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Double Nicking by RNA-Guided CRISPR Cas9 for Enhanced Genome
Transcript of Double Nicking by RNA-Guided CRISPR Cas9 for Enhanced Genome
able to cleave only a single strand of the RNA-guided DNA target sequence
(D10A mutant nickase version of Cas9)
Clustered Regularly Interspaced Short Palindromic Repeats
CRISPR Associated System
Discovered in bacteria
defense against foreign DNA (viral or plasmid)
"This new system uses a short RNA to guide a nuclease to the DNA target"
Cas9 Nickase Generates Efficient NHEJ with Paired, Offset Guide RNAs
Pair of offset sgRNAs
(located in opposite strands of the target site)
"Two Cas9-nicking enzymes directed by a pair of sgRNAs targeting opposite strands of a target locus could mediate DSBs and may minimize off-target activity"
Double Nicking Mediates Efficient Genome Editing with Improved Specificity
DN can potentially reduce the likelihood of off-target modifications
"Cas9n minimizes off-target mutagenesis and is suitable for genome editing with increased specificity"
Mc Fonseca's Lab
Efficiency of double-nicking-induced NHEJ
offset no more than 24bp
SURVEYOR nuclease assay
Cas9n didn't generate detectable modification
Ryan M. Walsh & Konrad Hochedlinger
PNAS September 24, 2013 vol. 110 no. 39
Prashant Mali, Kevin M Esvelt & George M Church Nature Methods, October 2013 vol.10 no.10
Using a double nicking system (Cas9n with a pair of sgRNA),
we can reduce the off-target activity in CRISPR systems and
improve specificity of genome editing events