protein-folding catalysts.