Abstract
The CRISPR/Cas9 system is unable to edit all targetable genomic sites with full efficiency in vivo. We show that Cas9-mediated editing is more efficient in open chromatin regions than in closed chromatin regions in rice. A construct (Cas9-TV) formed by fusing a synthetic transcription activation domain to Cas9 edits target sites more efficiently, even in closed chromatin regions. Moreover, combining Cas9-TV with a proximally binding dead sgRNA (dsgRNA) further improves editing efficiency up to several folds. The use of Cas9-TV/dsgRNA thus provides a novel strategy for obtaining efficient genome editing in vivo, especially at nuclease-refractory target sites.
Highlights
The CRISPR/Cas9 system has been widely and successfully used for genome engineering in diverse eukaryotic species and is revolutionizing biology and providing powerful tools for gene therapy and plant breeding [1]
Cas9 genome editing is more efficient in open chromatin regions of rice We used the CRISPR/Cas9 system to edit 41 rice genes with 70 sgRNAs (Additional file 1: Table S1)
We found that the frequencies of Cas9-induced indels at the target sites we tested were significantly higher at DNase I hypersensitive (DH) sites (Fig. 1a), suggesting that CRISPR/Cas9 activity in rice is affected by chromatin openness
Summary
The CRISPR/Cas system has been widely and successfully used for genome engineering in diverse eukaryotic species and is revolutionizing biology and providing powerful tools for gene therapy and plant breeding [1]. The efficiency of editing at different genomic loci varies greatly, both in animal and plant cells [2, 3]. Unlike the prokaryotic DNA that Cas evolved to target, eukaryotic genomic DNA is wrapped around histones and further compacted to form higher-order chromatin structures [5] that may hinder the binding of Cas to its targets. Genome-wide mapping of the binding sites of catalytically inactive Cas (dCas9) in mammalian cells revealed that they were enriched in open chromatin regions [6, 7]. The production of CRISPR/Cas9-induced insertions and deletions (indels) in human cells was higher at sites in open chromatin
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