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1. A modular approach for dCas9-mediated enzyme cascading via orthogonal bioconjugation

   https://doi.org/10.1039/d0cc04196c  

   Berckman, Emily A.; Chen, Wilfred (September 2020, Chemical Communications)

   null (Ed.)

   We report a new modular strategy to assemble dCas9-guided enzyme cascades by employing orthogonal post-translation chemistry. Two orthogonal SpyCatcher and SnoopCatcher pairs were used for the one-pot enzyme bioconjugation onto two different dCas9 proteins to enable their guided assembly onto a DNA scaffold. The resulting two-component cellulosomes exhibited 2.8-fold higher reducing sugar production over unassembled enzymes. This platform retains the high binding affinity afforded by dCas9 proteins for easy control over enzyme assembly while offering the flexibility for both in vivo and in vitro assembly of a wide array of enzyme cascades with minimal optimization. 

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2. A CRISPRi-dCas9 System for Archaea and Its Use To Examine Gene Function during Nitrogen Fixation by Methanosarcina acetivorans

   https://doi.org/10.1128/AEM.01402-20  

   Dhamad, Ahmed E.; Lessner, Daniel J. (October 2020, Applied and Environmental Microbiology)

   Atomi, Haruyuki (Ed.)

   ABSTRACT CRISPR-based systems are emerging as the premier method to manipulate many cellular processes. In this study, a simple and efficient CRISPR interference (CRISPRi) system for targeted gene repression in archaea was developed. The Methanosarcina acetivorans CRISPR-Cas9 system was repurposed by replacing Cas9 with the catalytically dead Cas9 (dCas9) to generate a CRISPRi-dCas9 system for targeted gene repression. To test the utility of the system, genes involved in nitrogen (N 2 ) fixation were targeted for dCas9-mediated repression. First, the nif operon ( nifHI 1 I 2 DKEN ) that encodes molybdenum nitrogenase was targeted by separate guide RNAs (gRNAs), one targeting the promoter and the other targeting nifD . Remarkably, growth of M. acetivorans with N 2 was abolished by dCas9-mediated repression of the nif operon with each gRNA. The abundance of nif transcripts was >90% reduced in both strains expressing the gRNAs, and NifD was not detected in cell lysate. Next, we targeted NifB, which is required for nitrogenase cofactor biogenesis. Expression of a gRNA targeting the coding sequence of NifB decreased nifB transcript abundance >85% and impaired but did not abolish growth of M. acetivorans with N 2 . Finally, to ascertain the ability to study gene regulation using CRISPRi-dCas9, nrpR1 , encoding a subunit of the repressor of the nif operon, was targeted. The nrpR1 repression strain grew normally with N 2 but had increased nif operon transcript abundance, consistent with NrpR1 acting as a repressor. These results highlight the utility of the system, whereby a single gRNA when expressed with dCas9 can block transcription of targeted genes and operons in M. acetivorans . IMPORTANCE Genetic tools are needed to understand and manipulate the biology of archaea, which serve critical roles in the biosphere. Methanogenic archaea (methanogens) are essential for the biological production of methane, an intermediate in the global carbon cycle, an important greenhouse gas, and a biofuel. The CRISPRi-dCas9 system in the model methanogen Methanosarcina acetivorans is, to our knowledge, the first Cas9-based CRISPR interference system in archaea. Results demonstrate that the system is remarkably efficient in targeted gene repression and provide new insight into nitrogen fixation by methanogens, the only archaea with nitrogenase. Overall, the CRISPRi-dCas9 system provides a simple, yet powerful, genetic tool to control the expression of target genes and operons in methanogens. 

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3. A bacterial dual positive and negative selection system for dCas9 activity

   https://doi.org/10.1371/journal.pone.0269270  

   Spisak, Shaun; O’Brien, Brett; Ostermeier, Marc (June 2022, PLOS ONE)

   Fujii, Hodaka (Ed.)

   The engineering of switchable or activatable dCas9 proteins would benefit from a single system for both positive and negative selection of dCas9 activity. Most systems that are used to interrogate dCas9 libraries use a fluorescent protein screen or an antibiotic selection for active dCas9 variants. To avoid some of the limitations of these systems, we have developed a single system capable of selecting for either active or inactive dCas9 variants. E . coli expressing active dCas9 variants are isolated in the positive selection system through growth in the presence of ampicillin. The negative selection can isolate cells lacking dCas9 activity through two separate mechanisms: growth in M9 minimal media or growth in media containing streptomycin. This system is capable of enriching for rare dCas9 variants up to 9,000-fold and possesses potential utility in directed evolution experiments to create switchable dCas9 proteins. 

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4. Targeted Insertion of the mPing Transposable Element

   Strother, Ashley E.; Diaz, Stephanie S.; Baker, Mary E.; and Hancock, C. Nathan (January 2018, Journal of the South Carolina Academy of Science)

   Class II DNA Transposable Elements (TEs) are moved from one location to another in the genome by the action of transposase proteins that bind to repeat sequences at the ends of the elements. Although the location TE insertion is mostly random, the addition of DNA binding domains to the transposase proteins has allowed for targeted insertion of some elements. In this study, the Gal4 binding domain was added to the transposase proteins, ORF1 and TPase, which mobilize the mPing element from rice. The Gal4:TPase construct was capable of increasing the number of mPing insertions into the Gal2 and Gal4 promoter sequences in yeast. While this confirms that mPing insertion preference can be manipulated, the target specificity is relatively low. Thus, the CRISPR/Cas9 system was tested for its ability to generate targeted insertion of mPing. A dCas9:TPase fusion protein had a low transposition rate suggesting that the addition of this large protein disrupts TPase function. Unfortunately, the use of a MS2 binding domain to localize the TPase to the MS2 hairpin containing gRNA failed to produce targeted insertion. Thus, our results suggest that the addition of small DNA binding domain to the N-terminal of TPase is the best strategy for targeted insertion of mPing. 

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5. DASH : a versatile and high‐capacity gene stacking system for plant synthetic biology

   https://doi.org/10.1111/pbi.70179  

   Zhao, Chengsong; Stepanova, Anna_N; Alonso, Jose_M (June 2025, Plant Biotechnology Journal)

   Summary DNA assembly systems based on the Golden Gate method are popular in synthetic biology but have several limitations: small insert size, incompatibility with other cloning platforms, DNA domestication requirement, generation of fusion scars, and lack of post‐assembly modification. To address these obstacles, we present the DASH assembly toolset, which combines features of Golden Gate‐based cloning, recombineering, and site‐specific recombinase systems. We developed (1) a set of donor vectors based on the GoldenBraid platform, (2) an acceptor vector derived from the plant transformation‐competent artificial chromosome (TAC) vector, pYLTAC17, and (3) a re‐engineered recombineering‐readyE. colistrain, CZ105, based on SW105. The initial assembly steps are carried out using the donor vectors following standard GoldenBraid assembly procedures. Importantly, existing parts and transcriptional units created using compatible Golden Gate‐based systems can be transferred to the DASH donor vectors using standard single‐tube restriction/ligation reactions. The cargo DNA from a DASH donor vector is then efficiently transferredin vivoinE. coliinto the acceptor vector by the sequential action of a rhamnose‐inducible phage‐derived PhiC31 integrase and arabinose‐inducible yeast‐derived Flippase (FLP) recombinase using CZ105. Furthermore, recombineering‐based post‐assembly modification, including the removal of undesirable scars, is greatly simplified. To demonstrate the utility of the DASH system, a 116 kilobase (kb) DNA construct harbouring a 97 kb cargo consisting of 35 transcriptional units was generated. One of the coding DNA sequences (CDSs) in the final assembly was replaced through recombineering, and thein plantafunctionality of the entire construct was tested in both transient and stable transformants. 

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