Abstract
AbstractThe first step in CRISPR-Cas9-mediated genome editing is the cleavage of target DNA sequences that are complementary to so-called spacer sequences in CRISPR guide RNAs (gRNAs). However, some DNA sequences are refractory to CRISPR-Cas9 cleavage, which is at least in part due to gRNA misfolding. To overcome this problem, we have engineered gRNAs with highly stable hairpins in their constant parts and further enhanced their stability by chemical modifications. The ‘Genome-editing Optimized Locked Design’ (GOLD)-gRNA increases genome editing efficiency up to around 1000-fold (from 0.08 to 80.5%) with a mean increase across different other targets of 7.4-fold. We anticipate that this improved gRNA will allow efficient editing regardless of spacer sequence composition and will be especially useful if a desired genomic site is difficult to edit.
Funder
Funding was provided by the Max Planck Society and the NOMIS foundation.
Publisher
Springer Science and Business Media LLC
Subject
General Physics and Astronomy,General Biochemistry, Genetics and Molecular Biology,General Chemistry,Multidisciplinary
Cited by
40 articles.
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