Browsing by keyword "CRISPR/Cas9 genome editing"
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CRISPR/Cas9-mediated genome editing induces exon skipping by alternative splicing or exon deletionCRISPR is widely used to disrupt gene function by inducing small insertions and deletions. Here, we show that some single-guide RNAs (sgRNAs) can induce exon skipping or large genomic deletions that delete exons. For example, CRISPR-mediated editing of beta-catenin exon 3, which encodes an autoinhibitory domain, induces partial skipping of the in-frame exon and nuclear accumulation of beta-catenin. A single sgRNA can induce small insertions or deletions that partially alter splicing or unexpected larger deletions that remove exons. Exon skipping adds to the unexpected outcomes that must be accounted for, and perhaps taken advantage of, in CRISPR experiments.
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Engineering essential genes with a "jump board" strategy using CRISPR/Cas9Here, we describe a platformed “jump board” strategy and its application in systematically engineering the essential microRNA let-7 (Fig. 1A-E) and protein coding gene lin-28 (Fig. 1F) in C. elegans. We chose the jump board protospacer sequence (INPP4A) which is (1) comprised of a PAM site and a protospacer antisense to a crRNA with experimentally confirmed high editing efficiency (INPP4A-crRNA), and (2) non-homologous to C. elegans genome, including the genetic balancer we used (mnDp1). Notably, the jump board protospacer contains an EcoRV restriction site, which can be utilized for rapid large-scale genotyping by which HDR events can be identified in the F1 generation (Fig. 1C). Using the jump board strategy, we have so far created 28 let-7 alleles for various experimental purposes, among which 15 alleles showed lethality and require rescue by mnDp1. Note that the let-7 jump board allele (ma393) itself is a new let-7 null allele in which the precursor-let-7 is completely removed.
