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Reference
Citation
Koreman, G.T., Xu, Y., Hu, Q., Zhang, Z., Allen, S.E., Wolfner, M.F., Wang, B., Han, C. (2021). Upgraded CRISPR/Cas9 tools for tissue-specific mutagenesis in Drosophila.  Proc. Natl. Acad. Sci. U.S.A. 118(14): e2014255118.
FlyBase ID
FBrf0248562
Publication Type
Research paper
Abstract

CRISPR/Cas9 has emerged as a powerful technology for tissue-specific mutagenesis. However, tissue-specific CRISPR/Cas9 tools currently available in Drosophila remain deficient in three significant ways. First, many existing gRNAs are inefficient, such that further improvements of gRNA expression constructs are needed for more efficient and predictable mutagenesis in both somatic and germline tissues. Second, it has been difficult to label mutant cells in target tissues with current methods. Lastly, application of tissue-specific mutagenesis at present often relies on Gal4-driven Cas9, which hampers the flexibility and effectiveness of the system. Here, we tackle these deficiencies by building upon our previous CRISPR-mediated tissue-restricted mutagenesis (CRISPR-TRiM) tools. First, we significantly improved gRNA efficiency in somatic tissues by optimizing multiplexed gRNA design. Similarly, we also designed efficient dual-gRNA vectors for the germline. Second, we developed methods to positively and negatively label mutant cells in tissue-specific mutagenesis by incorporating co-CRISPR reporters into gRNA expression vectors. Lastly, we generated genetic reagents for convenient conversion of existing Gal4 drivers into tissue-specific Cas9 lines based on homology-assisted CRISPR knock-in. In this way, we expand the choices of Cas9 for CRISPR-TRiM analysis to broader tissues and developmental stages. Overall, our upgraded CRISPR/Cas9 tools make tissue-specific mutagenesis more versatile, reliable, and effective in Drosophila These improvements may be also applied to other model systems.

PubMed ID
PubMed Central ID
PMC8040800 (PMC) (EuropePMC)
Related Publication(s)
Personal communication to FlyBase

elav-Cas9[C155], repo-Cas9 and UAS-tdTom insertions.
Han, 2021.4.7, elav-Cas9[C155], repo-Cas9 and UAS-tdTom insertions. [FBrf0249069]

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Secondary IDs
    Language of Publication
    English
    Additional Languages of Abstract
    Parent Publication
    Publication Type
    Journal
    Abbreviation
    Proc. Natl. Acad. Sci. U.S.A.
    Title
    Proceedings of the National Academy of Sciences of the United States of America
    Publication Year
    1915-
    ISBN/ISSN
    0027-8424
    Data From Reference
    Alleles (63)
    Genes (16)
    Natural transposons (2)
    Insertions (55)
    Experimental Tools (8)
    Transgenic Constructs (58)