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CRISPR-associated transposons (CASTs) are RNA-guided mobile genetic elements that are widespread in bacterial genomes. Here, we describe the UltraCAST, a suicide vector with the Vibrio cholerae Type I-F CAST system and Golden Gate assembly sites with fluorescent protein gene dropouts for guide RNA and a mini-transposon cargo cloning. We show an example of UltraCAST genome editing by disrupting a gene in the chromosome of Serratia symbiotica CWBI-2.3T, a culturable relative of aphid endosymbionts. The UltraCAST can be used to flexibly insert DNA into specific genomic sites and facilitates testing this genome editing platform in non-model bacterial species that lack genetic tools.
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DASH : a versatile and high‐capacity gene stacking system for plant synthetic biology
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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- PAR ID:
- 10600260
- Publisher / Repository:
- Wiley-Blackwell
- Date Published:
- Journal Name:
- Plant Biotechnology Journal
- Volume:
- 23
- Issue:
- 9
- ISSN:
- 1467-7644
- Format(s):
- Medium: X Size: p. 3697-3712
- Size(s):
- p. 3697-3712
- Sponsoring Org:
- National Science Foundation
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