Dataset Information

Split selectable markers.

ABSTRACT: Selectable markers are widely used in transgenesis and genome editing for selecting engineered cells with a desired genotype but the variety of markers is limited. Here we present split selectable markers that each allow for selection of multiple "unlinked" transgenes in the context of lentivirus-mediated transgenesis as well as CRISPR-Cas-mediated knock-ins. Split marker gene segments fused to protein splicing elements called "inteins" can be separately co-segregated with different transgenic vectors, and rejoin via protein trans-splicing to reconstitute a full-length marker protein in host cells receiving all intended vectors. Using a lentiviral system, we create and validate 2-split Hygromycin, Puromycin, Neomycin and Blasticidin resistance genes as well as mScarlet fluorescent proteins. By combining split points, we create 3- and 6-split Hygromycin resistance genes, demonstrating that higher-degree split markers can be generated by a "chaining" design. We adapt the split marker system for selecting biallelically engineered cells after CRISPR gene editing. Future engineering of split markers may allow selection of a higher number of genetic modifications in target cells.

SUBMITTER: Jillette N

PROVIDER: S-EPMC6823381 | biostudies-literature | 2019 Oct

REPOSITORIES: biostudies-literature

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Split selectable markers.

Jillette Nathaniel N Du Menghan M Zhu Jacqueline Jufen JJ Cardoz Peter P Cheng Albert Wu AW

Nature communications 20191031 1

Selectable markers are widely used in transgenesis and genome editing for selecting engineered cells with a desired genotype but the variety of markers is limited. Here we present split selectable markers that each allow for selection of multiple "unlinked" transgenes in the context of lentivirus-mediated transgenesis as well as CRISPR-Cas-mediated knock-ins. Split marker gene segments fused to protein splicing elements called "inteins" can be separately co-segregated with different transgenic v ...[more]

PMID: 31672965

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Project description:Lyme disease is the most widely reported vector-borne disease in the United States. Its incidence is rapidly increasing, and disease symptoms can be debilitating. The need to understand the biology of the disease agent, the spirochete Borrelia burgdorferi, is thus evermore pressing. Despite important advances in B. burgdorferi genetics, the array of molecular tools available for use in this organism remains limited, especially for cell biological studies. Here, we adapt a palette of bright and mostly monomeric fluorescent proteins for versatile use and multicolor imaging in B. burgdorferi We also characterize two novel antibiotic selection markers and establish the feasibility of their use in conjunction with extant markers. Last, we describe a set of promoters of low and intermediate strengths that allow fine-tuning of gene expression levels. These molecular tools complement and expand current experimental capabilities in B. burgdorferi, which will facilitate future investigation of this important human pathogen. To showcase the usefulness of these reagents, we used them to investigate the subcellular localization of BB0323, a B. burgdorferi lipoprotein essential for survival in the host and vector environments. We show that BB0323 accumulates at the cell poles and future division sites of B. burgdorferi cells, highlighting the complex subcellular organization of this spirochete.IMPORTANCE Genetic manipulation of the Lyme disease spirochete B. burgdorferi remains cumbersome, despite significant progress in the field. The scarcity of molecular reagents available for use in this pathogen has slowed research efforts to study its unusual biology. Of interest, B. burgdorferi displays complex cellular organization features that have yet to be understood. These include an unusual morphology and a highly fragmented genome, both of which are likely to play important roles in the bacterium's transmission, infectivity, and persistence. Here, we complement and expand the array of molecular tools available for use in B. burgdorferi by generating and characterizing multiple fluorescent proteins, antibiotic selection markers, and promoters of varied strengths. These tools will facilitate investigations in this important human pathogen, as exemplified by the polar and midcell localization of the cell envelope regulator BB0323, which we uncovered using these reagents.

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Split selectable markers.

Publications

Split selectable markers.

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