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Recombineering

About: Recombineering is a research topic. Over the lifetime, 602 publications have been published within this topic receiving 30799 citations.


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Journal ArticleDOI
TL;DR: The exhaustively analyze dual-RNA:Cas9 target requirements to define the range of targetable sequences and show strategies for editing sites that do not meet these requirements, suggesting the versatility of this technique for bacterial genome engineering.
Abstract: The targeting of nucleases to specific DNA sequences facilitates genome editing. Recent work demonstrated that the CRISPR-associated (Cas) nuclease Cas9 can be targeted to sequences in vitro simply by modifying a short7 CRISPR RNA (crRNA) guide. Here we use this CRISPR-Cas system to introduce marker-free mutations in Streptococcus pneumoniae and Escherichia coli. The approach involves re-programming Cas9 by using a crRNA complementary to a target chromosomal locus and introducing a template DNA harboring a desired mutation and an altered crRNA recognition site for recombination with the target locus. We exhaustively analyze Cas9 target requirements to define the range of targetable sequences and show strategies for editing sites that do not meet these requirements. Alone or together with recombineering, CRISPR assisted editing induces recombination at the targeted locus and kills non-edited cells leading to a recovery of close to a 100% of edited cells. Multiple crRNA can be used to modify several loci simultaneously. Our results show that CRISPR-mediated genome editing only requires programming of the crRNA and template sequences and thus constitutes a useful tool for genetic engineering.

2,394 citations

Journal ArticleDOI
TL;DR: A recombination system has been developed for efficient chromosome engineering in Escherichia coli by using electroporated linear DNA using a defective lambda prophage, which will be especially useful for the engineering of large bacterial plasmids such as those from bacterial artificial chromosome libraries.
Abstract: A recombination system has been developed for efficient chromosome engineering in Escherichia coli by using electroporated linear DNA. A defective lambda prophage supplies functions that protect and recombine an electroporated linear DNA substrate in the bacterial cell. The use of recombination eliminates the requirement for standard cloning as all novel joints are engineered by chemical synthesis in vitro and the linear DNA is efficiently recombined into place in vivo. The technology and manipulations required are simple and straightforward. A temperature-dependent repressor tightly controls prophage expression, and, thus, recombination functions can be transiently supplied by shifting cultures to 42 degrees C for 15 min. The efficient prophage recombination system does not require host RecA function and depends primarily on Exo, Beta, and Gam functions expressed from the defective lambda prophage. The defective prophage can be moved to other strains and can be easily removed from any strain. Gene disruptions and modifications of both the bacterial chromosome and bacterial plasmids are possible. This system will be especially useful for the engineering of large bacterial plasmids such as those from bacterial artificial chromosome libraries.

1,790 citations

Journal ArticleDOI
TL;DR: A straightforward way to engineer DNA in E. coli using homologous recombination is described in this article, which uses RecE and RecT and is transferable between different E coli strains.
Abstract: A straightforward way to engineer DNA in E. coli using homologous recombination is described. The homologous recombination reaction uses RecE and RecT and is transferable between E. coli strains. Several target molecules were manipulated, including high copy plasmids, a large episome and the E. coli chromosome. Sequential steps of homologous or site-specific recombination were used to demonstrate a new logic for engineering DNA, unlimited by the disposition of restriction endonuclease cleavage sites or the size of the target DNA.

1,294 citations

Journal ArticleDOI
01 Apr 2001-Genomics
TL;DR: The ability to modify or subclone large fragments of genomic DNA with precision should facilitate many kinds of genomic experiments that were difficult or impossible to perform previously and aid in studies of gene function in the postgenomic era.

1,267 citations

Journal ArticleDOI
TL;DR: Three new recombineering strains are described that allow bacterial artificial chromosomes (BACs) to be modified using galK positive/negative selection, and it is shown how galK selection can be used to rapidly introduce point mutations, deletions and loxP sites into BAC DNA and thus facilitate functional studies of SNP and/or disease-causing point mutations.
Abstract: Recombineering allows DNA cloned in Escherichia coli to be modified via lambda (l) Red-mediated homologous recombination, obviating the need for restriction enzymes and DNA ligases to modify DNA Here, we describe the construction of three new recombineering strains (SW102, SW105 and SW106) that allow bacterial artificial chromosomes (BACs) to be modified using galK positive/negative selection This two-step selection procedure allows DNA to be modified without introducing an unwanted selectable marker at the modification site All three strains contain an otherwise complete galactose operon, except for a precise deletion of the galK gene, and a defective temperature-sensitive l prophage that makes recombineering possible SW105 and SW106 cells in addition carry L-arabinose-inducible Cre or Flp genes, respectively The galK function can be selected both for and against This feature greatly reduces the background seen in other negative-selection schemes, and galK selection is considerably more efficient than other related selection methods published We also show how galK selection can be used to rapidly introduce point mutations, deletions and loxP sites into BAC DNA and thus facilitate functional studies of SNP and/or disease-causing point mutations, the identification of long-range regulatory elements and the construction of conditional targeting vectors

1,261 citations


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Performance
Metrics
No. of papers in the topic in previous years
YearPapers
202326
202252
202134
202042
201946
201831