BiooTools
NgAgo-gDNA tool

NgAgo Gene Editing -horizondiscovery.com

www.horizondiscovery.com/gene-editing/ngago

In May 2016, a DNA-guided nucleasesuitable for genome editing in cells was reported by Gao et al in NatureBiotechnology.  Like CRISPR-Cas9, NgAgo can introduce targeteddouble strand breaks in mammalian cells (the paper reports targeting in MCF7,K562 and HeLa cells). However, NgAgo differs from the CRISPR Cas9 system in anumber of ways:


·        It uses a DNA guide and not RNA as a guide for targetinga genomic site

·        It removes several nucleotides within its target regionrather than just introducing a break

·        The guide can be 24 nucleotides long, while sgRNAs forCas9 are limited to 20 nucleotides of homology

·        NgAgo has no requirement for a PAM site - meaning thereare potentiall no target sequence restrictions


Alongside these differences the systemhas a number of other nice features that lend it to genome editing withpotentially high fidelity:


·        It requires 5' phosphorylated ssDNA as a guide. Thesetypes of nucleotide are not found in high abundance in human cells

·        Once loaded with its guide it will not swap it for unboundfree oligos - further minimizing the possibility it will be loaded withunexpected guides.

·        The system is highly sensitive to single base mismatchesalong the length of the guide

·        Evidence from Gao et al suggests NgAgocan drive HDR with high efficiency (>10%)

·        It's smaller than Cas9 - 887aa vs 1368aa


Finally, Gao et al comparedthe targeting efficiency of NgAgo with Cas9 directly. At a well studied genetarget (DYRK1A) the efficiency of the two systems was comparable (31.97% forNgAgo vs 32.2% for Cas9). However in GC rich loci NgAgo was found to performsignificantly better. This observation, coupled with the lack ofrequirement for PAM makes this a potentially very exciting expansion to thegenome editors tool box.