Browsing by Subject "CRISPR-Cas9"

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    CRISPR-Cas9-mediated homology-directed repair for precise gene editing
    (Elsevier, 2024-09-26) Liao, Hongyu; Wu, Jiahao; VanDusen, Nathan J.; Li, Yifei; Zheng, Yanjiang; Pediatrics, School of Medicine
    CRISPR-Cas9-mediated homology-directed repair (HDR) is a versatile platform for creating precise site-specific DNA insertions, deletions, and substitutions. These precise edits are made possible through the use of exogenous donor templates that carry the desired sequence. CRISPR-Cas9-mediated HDR can be widely used to study protein functions, disease modeling, and gene therapy. However, HDR is limited by its low efficiency, especially in postmitotic cells. Here, we review CRISPR-Cas9-mediated HDR, with a focus on methodologies for boosting HDR efficiency, and applications of precise editing via HDR. First, we describe two common mechanisms of DNA repair, non-homologous end joining (NHEJ), and HDR, and discuss their impact on CRISPR-Cas9-mediated precise genome editing. Second, we discuss approaches for improving HDR efficiency through inhibition of the NHEJ pathway, activation of the HDR pathway, modification of donor templates, and delivery of Cas9/sgRNA reagents. Third, we summarize the applications of HDR for protein labeling in functional studies, disease modeling, and ex vivo and in vivo gene therapies. Finally, we discuss alternative precise editing platforms and their limitations, and describe potential avenues to improving CRISPR-Cas9-mediated HDR efficiency and fidelity in future research.
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    One-step generation of a conditional allele in mice using a short artificial intron
    (Elsevier, 2022-12-24) Cassidy, Annelise M.; Thomas, Destinée B.; Kuliyev, Emin; Chen, Hanying; Pelletier, Stephane; Medical and Molecular Genetics, School of Medicine
    Despite tremendous advances in genome editing technologies, generation of conditional alleles in mice has remained challenging. Recent studies in cells have successfully made use of short artificial introns to engineer conditional alleles. The approach consists of inserting a small cassette within an exon of a gene using CRISPR-Cas9 technology. The cassette, referred to as Artificial Intron version 4 (AIv4), contains sequences encoding a splice donor, essential intronic sequences flanked by loxP sites and a splice acceptor site. Under normal conditions, the artificial intron is removed by the splicing machinery, allowing for proper expression of the gene product. Following Cre-mediated recombination of the two loxP sites, the intron is disabled, and splicing can no longer occur. The remaining intronic sequences create a frameshift and early translation termination. Here we describe the application of this technology to engineer a conditional allele in mice using Scyl1 as a model gene. Insertion of the cassette occurred in 17% of edited mice obtained from pronuclear stage zygote microinjection. Mice homozygous for the insertion expressed SCYL1 at levels comparable to wild-type mice and showed no overt abnormalities associated with the loss of Scyl1 function, indicating the proper removal of the artificial intron. Inactivation of the cassette via Cre-mediated recombination in vivo occurred at high frequency, abrogated SCYL1 protein expression, and resulted in loss-of-function phenotypes. Our results broaden the applicability of this approach to engineering conditional alleles in mice.