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  • 1
    Online Resource
    Online Resource
    An educational module to explore CRISPR technologies with a cell-free transcription-translation system
    Oxford University Press (OUP) ; 2019
    In:  Synthetic Biology Vol. 4, No. 1 ( 2019-01-01)
    Details
    In: Synthetic Biology, Oxford University Press (OUP), Vol. 4, No. 1 ( 2019-01-01)
    Abstract: Within the last 6 years, CRISPR-Cas systems have transitioned from adaptive defense systems in bacteria and archaea to revolutionary genome-editing tools. The resulting CRISPR technologies have driven innovations for treating genetic diseases and eradicating human pests while raising societal questions about gene editing in human germline cells as well as crop plants. Bringing CRISPR into the classroom therefore offers a means to expose students to cutting edge technologies and to promote discussions about ethical questions at the intersection of science and society. However, working with these technologies in a classroom setting has been difficult because typical experiments rely on cellular systems such as bacteria or mammalian cells. We recently reported the use of an E. coli cell-free transcription-translation (TXTL) system that simplifies the demonstration and testing of CRISPR technologies with shorter experiments and limited equipment. Here, we describe three educational modules intended to expose undergraduate students to CRISPR technologies using TXTL. The three sequential modules comprise (i) designing the RNAs that guide DNA targeting, (ii) measuring DNA cleavage activity in TXTL and (iii) testing how mutations to the targeting sequence or RNA backbone impact DNA binding and cleavage. The modules include detailed protocols, questions for group discussions or individual evaluation, and lecture slides to introduce CRISPR and TXTL. We expect these modules to allow students to experience the power and promise of CRISPR technologies in the classroom and to engage with their instructor and peers about the opportunities and potential risks for society.
    Type of Medium: Online Resource
    ISSN: 2397-7000
    URL: Article
    DOI: 10.1093/synbio/ysz005
    Language: English
    Publisher: Oxford University Press (OUP)
    Publication Date: 2019
    detail.hit.zdb_id: 2864609-5
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  • 2
    Online Resource
    Online Resource
    Advancing the design and delivery of CRISPR antimicrobials
    Elsevier BV ; 2017
    In:  Current Opinion in Biomedical Engineering Vol. 4 ( 2017-12), p. 57-64
    Details
    In: Current Opinion in Biomedical Engineering, Elsevier BV, Vol. 4 ( 2017-12), p. 57-64
    Type of Medium: Online Resource
    ISSN: 2468-4511
    URL: Article
    DOI: 10.1016/j.cobme.2017.10.001
    Language: English
    Publisher: Elsevier BV
    Publication Date: 2017
    detail.hit.zdb_id: 2891622-0
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  • 3
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    Online Resource
    Systematically attenuating DNA targeting enables CRISPR-driven editing in bacteria
    Springer Science and Business Media LLC ; 2023
    In:  Nature Communications Vol. 14, No. 1 ( 2023-02-08)
    Details
    In: Nature Communications, Springer Science and Business Media LLC, Vol. 14, No. 1 ( 2023-02-08)
    Abstract: Bacterial genome editing commonly relies on chromosomal cleavage with Cas nucleases to counter-select against unedited cells. However, editing normally requires efficient recombination and high transformation efficiencies, which are unavailable in most strains. Here, we show that systematically attenuating DNA targeting activity enables RecA-mediated repair in different bacteria, allowing chromosomal cleavage to drive genome editing. Attenuation can be achieved by altering the format or expression strength of guide (g)RNAs; using nucleases with reduced cleavage activity; or engineering attenuated gRNAs (atgRNAs) with disruptive hairpins, perturbed nuclease-binding scaffolds, non-canonical PAMs, or guide mismatches. These modifications greatly increase cell counts and even improve the efficiency of different types of edits for Cas9 and Cas12a in Escherichia coli and Klebsiella oxytoca . We further apply atgRNAs to restore ampicillin sensitivity in Klebsiella pneumoniae , establishing a resistance marker for genetic studies. Attenuating DNA targeting thus offers a counterintuitive means to achieve CRISPR-driven editing across bacteria.
    Type of Medium: Online Resource
    ISSN: 2041-1723
    URL: Article
    DOI: 10.1038/s41467-023-36283-9
    Language: English
    Publisher: Springer Science and Business Media LLC
    Publication Date: 2023
    detail.hit.zdb_id: 2553671-0
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  • 4
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    Online Resource
    CRISPR technologies and the search for the PAM-free nuclease
    Springer Science and Business Media LLC ; 2021
    In:  Nature Communications Vol. 12, No. 1 ( 2021-01-22)
    Details
    In: Nature Communications, Springer Science and Business Media LLC, Vol. 12, No. 1 ( 2021-01-22)
    Abstract: The ever-expanding set of CRISPR technologies and their programmable RNA-guided nucleases exhibit remarkable flexibility in DNA targeting. However, this flexibility comes with an ever-present constraint: the requirement for a protospacer adjacent motif (PAM) flanking each target. While PAMs play an essential role in self/nonself discrimination by CRISPR-Cas immune systems, this constraint has launched a far-reaching expedition for nucleases with relaxed PAM requirements. Here, we review ongoing efforts toward realizing PAM-free nucleases through natural ortholog mining and protein engineering. We also address potential consequences of fully eliminating PAM recognition and instead propose an alternative nuclease repertoire covering all possible PAM sequences.
    Type of Medium: Online Resource
    ISSN: 2041-1723
    URL: Article
    DOI: 10.1038/s41467-020-20633-y
    Language: English
    Publisher: Springer Science and Business Media LLC
    Publication Date: 2021
    detail.hit.zdb_id: 2553671-0
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