Journal
CHEMICAL COMMUNICATIONS
Volume 54, Issue 19, Pages 2377-2380Publisher
ROYAL SOC CHEMISTRY
DOI: 10.1039/c7cc08893k
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- Council of Scientific and Industrial Research (CSIR), Government of India [HCP0008]
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Originating as a component of prokaryotic adaptive immunity, the type II CRISPR/Cas9 system has been repurposed for targeted genome editing in various organisms. Although Cas9 can bind and cleave DNA efficiently under in vitro conditions, its activity inside a cell can vary dramatically between targets owing to the differences between genomic loci and the availability of enough Cas9/sgRNA (single guide RNA) complex molecules for cleavage. Most methods have so far relied on Cas9 protein engineering or base modifications in the sgRNA sequence to improve CRISPR/Cas9 activity. Here we demonstrate that a structure based rational design of sgRNAs can enhance the efficiency of Cas9 cleavage in vivo. By appending a naturally forming RNA G-quadruplex motif to the 3 ' end of sgRNAs we can improve its stability and target cleavage efficiency in zebrafish embryos without inducing off-target activity, thereby underscoring its value in the design of better and optimized genome editing triggers.
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