Abstract
The 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.
Highlights
The 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
The CRISPR-Cas[9] system is an invaluable tool for genome modification. It uses the enzyme Cas[9] that introduces a DNA double-strand break (DSB) in a target DNA sequence that is complementary to a 20-nt ‘spacer sequence’ in a guide RNA that is non-covalently bound to the enzyme
Each guide RNAs (gRNAs) consists of a target-specific spacer sequence and a constant part comprised of distinct conserved motifs including the nexus, and two hairpins[2]
Summary
The 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). Genome editing efficiency can be further increased by so-called ‘non-homologous oligonucleotide enhancement’ (NOE) when an external non-homologous DNA is provided which may divert the cells towards error-prone instead of error-free repair pathways[5] This can be achieved by using DNA as electroporation enhancer or a DNA donor as a template for HDR. Another approach to improve genome editing efficiency is to extend the length of complementary sequences where crRNA and tracrRNA hybridize with each other, which possibly increases the binding of Cas[9] to the sgRNA6. Some targets are intractable to genome editing even when chemically stabilized gRNAs, HEAT sgRNAs, and NOE are used
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