Showing papers on "Recombineering published in 2021"

High-throughput functional variant screens via in vivo production of single-stranded DNA.

Abstract: Creating and characterizing individual genetic variants remains limited in scale, compared to the tremendous variation both existing in nature and envisioned by genome engineers. Here we introduce retron library recombineering (RLR), a methodology for high-throughput functional screens that surpasses the scale and specificity of CRISPR-Cas methods. We use the targeted reverse-transcription activity of retrons to produce single-stranded DNA (ssDNA) in vivo, incorporating edits at >90% efficiency and enabling multiplexed applications. RLR simultaneously introduces many genomic variants, producing pooled and barcoded variant libraries addressable by targeted deep sequencing. We use RLR for pooled phenotyping of synthesized antibiotic resistance alleles, demonstrating quantitative measurement of relative growth rates. We also perform RLR using the sheared genomic DNA of an evolved bacterium, experimentally querying millions of sequences for causal variants, demonstrating that RLR is uniquely suited to utilize large pools of natural variation. Using ssDNA produced in vivo for pooled experiments presents avenues for exploring variation across the genome.

Recombineering and MAGE

Abstract: Recombination-mediated genetic engineering, also known as recombineering, is the genomic incorporation of homologous single-stranded or double-stranded DNA into bacterial genomes Recombineering and its derivative methods have radically improved genome engineering capabilities, perhaps none more so than multiplex automated genome engineering (MAGE) MAGE is representative of a set of highly multiplexed single-stranded DNA-mediated technologies First described in Escherichia coli, both MAGE and recombineering are being rapidly translated into diverse prokaryotes and even into eukaryotic cells Together, this modern set of tools offers the promise of radically improving the scope and throughput of experimental biology by providing powerful new methods to ease the genetic manipulation of model and non-model organisms In this Primer, we describe recombineering and MAGE, their optimal use, their diverse applications and methods for pairing them with other genetic editing tools We then look forward to the future of genetic engineering This Primer by Wannier and colleagues summarizes the methodology, analysis and utility of recombineering and multiplex automated genome engineering (MAGE) in microbial species In addition, this Primer examines advanced techniques that pair MAGE with other tools to improve editing efficiency

CRISPY-BRED and CRISPY-BRIP: efficient bacteriophage engineering

Abstract: Genome engineering of bacteriophages provides opportunities for precise genetic dissection and for numerous phage applications including therapy. However, few methods are available for facile construction of unmarked precise deletions, insertions, gene replacements and point mutations in bacteriophages for most bacterial hosts. Here we describe CRISPY-BRED and CRISPY-BRIP, methods for efficient and precise engineering of phages in Mycobacterium species, with applicability to phages of a variety of other hosts. This recombineering approach uses phage-derived recombination proteins and Streptococcus thermophilus CRISPR-Cas9.

Development and application of an efficient recombineering system for Burkholderia glumae and Burkholderia plantarii

Abstract: The lambda phage Red proteins Redα/Redβ/Redγ and Rac prophage RecE/RecT proteins are powerful tools for precise and efficient genetic manipulation but have been limited to only a few prokaryotes. Here, we report the development and application of a new recombineering system for Burkholderia glumae and Burkholderia plantarii based on three Rac bacteriophage RecET-like operons, RecETheBDU8 , RecEThTJI49 and RecETh1h2eYI23 , which were obtained from three different Burkholderia species. Recombineering experiments indicated that RecEThTJI49 and RecETh1h2eYI23 showed higher recombination efficiency compared to RecETheBDU8 in Burkholderia glumae PG1. Furthermore, all of the proteins currently categorized as hypothetical proteins in RecETh1h2eYI23, RecEThTJI49 and RecETheBDU8 may have a positive effect on recombination in B. glumae PG1 except for the h2 protein in RecETh1h2eYI23 . Additionally, RecETYI23 combined with exonuclease inhibitors Pluγ or Redγ exhibited equivalent recombination efficiency compared to Redγβα in Escherichia coli, providing potential opportunity of recombineering in other Gram-negative bacteria for its loose host specificity. Using recombinase-assisted in situ insertion of promoters, we successfully activated three cryptic non-ribosomal peptide synthetase biosynthetic gene clusters in Burkholderia strains, resulting in the generation of a series of lipopeptides that were further purified and characterized. Compound 7 exhibited significant potential anti-inflammatory activity by inhibiting lipopolysaccharide-stimulated nitric oxide production in RAW 264.7 macrophages. This recombineering system may greatly enhance functional genome research and the mining of novel natural products in the other species of the genus Burkholderia after optimization of a protocol.

A recombineering pipeline to clone large and complex genes in Chlamydomonas

Abstract: The ability to clone genes has greatly advanced cell and molecular biology research, enabling researchers to generate fluorescent protein fusions for localization and confirm genetic causation by mutant complementation Most gene cloning is polymerase chain reaction (PCR)�or DNA synthesis-dependent, which can become costly and technically challenging as genes increase in size, particularly if they contain complex regions This has been a long-standing challenge for the Chlamydomonas reinhardtii research community, as this alga has a high percentage of genes containing complex sequence structures Here we overcame these challenges by developing a recombineering pipeline for the rapid parallel cloning of genes from a Chlamydomonas bacterial artificial chromosome collection To generate fluorescent protein fusions for localization, we applied the pipeline at both batch and high-throughput scales to 203 genes related to the Chlamydomonas CO2 concentrating mechanism (CCM), with an overall cloning success rate of 77% Cloning success was independent of gene size and complexity, with cloned genes as large as 23 kb Localization of a subset of CCM targets confirmed previous mass spectrometry data, identified new pyrenoid components, and enabled complementation of mutants We provide vectors and detailed protocols to facilitate easy adoption of this technology, which we envision will open up new possibilities in algal and plant research

Optimizing Recombineering in Corynebacterium glutamicum.

Abstract: Owing to the increasing demand for amino acids and valuable commodities that can be produced by Corynebacterium glutamicum, there is a pressing need for new rapid genome engineering tools that improve the speed and efficiency of genomic insertions, deletions, and mutations. Recombineering using the λ Red system in Escherichia coli has proven very successful at genetically modifying this organism in a quick and efficient manner, suggesting that optimizing a recombineering system for C. glutamicum will also improve the speed for genomic modifications. Here, we maximized the recombineering efficiency in C. glutamicum by testing the efficacy of seven different recombinase/exonuclease pairs for integrating single-stranded DNA and double-stranded DNA (dsDNA) into the genome. By optimizing the homologous arm length and the amount of dsDNA transformed, as well as eliminating codon bias, a dsDNA recombineering efficiency of 13,250 transformed colonies/109 viable cells was achieved, the highest efficiency currently reported in the literature. Using this optimized system, over 40,000 bp could be deleted in one transformation step. This recombineering strategy will greatly improve the speed of genetic modifications in C. glutamicum and assist other systems, such as clustered regularly interspaced short palindromic repeats and multiplexed automated genome engineering, in improving targeted genome editing.

Genomics-Driven Activation of Silent Biosynthetic Gene Clusters in Burkholderia gladioli by Screening Recombineering System.

Abstract: The Burkholderia genus possesses ecological and metabolic diversities. A large number of silent biosynthetic gene clusters (BGCs) in the Burkholderia genome remain uncharacterized and represent a promising resource for new natural product discovery. However, exploitation of the metabolomic potential of Burkholderia is limited by the absence of efficient genetic manipulation tools. Here, we screened a bacteriophage recombinase system Redγ-BAS, which was functional for genome modification in the plant pathogen Burkholderia gladioli ATCC 10248. By using this recombineering tool, the constitutive promoters were precisely inserted in the genome, leading to activation of two silent nonribosomal peptide synthetase gene clusters (bgdd and hgdd) and production of corresponding new classes of lipopeptides, burriogladiodins A–H (1–8) and haereogladiodins A–B (9–10). Structure elucidation revealed an unnatural amino acid Z- dehydrobutyrine (Dhb) in 1–8 and an E-Dhb in 9–10. Notably, compounds 2–4 and 9 feature an unusual threonine tag that is longer than the predicted collinearity assembly lines. The structural diversity of burriogladiodins was derived from the relaxed substrate specificity of the fifth adenylation domain as well as chain termination conducted by water or threonine. The recombinase-mediating genome editing system is not only applicable in B. gladioli, but also possesses great potential for mining meaningful silent gene clusters from other Burkholderia species.

Rapid Genome Engineering of Pseudomonas Assisted by Fluorescent Markers and Tractable Curing of Plasmids

Abstract: Precise genome engineering has become a commonplace technique for metabolic engineering. Also, insertion, deletion and alteration of genes and other functional DNA sequences are essential for understanding and engineering cells. Several techniques have been developed to this end (e.g., CRISPR/Cas-assisted methods, homologous recombination, or λ Red recombineering), yet most of them rely on the use of auxiliary plasmids, which have to be cured after the editing procedure. Temperature-sensitive replicons, counter-selectable markers or repeated passaging of plasmid-bearing cells have been traditionally employed to circumvent this hurdle. While these protocols work reasonably well in some bacteria, they are not applicable for other species or are time consuming and laborious. Here, we present a fast and versatile protocol of fluorescent marker-assisted genome editing in Pseudomonas putida, followed by clean curing of auxiliary plasmids through user-controlled plasmid replication. One fluorescent marker facilitates identification of genome-edited colonies, while the second reporter enables detection of plasmid-free bacterial clones. Not only is this protocol the fastest available for Pseudomonas species, but it can be easily adapted to any type of genome modifications, including sequence deletions, insertions, and replacements. Graphical abstract: Rapid genome engineering of Pseudomonas with curable plasmids.

RecT Recombinase Expression Enables Efficient Gene Editing in Enterococcus spp.

Abstract: Enterococcus faecium is a ubiquitous Gram-positive bacterium that has been recovered from the environment, food, and microbiota of mammals. Commensal strains of E. faecium can confer beneficial effects on host physiology and immunity, but antibiotic usage has afforded antibiotic-resistant and pathogenic isolates from livestock and humans. However, the dissection of E. faecium functions and mechanisms has been restricted by inefficient gene-editing methods. To address these limitations, here, we report that the expression of E. faecium RecT recombinase significantly improves the efficiency of recombineering technologies in both commensal and antibiotic-resistant strains of E. faecium and other Enterococcus species such as E. durans and E. hirae. Notably, the expression of RecT in combination with clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 and guide RNAs (gRNAs) enabled highly efficient scarless single-stranded DNA recombineering to generate specific gene-editing mutants in E. faecium. Moreover, we demonstrate that E. faecium RecT expression facilitated chromosomal insertions of double-stranded DNA templates encoding antibiotic-selectable markers to generate gene deletion mutants. As a further proof of principle, we use CRISPR-Cas9-mediated recombineering to knock out both sortase A genes in E. faecium for downstream functional characterization. The general RecT-mediated recombineering methods described here should significantly enhance genetic studies of E. faecium and other closely related species for functional and mechanistic studies. IMPORTANCE Enterococcus faecium is widely recognized as an emerging public health threat with the rise of drug resistance and nosocomial infections. Nevertheless, commensal Enterococcus strains possess beneficial health functions in mammals to upregulate host immunity and prevent microbial infections. This functional dichotomy of Enterococcus species and strains highlights the need for in-depth studies to discover and characterize the genetic components underlying its diverse activities. However, current genetic engineering methods in E. faecium still require passive homologous recombination from plasmid DNA. This involves the successful cloning of multiple homologous fragments into a plasmid, introducing the plasmid into E. faecium, and screening for double-crossover events that can collectively take up to multiple weeks to perform. To alleviate these challenges, we show that RecT recombinase enables the rapid and efficient integration of mutagenic DNA templates to generate substitutions, deletions, and insertions in the genomic DNA of E. faecium. These improved recombineering methods should facilitate functional and mechanistic studies of Enterococcus.

An efficient and versatile CRISPR-Cas9 system for genetic manipulation of multi-drug resistant Klebsiella pneumoniae.

Abstract: Summary Multi-drug resistant (MDR) Klebsiella pneumoniae remains an urgent public health threat. While whole-genome sequencing has helped identify genetic changes underlying resistance, functional validation remains difficult due to a lack of genetic manipulation systems for MDR K. pneumoniae. CRISPR-Cas9 has revolutionized molecular biology, but its use was only recently adapted in bacteria by overcoming the lack of genetic repair systems. We describe a CRISPR-Cas9/lambda recombineering system utilizing a zeocin resistance cassette allowing efficient and versatile genetic manipulation of K. pneumoniae. For complete details on the use and execution of this protocol, please refer to McConville et al. (2020) .

Reverse genetics systems for contemporary isolates of respiratory syncytial virus enable rapid evaluation of antibody escape mutants.

Abstract: Human respiratory syncytial virus (RSV) is the leading cause of acute lower respiratory infection in children under 5 y of age. In the absence of a safe and effective vaccine and with limited options for therapeutic interventions, uncontrolled epidemics of RSV occur annually worldwide. Existing RSV reverse genetics systems have been predominantly based on older laboratory-adapted strains such as A2 or Long. These strains are not representative of currently circulating genotypes and have a convoluted passage history, complicating their use in studies on molecular determinants of viral pathogenesis and intervention strategies. In this study, we have generated reverse genetics systems for clinical isolates of RSV-A (ON1, 0594 strain) and RSV-B (BA9, 9671 strain) in which the full-length complementary DNA (cDNA) copy of the viral antigenome is cloned into a bacterial artificial chromosome (BAC). Additional recombinant (r) RSVs were rescued expressing enhanced green fluorescent protein (EGFP), mScarlet, or NanoLuc luciferase from an additional transcription unit inserted between the P and M genes. Mutations in antigenic site II of the F protein conferring escape from palivizumab neutralization (K272E, K272Q, S275L) were investigated using quantitative cell-fusion assays and rRSVs via the use of BAC recombineering protocols. These mutations enabled RSV-A and -B to escape palivizumab neutralization but had differential impacts on cell-to-cell fusion, as the S275L mutation resulted in an almost-complete ablation of syncytium formation. These reverse genetics systems will facilitate future cross-validation efficacy studies of novel RSV therapeutic intervention strategies and investigations into viral and host factors necessary for virus entry and cell-to-cell spread.

Comparison of CRISPR-Cas9 Tools for Transcriptional Repression and Gene Disruption in the BEVS.

Abstract: The generation of knock-out viruses using recombineering of bacmids has greatly accelerated scrutiny of baculovirus genes for a variety of applications. However, the CRISPR–Cas9 system is a powerful tool that simplifies sequence-specific genome editing and effective transcriptional regulation of genes compared to traditional recombineering and RNAi approaches. Here, the effectiveness of the CRISPR–Cas9 system for gene disruption and transcriptional repression in the BEVS was compared. Cell lines constitutively expressing the cas9 or dcas9 gene were developed, and recombinant baculoviruses delivering the sgRNA were evaluated for disruption or repression of a reporter green fluorescent protein gene. Finally, endogenous AcMNPV genes were targeted for disruption or downregulation to affect gene expression and baculovirus replication. This study provides a proof-of-concept that CRISPR–Cas9 technology may be an effective tool for efficient scrutiny of baculovirus genes through targeted gene disruption and transcriptional repression.

Genetic Dissection of a Prevalent Plasmid-Encoded Conjugation System in Lactococcus lactis .

Abstract: Plasmid pNP40, which was first identified nearly 40 years ago in Lactococcus lactis subsp. lactis biovar diacetylactis DRC3, encodes functions such as heavy metal-, bacteriophage-, and nisin-resistance, as well as plasmid transfer ability by conjugation. Here, we report an optimized conjugation protocol for this plasmid, yielding a transfer frequency that is approximately 4,000-fold higher than those previously reported in literature, while we also observed high-frequency plasmid co-mobilization. Individual mutations in 18 genes that encompass the presumed conjugation cluster of pNP40 were generated using ssDNA recombineering to evaluate the role of each gene in the conjugation process. A possible transcriptional repressor of this conjugation cluster, the product of the traR gene, was identified in this manner. This mutational analysis, paired with bioinformatic predictions as based on sequence and structural similarities, allowed us to generate a preliminary model of the pNP40 conjugation machinery.

ReScribe: An Unrestrained Tool Combining Multiplex Recombineering and Minimal-PAM ScCas9 for Genome Recoding Pseudomonas putida

Abstract: Genome recoding enables incorporating new functions into the DNA of microorganisms. By reassigning codons to noncanonical amino acids, the generation of new-to-nature proteins offers countless opportunities for bioproduction and biocontainment in industrial chassis. A key bottleneck in genome recoding efforts, however, is the low efficiency of recombineering, which hinders large-scale applications at acceptable speed and cost. To relieve this bottleneck, we developed ReScribe, a highly optimized recombineering tool enhanced by CRISPR-Cas9-mediated counterselection built upon the minimal PAM 5′-NNG-3′ of the Streptococcus canis Cas9 (ScCas9). As a proof of concept, we used ReScribe to generate a minimally recoded strain of the industrial chassis Pseudomonas putida by replacing TAG stop codons (functioning as PAMs) of essential metabolic genes with the synonymous TAA. We showed that ReScribe enables nearly 100% engineering efficiency of multiple loci in P. putida, opening promising avenues for genome editing and applications thereof in this bacterium and beyond.

Recombineering facilitates the discovery of natural product biosynthetic pathways in Pseudomonas parafulva.

Abstract: Microbial natural products among other functions they play a vital role in the disease prevention in humans, animals and plants. Pseudomonas parafulva CRS01-1 is a broad-spectrum antagonistic bacterium present in plants. However, no natural products have been isolated from this strain till date. Corresponding biosynthetic gene clusters to natural products is an effective method for bioprospecting, for which, genome manipulation tools are essential. We previously developed Pseudomonas-specific phage-derived homologous recombination systems for genetic engineering in four Pseudomonas species. Herein, we report the application of these recombineering systems in Pseudomonas parafulva CRS01-1, along with structural elucidation and bioactivity evaluation of natural products. The Pseudomonas recombineering toolbox established before in different four species is efficient for genome mining and bioactive metabolite discovery from more distant species.

ssDNA recombineering boosts in vivo evolution of nanobodies displayed on bacterial surfaces.

Abstract: ssDNA recombineering has been exploited to hyperdiversify genomically-encoded nanobodies displayed on the surface of Escherichia coli for originating new binding properties. As a proof-of-principle a nanobody recognizing the antigen TirM from enterohaemorrhagic E. coli (EHEC) was evolved towards the otherwise not recognized TirM antigen from enteropathogenic E. coli (EPEC). To this end, E. coli cells displaying this nanobody fused to the intimin outer membrane-bound domain were subjected to multiple rounds of mutagenic oligonucleotide recombineering targeting the complementarity determining regions (CDRs) of the cognate VHH gene sequence. Binders to the EPEC-TirM were selected upon immunomagnetic capture of bacteria bearing active variants and nanobodies identified with a new ability to strongly bind the new antigen. The results highlight the power of combining evolutionary properties of bacteria in vivo with oligonucleotide synthesis in vitro for the sake of focusing diversification to specific segments of a gene (or protein thereof) of interest. Yamal Al-ramahi et al. describe an accelerated laboratory evolution method that applies ssDNA recombineering and bacterial surface display to engineer nanobody epitope recognition. As a proof-of-concept, they evolved TD4 recognition to bind the translocated intimin receptor (TirM) of EPEC pathogens, with this method bypassing the need for laborious cloning and mutagenesis procedures.

Oligo-Mediated Recombineering and its Use for Making SNPs, Knockouts, Insertions, and Fusions in Mycobacterium tuberculosis

Abstract: Phage recombination systems have been instrumental in the development of gene modification technologies for bacterial pathogens. In particular, the Che9 phage RecET system has been used successfully for over 10 years for making gene knockouts and fusions in Mycobacterium tuberculosis. This "recombineering" technology typically uses linear dsDNA substrates that contain a drug-resistance marker flanked by (up to) 500 base pairs of DNA homologous to the target site. Less often employed in mycobacterial recombineering is the use of oligonucleotides, which require only the action of the RecT annealase to align oligos to ssDNA regions of the replication fork, for subsequent incorporation into the chromosome. Despite the higher frequency of such events relative to dsDNA-promoted recombineering, oligo-mediated changes generally suffer from the disadvantage of not being selectable, thus making them harder to isolate. This chapter discusses steps and methodologies that increase the frequencies of finding oligo-mediated events, including the transfer of single nucleotide polymorphisms (SNPs) to mycobacterial chromosomes, and the use of oligos in conjunction with the mycobacterial phage Bxb1 site-specific recombination system for the easy generation of knockouts, insertion, and fusions, in a protocol known as ORBIT.

Rapid Genome Modification in Serratia marcescens Through Red Homologous Recombination

Abstract: Despite the great potential of Serratia marcescens in industrial applications, lack of powerful genetic modification tools limits understanding of the regulatory networks of the useful metabolites and therefore restricts their mass production. To meet the urgent demand, we established a genome-editing strategy for S. marcescens based on Red recombineering in this study. Without host modification in advance, nucA and pigA were substituted by PCR-amplified resistance genes. No long homologous arms were required at the two sides of resistance genes. Using this procedure, the fragment at the S. marcescens as large as 20 kb was easily deleted. Then we constructed a counter-selection gene kil constructed under the control of inducible PBAD operon, which demonstrates obvious lethality to S. marcescens. Subsequently, GmR-kil double selection cassette was inserted into the CDS of pigA gene. Using single-stranded DNA-mediated recombination, this insertion mutation was efficiently repaired through kil counter-selection. A powerful genetic modification platform based on Red recombineering system was successfully established for S. marcescens. Multiple types of modification and multiple recombination strategies can all be performed easily in this species. We hope this study will be useful for the theoretical research and the research of metabolic engineering in S. marcescens.

ESI mutagenesis: a one-step method for introducing mutations into bacterial artificial chromosomes.

Abstract: Bacterial artificial chromosome (BAC)-based transgenes have emerged as a powerful tool for controlled and conditional interrogation of protein function in higher eukaryotes. Although homologous recombination-based recombineering methods have streamlined the efficient integration of protein tags onto BAC transgenes, generating precise point mutations has remained less efficient and time-consuming. Here, we present a simplified method for inserting point mutations into BAC transgenes requiring a single recombineering step followed by antibiotic selection. This technique, which we call exogenous/synthetic intronization (ESI) mutagenesis, relies on co-integration of a mutation of interest along with a selectable marker gene, the latter of which is harboured in an artificial intron adjacent to the mutation site. Cell lines generated from ESI-mutated BACs express the transgenes equivalently to the endogenous gene, and all cells efficiently splice out the synthetic intron. Thus, ESI mutagenesis provides a robust and effective single-step method with high precision and high efficiency for mutating BAC transgenes.

Identification of phage recombinase function unit in genus Corynebacterium

Abstract: Phage recombinase function unit (PRFU) plays a key role in the life cycle of phage. Repurposing this system such as lambda-Redαβ or Rac-RecET for recombineering has gained success in Escherichia coli. Previous studies have showed that most PRFUs only worked well in its native hosts but poorly in the distant species. Thus, identification of new PRFUs in specific species is necessary for the development of its corresponding genetic engineering tools. Here, we present a thorough study of PRFUs in the genomes of genus Corynebacterium. We first used a database to database searching method to facilitate accurate prediction of novel PRFUs in 423 genomes. A total number of 60 sets of unique PRFUs were identified and divided into 8 types based on evolution affinities. Recombineering ability of the 8 representative PRFUs was experimentally verified in the Corynebacterium glutamicum ATCC 13032 strain. In particular, PRFU from C. aurimucosum achieved highest efficiency in both ssDNA and dsDNA mediated recombineering, which is expected to greatly facilitate genome engineering in genus Corynebacterium. These results will provide new insights for the study and application of PRFUs. KEY POINTS: • First report of bioinformatic mining and systematic analysis of Phage recombinase function unit (PRFU) in Corynebacterium genomes. • Recombineering ability of the representative PRFUs was experimentally verified in Corynebacterium glutamicum ATCC 13032 strain. • PRFU with the highest recombineering efficiency at 10-2 magnitude was identified from Corynebacterium aurimucosum.

Analysing Parallel Strategies to Alter the Host Specificity of Bacteriophage T7

Abstract: The recognition and binding of host bacteria by bacteriophages is most often enabled by a highly specific receptor-ligand type of interaction, with the receptor-binding proteins (RBPs) of phages being the primary determinants of host specificity. Specifically modifying the RBPs could alter or extend the host range of phages otherwise exhibiting desired phenotypic properties. This study employed two different strategies to reprogram T7 phages ordinarily infecting commensal K12 Escherichia coli strains to infect pathogen-associated K1-capsule-expressing strains. The strategies were based on either plasmid-based homologous recombination or bacteriophage recombineering using electroporated DNA (BRED). Our work pursued the construction of two genetic designs: one replacing the gp17 gene of T7, the other replacing gp11, gp12, and gp17 of T7 with their K1F counterparts. Both strategies displayed successful integration of the K1F sequences into the T7 genome, detected by PCR screening. Multiple methods were utilised to select or enrich for chimeric phages incorporating the K1F gp17 alone, including trxA, host-specificity, and CRISPR-Cas-based selection. Irrespective of the selection method, the above strategy yielded poorly reproducible phage propagation on the new host, indicating that the chimeric phage was less fit than the wild type and could not promote continual autonomous reproduction. Chimeric phages obtained from BRED incorporating gp11-12 and gp17, however, all displayed infection in a 2-stage pattern, indicating the presence of both K1F and T7 phenotypes. This study shows that BRED can be used as a tool to quickly access the potential of new RBP constructs without the need to engineer sustainably replicating phages. Additionally, we show that solely repurposing the primary RBP is, in some cases, insufficient to produce a viable chimeric phage.

Knockout of a highly GC-rich gene in Burkholderia pyrrocinia by recombineering with freeze-thawing transformation.

Abstract: Genetic transformation is a valuable and essential method that provides powerful insights into the gene function of microorganisms and contributes to the construction of engineered bacteria. Here, we developed a novel genetic transformation system to easily knock out a highly GC-rich gene (74.71% GC) from Burkholderia pyrrocinia JK-SH007, a biocontrol strain of poplar canker disease. This system revealed a reliable selectable marker (trimethoprim resistance gene, Tmp) and a simplified, efficient transformation method (6,363.64 CFU/μg, pHKT2) that was developed via freeze-thawing. The knockout recombineering of B. pyrrocinia JK-SH007 was achieved through a suicide plasmid with a three-fragment mutagenesis construct. The three-fragment cassette for mutagenesis was generated by overlap extension and touchdown PCRs and composed of Tmp flanked by GC-rich upstream and downstream fragments from B. pyrrocinia JK-SH007. The mutant strain (ΔBpEG), which was verified by PCR, lost 93.3% of its ability to degrade carboxymethyl cellulose over 40 days. Overall, this system may contribute to future research on B. pyrrocinia traits.

Prioritization of antimicrobial targets by CRISPR-based oligo recombineering

Abstract: Nucleophilic amino acids are important in covalent drug development yet underutilized as antimicrobial targets. Over recent years, several chemoproteomic technologies have been developed to mine chemically-accessible residues via their intrinsic reactivity toward electrophilic probes. However, these approaches cannot discern which reactive sites contribute to protein function and should therefore be prioritized for drug discovery. To address this, we have developed a CRISPR-based Oligo Recombineering (CORe) platform to systematically prioritize reactive amino acids according to their contribution to protein function. Our approach directly couples protein sequence and function with biological fitness. Here, we profile the reactivity of >1,000 cysteines on ~700 proteins in the eukaryotic pathogen Toxoplasma gondii and prioritize functional sites using CORe. We competitively compared the fitness effect of 370 codon switches at 74 cysteines and identify functional sites in a diverse range of proteins. In our proof of concept, CORe performed >800 times faster than a standard genetic workflow. Reactive cysteines decorating the ribosome were found to be critical for parasite growth, with subsequent target-based screening validating the apicomplexan translation machinery as a target for covalent ligand development. CORe is system-agnostic, and supports expedient identification, functional prioritization, and rational targeting of reactive sites in a wide range of organisms and diseases.

Guide-directed DNA cleavage by a prokaryotic Argonaute protein induces chromosome recombination in Escherichia coli

Abstract: Emerging evidence supports the argument that some prokaryotic argonautes (pAgos) serve as a defensive system against invasion of viruses and plasmids through guide DNAs (gDNAs) directed DNA cleavage. This DNA-guided DNA interference motivates research to induce genomic mutations via pAgo mediated cleavage. Here we demonstrate that CbAgo, a pAgo from Clostridium butyricum, is able to induce chromosomal recombination between direct repeat sequences via its gDNA-directed cleavage in Escherichia coli chromosome. We also show that CbAgo targeting can assist Lambda-Red recombineering in RecA-deficient strain. Our study reveals that cleavage by CbAgo in E. coli chromosome can be mutagenic and suggests its broader application in genetic manipulation.

ssDNA recombineering boosts in vivo evolution of nanobodies displayed on bacterial surfaces

Abstract: SUMMARY In vivo evolution of antibodies facilitates emergence of novel target specificities from pre-existing clones. In this work we show how mutagenic ssDNA recombineering of camel-derived nanobodies encoded in a bacterial genome enables clonal hyper-diversification and the rise of new properties. As a proof-of-principle we used a nanobody recognizing the antigen TirM from enterohaemorrhagic E. coli (EHEC) and evolved it towards the otherwise not recognized TirM antigen from enteropathogenic E. coli (EPEC). To this end, E. coli cells displaying on their surface this nanobody fused to the intimin outer membrane anchor domain were subjected to multiple rounds of mutagenic ssDNA recombineering targeted to the CDR1, CDR2 and CDR3 regions of its genomically encoded VHH sequence. Binders to the new antigen (EPEC TirM) were then selected upon immunomagnetic capture of bacteria bearing the corresponding nanobody variants. As a result, several modified nanobodies were identified which maintained recognition of EHEC TirM but acquired the ability to bind the new antigen with high affinity (Kd ~20 nM). The results highlight the power of combining evolutionary properties of bacteria in vivo with oligonucleotide synthesis in vitro for the sake of focusing diversification to specific segments of a gene (or protein thereof) of interest. Our experimental workflow empowers the evolution of nanobodies displayed on the surface of bacterial cells for a large number of potential applications in medical and industrial biotechnology.