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Nature Cell Biology Feb 2022Gene editing is a powerful tool for genome and cell engineering. Exemplified by CRISPR-Cas, gene editing could cause DNA damage and trigger DNA repair processes that are...
Gene editing is a powerful tool for genome and cell engineering. Exemplified by CRISPR-Cas, gene editing could cause DNA damage and trigger DNA repair processes that are often error-prone. Such unwanted mutations and safety concerns can be exacerbated when altering long sequences. Here we couple microbial single-strand annealing proteins (SSAPs) with catalytically inactive dCas9 for gene editing. This cleavage-free gene editor, dCas9-SSAP, promotes the knock-in of long sequences in mammalian cells. The dCas9-SSAP editor has low on-target errors and minimal off-target effects, showing higher accuracy than canonical Cas9 methods. It is effective for inserting kilobase-scale sequences, with an efficiency of up to approximately 20% and robust performance across donor designs and cell types, including human stem cells. We show that dCas9-SSAP is less sensitive to inhibition of DNA repair enzymes than Cas9 references. We further performed truncation and aptamer engineering to minimize its size to fit into a single adeno-associated-virus vector for future application. Together, this tool opens opportunities towards safer long-sequence genome engineering.
Topics: Actins; Aptamers, Nucleotide; CRISPR-Associated Protein 9; CRISPR-Cas Systems; Clustered Regularly Interspaced Short Palindromic Repeats; DNA-Binding Proteins; Dyneins; Escherichia coli Proteins; Gene Editing; Gene Knock-In Techniques; HEK293 Cells; HSP90 Heat-Shock Proteins; HeLa Cells; Hep G2 Cells; Humans; Viral Proteins
PubMed: 35145221
DOI: 10.1038/s41556-021-00836-1 -
Advances in Virus Research 2019The first steps in phage lysis involve a temporally controlled permeabilization of the cytoplasmic membrane followed by enzymatic degradation of the peptidoglycan. For... (Review)
Review
The first steps in phage lysis involve a temporally controlled permeabilization of the cytoplasmic membrane followed by enzymatic degradation of the peptidoglycan. For Caudovirales of Gram-negative hosts, there are two different systems: the holin-endolysin and pinholin-SAR endolysin pathways. In the former, lysis is initiated when the holin forms micron-scale holes in the inner membrane, releasing active endolysin into the periplasm to degrade the peptidoglycan. In the latter, lysis begins when the pinholin causes depolarization of the membrane, which activates the secreted SAR endolysin. Historically, the disruption of the first two barriers of the cell envelope was thought to be necessary and sufficient for lysis of Gram-negative hosts. However, recently a third functional class of lysis proteins, the spanins, has been shown to be required for outer membrane disruption. Spanins are so named because they form a protein bridge that connects both membranes. Most phages produce a two-component spanin complex, composed of an outer membrane lipoprotein (o-spanin) and an inner membrane protein (i-spanin) with a predominantly coiled-coil periplasmic domain. Some phages have a different type of spanin which spans the periplasm as a single molecule, by virtue of an N-terminal lipoprotein signal and a C-terminal transmembrane domain. Evidence is reviewed supporting a model in which the spanins function by fusing the inner membrane and outer membrane. Moreover, it is proposed that spanin function is inhibited by the meshwork of the peptidoglycan, thus coupling the spanin step to the first two steps mediated by the holin and endolysin.
Topics: Bacteriolysis; Bacteriophages; Cell Wall; DNA; Evolution, Molecular; Gene Expression Regulation, Bacterial; Gene Expression Regulation, Viral; Gram-Negative Bacteria; Membrane Fusion; N-Acetylmuramoyl-L-alanine Amidase; Signal Transduction; Viral Proteins
PubMed: 30635077
DOI: 10.1016/bs.aivir.2018.09.003 -
Molecular Microbiology May 2024The site-specific recombination pathway of bacteriophage λ encompasses isoenergetic but highly directional and tightly regulated integrative and excisive reactions that... (Review)
Review
The site-specific recombination pathway of bacteriophage λ encompasses isoenergetic but highly directional and tightly regulated integrative and excisive reactions that integrate and excise the vial chromosome into and out of the bacterial chromosome. The reactions require 240 bp of phage DNA and 21 bp of bacterial DNA comprising 16 protein binding sites that are differentially used in each pathway by the phage-encoded Int and Xis proteins and the host-encoded integration host factor and factor for inversion stimulation proteins. Structures of higher-order protein-DNA complexes of the four-way Holliday junction recombination intermediates provided clarifying insights into the mechanisms, directionality, and regulation of these two pathways, which are tightly linked to the physiology of the bacterial host cell. Here we review our current understanding of the mechanisms responsible for regulating and executing λ site-specific recombination, with an emphasis on key studies completed over the last decade.
Topics: Bacteriophage lambda; Recombination, Genetic; DNA, Viral; Viral Proteins; DNA, Bacterial; Binding Sites; Integration Host Factors
PubMed: 38372210
DOI: 10.1111/mmi.15241 -
ELife Dec 2023Nucleotide and force-dependent mechanisms control how the viral genome of lambda bacteriophage is inserted into capsids.
Nucleotide and force-dependent mechanisms control how the viral genome of lambda bacteriophage is inserted into capsids.
Topics: DNA, Viral; Bacteriophage lambda; Capsid; Genome, Viral; Nucleotides; Virus Assembly
PubMed: 38095555
DOI: 10.7554/eLife.94128 -
Cold Spring Harbor Protocols Jan 2018The Gateway recombinatorial cloning system was developed for cloning multiple DNA fragments in parallel (e.g., in 96-well formats) in a standardized manner using the...
The Gateway recombinatorial cloning system was developed for cloning multiple DNA fragments in parallel (e.g., in 96-well formats) in a standardized manner using the same enzymes. Gateway cloning is based on the highly specific integration and excision reactions of bacteriophage λ into and out of the genome. Because the sites of recombination ("" sites) are much longer (25-242 bp) than restriction sites, they are extremely unlikely to occur by chance in DNA fragments. Therefore, the same recombination enzyme can be used to robustly clone many different fragments of variable size in parallel reactions.
Topics: Attachment Sites, Microbiological; Bacteriophage lambda; Cloning, Molecular; Escherichia coli; Recombination, Genetic
PubMed: 29295908
DOI: 10.1101/pdb.top094912 -
Virology May 2015Molecular genetic research on bacteriophage lambda carried out during its golden age from the mid-1950s to mid-1980s was critically important in the attainment of our... (Review)
Review
Molecular genetic research on bacteriophage lambda carried out during its golden age from the mid-1950s to mid-1980s was critically important in the attainment of our current understanding of the sophisticated and complex mechanisms by which the expression of genes is controlled, of DNA virus assembly and of the molecular nature of lysogeny. The development of molecular cloning techniques, ironically instigated largely by phage lambda researchers, allowed many phage workers to switch their efforts to other biological systems. Nonetheless, since that time the ongoing study of lambda and its relatives has continued to give important new insights. In this review we give some relevant early history and describe recent developments in understanding the molecular biology of lambda's life cycle.
Topics: Bacteriophage lambda; History, 20th Century; History, 21st Century; Molecular Biology
PubMed: 25742714
DOI: 10.1016/j.virol.2015.02.010 -
Science (New York, N.Y.) Apr 2023Type VI CRISPR-Cas systems use RNA-guided ribonuclease (RNase) Cas13 to defend bacteria against viruses, and some of these systems encode putative membrane proteins that...
Type VI CRISPR-Cas systems use RNA-guided ribonuclease (RNase) Cas13 to defend bacteria against viruses, and some of these systems encode putative membrane proteins that have unclear roles in Cas13-mediated defense. We show that Csx28, of type VI-B2 systems, is a transmembrane protein that assists to slow cellular metabolism upon viral infection, increasing antiviral defense. High-resolution cryo-electron microscopy reveals that Csx28 forms an octameric pore-like structure. These Csx28 pores localize to the inner membrane in vivo. Csx28's antiviral activity in vivo requires sequence-specific cleavage of viral messenger RNAs by Cas13b, which subsequently results in membrane depolarization, slowed metabolism, and inhibition of sustained viral infection. Our work suggests a mechanism by which Csx28 acts as a downstream, Cas13b-dependent effector protein that uses membrane perturbation as an antiviral defense strategy.
Topics: CRISPR-Cas Systems; Cryoelectron Microscopy; Membrane Proteins; RNA, Viral; Bacterial Proteins; Endodeoxyribonucleases; CRISPR-Associated Proteins; RNA Cleavage; Bacteriophages; Bacteriophage lambda; Escherichia coli; Prevotella
PubMed: 37104586
DOI: 10.1126/science.abm1184 -
EcoSal Plus May 2016The bacteriophage λ Red homologous recombination system has been studied over the past 50 years as a model system to define the mechanistic details of how organisms... (Review)
Review
The bacteriophage λ Red homologous recombination system has been studied over the past 50 years as a model system to define the mechanistic details of how organisms exchange DNA segments that share extended regions of homology. The λ Red system proved useful as a system to study because recombinants could be easily generated by co-infection of genetically marked phages. What emerged from these studies was the recognition that replication of phage DNA was required for substantial Red-promoted recombination in vivo, and the critical role that double-stranded DNA ends play in allowing the Red proteins access to the phage DNA chromosomes. In the past 16 years, however, the λ Red recombination system has gained a new notoriety. When expressed independently of other λ functions, the Red system is able to promote recombination of linear DNA containing limited regions of homology (∼50 bp) with the Escherichia coli chromosome, a process known as recombineering. This review explains how the Red system works during a phage infection, and how it is utilized to make chromosomal modifications of E. coli with such efficiency that it changed the nature and number of genetic manipulations possible, leading to advances in bacterial genomics, metabolic engineering, and eukaryotic genetics.
Topics: Bacteriophage lambda; Chromosomes; DNA Replication; DNA, Bacterial; Escherichia coli; Genetic Engineering; Genomics; Plasmids; Recombination, Genetic
PubMed: 27223821
DOI: 10.1128/ecosalplus.ESP-0011-2015 -
Nucleic Acids Research Dec 2019Precision genome editing technologies have transformed modern biology. These technologies have arisen from the redirection of natural biological machinery, such as... (Review)
Review
Precision genome editing technologies have transformed modern biology. These technologies have arisen from the redirection of natural biological machinery, such as bacteriophage lambda proteins for recombineering and CRISPR nucleases for eliciting site-specific double-strand breaks. Less well-known is a widely distributed class of bacterial retroelements, retrons, that employ specialized reverse transcriptases to produce noncoding intracellular DNAs. Retrons' natural function and mechanism of genetic transmission have remained enigmatic. However, recent studies have harnessed their ability to produce DNA in situ for genome editing and evolution. This review describes retron biology and function in both natural and synthetic contexts. We also highlight areas that require further study to advance retron-based precision genome editing platforms.
Topics: Bacteria; DNA, Bacterial; Genes, Bacterial; Genetic Engineering; Retroelements
PubMed: 31598685
DOI: 10.1093/nar/gkz865 -
Microbiology Spectrum Jul 2018Although bacterial genomes are usually densely protein-coding, genome-wide mapping approaches of transcriptional start sites revealed that a significant fraction of the... (Review)
Review
Although bacterial genomes are usually densely protein-coding, genome-wide mapping approaches of transcriptional start sites revealed that a significant fraction of the identified promoters drive the transcription of noncoding RNAs. These can be -acting RNAs, mainly originating from intergenic regions and, in many studied examples, possessing regulatory functions. However, a significant fraction of these noncoding RNAs consist of natural antisense transcripts (asRNAs), which overlap other transcriptional units. Naturally occurring asRNAs were first observed to play a role in bacterial plasmid replication and in bacteriophage λ more than 30 years ago. Today's view is that asRNAs abound in all three domains of life. There are several examples of asRNAs in bacteria with clearly defined functions. Nevertheless, many asRNAs appear to result from pervasive initiation of transcription, and some data point toward global functions of such widespread transcriptional activity, explaining why the search for a specific regulatory role is sometimes futile. In this review, we give an overview about the occurrence of antisense transcription in bacteria, highlight particular examples of functionally characterized asRNAs, and discuss recent evidence pointing at global relevance in RNA processing and transcription-coupled DNA repair.
Topics: Bacteria; Bacterial Proteins; DNA Repair; Evolution, Molecular; Gene Expression Regulation, Bacterial; Genome, Bacterial; Plasmids; RNA, Antisense; RNA, Bacterial; RNA, Untranslated; Transcription, Genetic
PubMed: 30003872
DOI: 10.1128/microbiolspec.RWR-0029-2018