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NgAgo Gene Editing - horizondiscovery.com
罗列了其技术优势:
In May 2016, a DNA-guided nuclease suitable for genome editing in cells was reported by Gao et al in Nature Biotechnology. Like CRISPR-Cas9, NgAgo can introduce targeted double strand breaks in mammalian cells (the paper reports targeting in MCF7, K562 and HeLa cells). However, NgAgo differs from the CRISPR Cas9 system in a number of ways:
It uses a DNA guide and not RNA as a guide for targeting a genomic site
It removes several nucleotides within its target region rather than just introducing a break
The guide can be 24 nucleotides long, while sgRNAs for Cas9 are limited to 20 nucleotides of homology
NgAgo has no requirement for a PAM site - meaning there are potentiall no target sequence restrictions
Alongside these differences the system has a number of other nice features that lend it to genome editing with potentially high fidelity:
It requires 5' phosphorylated ssDNA as a guide. These types of nucleotide are not found in high abundance in human cells
Once loaded with its guide it will not swap it for unbound free oligos - further minimizing the possibility it will be loaded with unexpected guides.
The system is highly sensitive to single base mismatches along the length of the guide
Evidence from Gao et al suggests NgAgo can drive HDR with high efficiency (>10%)
It's smaller than Cas9 - 887aa vs 1368aa
Finally, Gao et al compared the targeting efficiency of NgAgo with Cas9 directly. At a well studied gene target (DYRK1A) the efficiency of the two systems was comparable (31.97% for NgAgo vs 32.2% for Cas9). However in GC rich loci NgAgo was found to perform significantly better. This observation, coupled with the lack of requirement for PAM makes this a potentially very exciting expansion to the genome editors tool box.
注:PAM:
Protospacer adjacent motif (PAM) is a DNA sequence immediately following the DNA sequence targeted by the Cas9 nuclease in the CRISPR bacterial adaptive immune system.
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