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1. One-step genome engineering in bee gut bacterial symbionts

   https://doi.org/10.1128/mbio.01392-24  

   Lariviere, Patrick J; Ashraf, A_H_M Zuberi; Navarro-Escalante, Lucio; Leonard, Sean P; Miller, Laurel G; Moran, Nancy A; Barrick, Jeffrey E (September 2024, mBio)

   Kaltenpoth, Martin (Ed.)

   ABSTRACT Mechanistic understanding of interactions in many host-microbe systems, including the honey bee microbiome, is limited by a lack of easy-to-use genome engineering approaches. To this end, we demonstrate a one-step genome engineering approach for making gene deletions and insertions in the chromosomes of honey bee gut bacterial symbionts. Electroporation of linear or non-replicating plasmid DNA containing an antibiotic resistance cassette flanked by regions with homology to a symbiont genome reliably results in chromosomal integration. This lightweight approach does not require expressing any exogenous recombination machinery. The high concentrations of large DNAs with long homology regions needed to make the process efficient can be readily produced using modern DNA synthesis and assembly methods. We use this approach to knock out genes, including genes involved in biofilm formation, and insert fluorescent protein genes into the chromosome of the betaproteobacterial bee gut symbiontSnodgrassella alvi. We are also able to engineer the genomes of multiple strains ofS. alviand another species,Snodgrassella communis, which is found in the bumble bee gut microbiome. Finally, we use the same method to engineer the chromosome of another bee symbiont,Bartonella apis, which is an alphaproteobacterium. As expected, gene knockout inS. alviusing this approach isrecA-dependent, suggesting that this straightforward procedure can be applied to other microbes that lack convenient genome engineering methods. IMPORTANCEHoney bees are ecologically and economically important crop pollinators with bacterial gut symbionts that influence their health. Microbiome-based strategies for studying or improving bee health have utilized wild-type or plasmid-engineered bacteria. We demonstrate that a straightforward, single-step method can be used to insert cassettes and replace genes in the chromosomes of multiple bee gut bacteria. This method can be used for investigating the mechanisms of host-microbe interactions in the bee gut community and stably engineering symbionts that benefit pollinator health. 

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2. Oca2 targeting using CRISPR/Cas9 in the Malawi cichlid Astatotilapia calliptera

   https://doi.org/10.1098/rsos.220077  

   Clark, Bethan; Elkin, Joel; Marconi, Aleksandra; Turner, George F.; Smith, Alan M.; Joyce, Domino; Miska, Eric A.; Juntti, Scott A.; Santos, M. Emília (April 2022, Royal Society Open Science)

   Identifying genetic loci underlying trait variation provides insights into the mechanisms of diversification, but demonstrating causality and characterizing the role of genetic loci requires testing candidate gene function, often in non-model species. Here we establish CRISPR/Cas9 editing in Astatotilapia calliptera , a generalist cichlid of the remarkably diverse Lake Malawi radiation. By targeting the gene oca2 required for melanin synthesis in other vertebrate species, we show efficient editing and germline transmission. Gene edits include indels in the coding region, probably a result of non-homologous end joining, and a large deletion in the 3′ untranslated region due to homology-directed repair. We find that oca2 knock-out A. calliptera lack melanin, which may be useful for developmental imaging in embryos and studying colour pattern formation in adults. As A. calliptera resembles the presumed generalist ancestor of the Lake Malawi cichlid radiation, establishing genome editing in this species will facilitate investigating speciation, adaptation and trait diversification in this textbook radiation. 

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3. Expanding the capabilities of MuGENT for large-scale genetic engineering of the fastest-replicating species, Vibrio natriegens

   https://doi.org/10.1128/spectrum.03964-23  

   Glasgo, Liz D; Lukasiak, Katie L; Zinser, Erik R (June 2024, Microbiology Spectrum)

   Cardona, Silvia T (Ed.)

   ABSTRACT The fastest replicating bacteriumVibrio natriegensis a rising workhorse for molecular and biotechnological research with established tools for efficient genetic manipulation. Here, we expand on the capabilities of multiplex genome editing by natural transformation (MuGENT) by identifying a neutral insertion site and showing how two selectable markers can be swapped at this site for sequential rounds of natural transformation. Second, we demonstrated that MuGENT can be used for complementation by gene insertion at an ectopic chromosomal locus. Additionally, we developed a robust method to cure the competence plasmid required to induce natural transformation. Finally, we demonstrated the ability of MuGENT to create massive deletions; the 280 kb deletion created in this study is one of the largest artificial deletions constructed in a single round of targeted mutagenesis of a bacterium. These methods each advance the genetic potential ofV. natriegensand collectively expand upon its utility as an emerging model organism for synthetic biology. IMPORTANCEVibrio natriegensis an emerging model organism for molecular and biotechnological applications. Its fast growth, metabolic versatility, and ease of genetic manipulation provide an ideal platform for synthetic biology. Here, we develop and apply novel methods that expand the genetic capabilities of theV. natriegensmodel system. Prior studies developed a method to manipulate multiple regions of the chromosome in a single step. Here, we provide new resources that diversify the utility of this method. We also provide a technique to remove the required genetic tools from the cell once the manipulation is performed, thus establishing “clean” derivative cells. Finally, we show the full extent of this technique’s capability by generating one of the largest chromosomal deletions reported in the literature. Collectively, these new tools will be beneficial broadly to theVibriocommunity and specifically to the advancement ofV. natriegensas a model system. 

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4. Efficient and modular CRISPR‐Cas9 vector system for Physcomitrella patens

   https://doi.org/10.1002/pld3.168  

   Mallett, Darren R.; Chang, Mingqin; Cheng, Xiaohang; Bezanilla, Magdalena (September 2019, Plant Direct)

   Abstract CRISPR‐Cas9 has been shown to be a valuable tool in recent years, allowing researchers to precisely edit the genome using an RNA‐guided nuclease to initiate double‐strand breaks. Until recently, classical RAD51‐mediated homologous recombination has been a powerful tool for gene targeting in the mossPhyscomitrella patens. However, CRISPR‐Cas9‐mediated genome editing inP. patenswas shown to be more efficient than traditional homologous recombination (Plant Biotechnology Journal, 15, 2017, 122). CRISPR‐Cas9 provides the opportunity to efficiently edit the genome at multiple loci as well as integrate sequences at precise locations in the genome using a simple transient transformation. To fully take advantage of CRISPR‐Cas9 genome editing inP. patens, here we describe the generation and use of a flexible and modular CRISPR‐Cas9 vector system. Without the need for gene synthesis, this vector system enables editing of up to 12 loci simultaneously. Using this system, we generated multiple lines that had null alleles at four distant loci. We also found that targeting multiple sites within a single locus can produce larger deletions, but the success of this depends on individual protospacers. To take advantage of homology‐directed repair, we developed modular vectors to rapidly generate DNA donor plasmids to efficiently introduce DNA sequences encoding for fluorescent proteins at the 5′ and 3′ ends of gene coding regions. With regard to homology‐directed repair experiments, we found that if the protospacer sequence remains on the DNA donor plasmid, then Cas9 cleaves the plasmid target as well as the genomic target. This can reduce the efficiency of introducing sequences into the genome. Furthermore, to ensure the generation of a null allele near the Cas9 cleavage site, we generated a homology plasmid harboring a “stop codon cassette” with downstream near‐effortless genotyping. 

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5. Efficient protein tagging and cis -regulatory element engineering via precise and directional oligonucleotide-based targeted insertion in plants

   https://doi.org/10.1093/plcell/koad139  

   Kumar, Jitesh; Char, Si Nian; Weiss, Trevor; Liu, Hua; Liu, Bo; Yang, Bing; Zhang, Feng (May 2023, The Plant Cell)

   Abstract Efficient and precise targeted insertion holds great promise but remains challenging in plant genome editing. An efficient nonhomologous end-joining-mediated targeted insertion method was recently developed by combining clustered regularly interspaced short palindromic repeat (CRISPR)/Streptococcus pyogenes CRISPR-associated nuclease 9 (SpCas9) gene editing with phosphorothioate modified double-stranded oligodeoxynucleotides (dsODNs). Yet, this approach often leads to imprecise insertions with no control over the insertion direction. Here, we compared the influence of chemical protection of dsODNs on efficiency of targeted insertion. We observed that CRISPR/SpCas9 frequently induced staggered cleavages with 1-nucleotide 5′ overhangs; we also evaluated the effect of donor end structures on the direction and precision of targeted insertions. We demonstrate that chemically protected dsODNs with 1-nucleotide 5′ overhangs significantly improved the precision and direction control of target insertions in all tested CRISPR targeted sites. We applied this method to endogenous gene tagging in green foxtail (Setaria viridis) and engineering of cis-regulatory elements for disease resistance in rice (Oryza sativa). We directionally inserted 2 distinct transcription activator-like effector binding elements into the promoter region of a recessive rice bacterial blight resistance gene with up to 24.4% efficiency. The resulting rice lines harboring heritable insertions exhibited strong resistance to infection by the pathogen Xanthomonas oryzae pv. oryzae in an inducible and strain-specific manner. 

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