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.

/pdf/rna-guided-editing-of-bacterial-genomes-using-crispr-cas-5b5bwzbrz4.pdf

RNA-guided editing of bacterial genomes using CRISPR-Cas systems

Caspase-1 activation by inflammasome scaffolds comprised of intracellular nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) and the adaptor ASC is believed to be essential for production of the pro-inflammatory cytokines interleukin (IL)-1β and IL-18 during the innate immune response. Here we show, with C57BL/6 Casp11 gene-targeted mice, that caspase-11 (also known as caspase-4) is critical for caspase-1 activation and IL-1β production in macrophages infected with Escherichia coli, Citrobacter rodentium or Vibrio cholerae. Strain 129 mice, like Casp11(-/-) mice, exhibited defects in IL-1β production and harboured a mutation in the Casp11 locus that attenuated caspase-11 expression. This finding is important because published targeting of the Casp1 gene was done using strain 129 embryonic stem cells. Casp1 and Casp11 are too close in the genome to be segregated by recombination; consequently, the published Casp1(-/-) mice lack both caspase-11 and caspase-1. Interestingly, Casp11(-/-) macrophages secreted IL-1β normally in response to ATP and monosodium urate, indicating that caspase-11 is engaged by a non-canonical inflammasome. Casp1(-/-)Casp11(129mt/129mt) macrophages expressing caspase-11 from a C57BL/6 bacterial artificial chromosome transgene failed to secrete IL-1β regardless of stimulus, confirming an essential role for caspase-1 in IL-1β production. Caspase-11 rather than caspase-1, however, was required for non-canonical inflammasome-triggered macrophage cell death, indicating that caspase-11 orchestrates both caspase-1-dependent and -independent outputs. Caspase-1 activation by non-canonical stimuli required NLRP3 and ASC, but caspase-11 processing and cell death did not, implying that there is a distinct activator of caspase-11. Lastly, loss of caspase-11 rather than caspase-1 protected mice from a lethal dose of lipopolysaccharide. These data highlight a unique pro-inflammatory role for caspase-11 in the innate immune response to clinically significant bacterial infections.

Non-canonical inflammasome activation targets caspase-11

Innate immunity provides the first line of defence against invading pathogens and provides important cues for the development of adaptive immunity. Type-2 immunity-responsible for protective immune responses to helminth parasites and the underlying cause of the pathogenesis of allergic asthma-consists of responses dominated by the cardinal type-2 cytokines interleukin (IL)4, IL5 and IL13 (ref. 5). T cells are an important source of these cytokines in adaptive immune responses, but the innate cell sources remain to be comprehensively determined. Here, through the use of novel Il13-eGFP reporter mice, we present the identification and functional characterization of a new innate type-2 immune effector leukocyte that we have named the nuocyte. Nuocytes expand in vivo in response to the type-2-inducing cytokines IL25 and IL33, and represent the predominant early source of IL13 during helminth infection with Nippostrongylus brasiliensis. In the combined absence of IL25 and IL33 signalling, nuocytes fail to expand, resulting in a severe defect in worm expulsion that is rescued by the adoptive transfer of in vitro cultured wild-type, but not IL13-deficient, nuocytes. Thus, nuocytes represent a critically important innate effector cell in type-2 immunity.

/pdf/nuocytes-represent-a-new-innate-effector-leukocyte-that-406cf5i7lc.pdf

Nuocytes represent a new innate effector leukocyte that mediates type-2 immunity

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.

/pdf/an-efficient-recombination-system-for-chromosome-engineering-4acmootzcr.pdf

An efficient recombination system for chromosome engineering in Escherichia coli

The breadth of genomic diversity found among organisms in nature allows populations to adapt to diverse environments. However, genomic diversity is difficult to generate in the laboratory and new phenotypes do not easily arise on practical timescales. Although in vitro and directed evolution methods have created genetic variants with usefully altered phenotypes, these methods are limited to laborious and serial manipulation of single genes and are not used for parallel and continuous directed evolution of gene networks or genomes. Here, we describe multiplex automated genome engineering (MAGE) for large-scale programming and evolution of cells. MAGE simultaneously targets many locations on the chromosome for modification in a single cell or across a population of cells, thus producing combinatorial genomic diversity. Because the process is cyclical and scalable, we constructed prototype devices that automate the MAGE technology to facilitate rapid and continuous generation of a diverse set of genetic changes (mismatches, insertions, deletions). We applied MAGE to optimize the 1-deoxy-D-xylulose-5-phosphate (DXP) biosynthesis pathway in Escherichia coli to overproduce the industrially important isoprenoid lycopene. Twenty-four genetic components in the DXP pathway were modified simultaneously using a complex pool of synthetic DNA, creating over 4.3 billion combinatorial genomic variants per day. We isolated variants with more than fivefold increase in lycopene production within 3 days, a significant improvement over existing metabolic engineering techniques. Our multiplex approach embraces engineering in the context of evolution by expediting the design and evolution of organisms with new and improved properties.

/pdf/programming-cells-by-multiplex-genome-engineering-and-2adz9ioaw6.pdf

Programming cells by multiplex genome engineering and accelerated evolution

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

/pdf/simple-and-highly-efficient-bac-recombineering-using-galk-2blhnj9fdj.pdf

Simple and highly efficient BAC recombineering using galK selection

This unit describes the procedure used to move portions of the E. coli genome from one genetic variant to another. Fragments of approximately 100 kb can be transferred by the P1 bacteriophage. The phage is first grown on a strain containing the elements to be moved, and the resulting phage lysate is used to infect a second recipient strain. The lysate will contain bacterial DNA as well as phage DNA, and genetic recombination, catalyzed by enzymes of the recipient strain, will incorporate the bacterial fragments into the recipient chromosome.

E. coli genome manipulation by P1 transduction.

https://redrecombineering.ncifcrf.gov/references-2/general-recombineering/datta-2006-gene.pdf

A set of recombineering plasmids for gram-negative bacteria

The bacterial chromosome and bacterial plasmids can be engineered in vivo by homologous recombination using PCR products and synthetic oligonucleotides as substrates. This is possible because bacteriophage-encoded recombination proteins efficiently recombine sequences with homologies as short as 35 to 50 bases. Recombineering allows DNA sequences to be inserted or deleted without regard to location of restriction sites. This unit first describes preparation of electrocompetent cells expressing the recombineering functions and their transformation with dsDNA or ssDNA. It then presents support protocols that describe several two-step selection/counter-selection methods of making genetic alterations without leaving any unwanted changes in the targeted DNA, and a method for retrieving onto a plasmid a genetic marker (cloning by retrieval) from the Escherichia coli chromosome or a co-electroporated DNA fragment. Additional protocols describe methods to screen for unselected mutations, removal of the defective prophage from recombineering strains, and other useful techniques.

/pdf/recombineering-genetic-engineering-in-bacteria-using-4n26lrv6vc.pdf

Recombineering: Genetic Engineering in Bacteria Using Homologous Recombination

Homologous recombination can be used to generate recombinants on episomes or directly on the Escherichia coli chromosome with PCR products or synthetic single-stranded DNA (ssDNA) oligonucleotides (oligos). Such recombination is possible because bacteriophage λ-encoded functions, called Red, efficiently recombine linear DNA with homologies as short as 20–70 bases. This technology, termed recombineering, provides ways to modify genes and segments of the chromosome as well as to study homologous recombination mechanisms. The Red Beta function, which binds and anneals ssDNA to complementary ssDNA, is able to recombine 70-base oligos with the chromosome. In E. coli, methyl-directed mismatch repair (MMR) can affect these ssDNA recombination events by eliminating the recombinant allele and restoring the original sequence. In so doing, MMR can reduce the apparent recombination frequency by >100-fold. In the absence of MMR, Red-mediated oligo recombination can incorporate a single base change into the chromosome in an unprecedented 25% of cells surviving electroporation. Our results show that Beta is the only bacteriophage function required for this level of recombination and suggest that Beta directs the ssDNA to the replication fork as it passes the target sequence.

https://www.pnas.org/content/pnas/100/26/15748.full.pdf

Nina Costantino

Papers

Simple and highly efficient BAC recombineering using galK selection

E. coli genome manipulation by P1 transduction.

A set of recombineering plasmids for gram-negative bacteria

Recombineering: Genetic Engineering in Bacteria Using Homologous Recombination

Enhanced levels of λ Red-mediated recombinants in mismatch repair mutants