Dataset Information

Comprehensive optimization of a reporter assay toolbox for three distinct CRISPR-Cas systems.

ABSTRACT: The clustered, regularly interspaced, short palindromic repeats-associated DNA nuclease (CRISPR-Cas) protein system allows programmable gene editing through inducing double-strand breaks. Reporter assays for DNA cleavage and DNA repair events play an important role in advancing the CRISPR technology and improving our understanding of the underlying molecular mechanisms. Here, we developed a series of reporter assays to probe mechanisms of action of various editing processes, including nonhomologous DNA end joining, homology-directed repair and single-strand annealing. With special target design, the reporter assays as an optimized toolbox can be used to take advantage of three distinct CRISPR-Cas systems (Streptococcus pyogenes Cas9, Staphylococcus aureus Cas9 and Francisella novicida U112 Cpf1) and two different reporters (GFP and Gaussia luciferase). We further validated the Gaussia reporter assays using a series of small molecules, including NU7441, RI-1 and Mirin, and showcased the use of a GFP reporter assay as an effective tool for enrichment of cells with edited genome.

SUBMITTER: Chen L

PROVIDER: S-EPMC8255852 | biostudies-literature | 2021 Jul

REPOSITORIES: biostudies-literature

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Comprehensive optimization of a reporter assay toolbox for three distinct CRISPR-Cas systems.

Chen Li L Gao Haoyuan H Zhou Bing B Wang Yu Y

FEBS open bio 20210609 7

The clustered, regularly interspaced, short palindromic repeats-associated DNA nuclease (CRISPR-Cas) protein system allows programmable gene editing through inducing double-strand breaks. Reporter assays for DNA cleavage and DNA repair events play an important role in advancing the CRISPR technology and improving our understanding of the underlying molecular mechanisms. Here, we developed a series of reporter assays to probe mechanisms of action of various editing processes, including nonhomolog ...[more]

PMID: 33999508

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Project description:CRISPR systems mediate adaptive immunity in bacteria and archaea through diverse effector mechanisms and have been repurposed for versatile applications in therapeutics and diagnostics thanks to their facile reprogramming with RNA guides. RNA-guided CRISPR-Cas targeting and interference are mediated by effectors that are either components of multisubunit complexes in class 1 systems or multidomain single-effector proteins in class 2. The compact class 2 CRISPR systems have been broadly adopted for multiple applications, especially genome editing, leading to a transformation of the molecular biology and biotechnology toolkit. The diversity of class 2 effector enzymes, initially limited to the Cas9 nuclease, was substantially expanded via computational genome and metagenome mining to include numerous variants of Cas12 and Cas13, providing substrates for the development of versatile, orthogonal molecular tools. Characterization of these diverse CRISPR effectors uncovered many new features, including distinct protospacer adjacent motifs (PAMs) that expand the targeting space, improved editing specificity, RNA rather than DNA targeting, smaller crRNAs, staggered and blunt end cuts, miniature enzymes, promiscuous RNA and DNA cleavage, etc. These unique properties enabled multiple applications, such as harnessing the promiscuous RNase activity of the type VI effector, Cas13, for supersensitive nucleic acid detection. class 1 CRISPR systems have been adopted for genome editing, as well, despite the challenge of expressing and delivering the multiprotein class 1 effectors. The rich diversity of CRISPR enzymes led to rapid maturation of the genome editing toolbox, with capabilities such as gene knockout, base editing, prime editing, gene insertion, DNA imaging, epigenetic modulation, transcriptional modulation, and RNA editing. Combined with rational design and engineering of the effector proteins and associated RNAs, the natural diversity of CRISPR and related bacterial RNA-guided systems provides a vast resource for expanding the repertoire of tools for molecular biology and biotechnology.

Dataset Information

Comprehensive optimization of a reporter assay toolbox for three distinct CRISPR-Cas systems.

Publications

Comprehensive optimization of a reporter assay toolbox for three distinct CRISPR-Cas systems.

Similar Datasets

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