On the island of genome editing, CRISPR is king. Rapidly rising from obscurity, the CRISPR nuclease Cas9 handily dispatched its predecessors, the clumsy TALENs and zinc-finger nucleases. Since then, there has been some strife within the royal family, as better Cas9 proteins and its relatives fought for dominance.
Despite this internal turmoil, the CRISPR dynasty itself seemed unassailable. Now, however, a new Argonaute protein has sailed in, boasting several advantages that could threaten the yet brief reign of CRISPR.
The Argonaute family of proteins can be found throughout all domains of life. Similar to CRISPR, they use nucleic acid guides to find complementary target sequences. Most Argonautes load miRNA guides to target the degradation of complementary mRNA. A few known Argonautes use single-stranded (ss)DNA guides to cleave DNA targets, but these are from thermophiles and require temperatures that would kill mammalian cells.
Sensing CRISPR competitor potential in these guided, DNA-cleaving Argonautes, Feng Gao and Chunyu Han from Hebei University, China, searched for lower-temperature homologs, hitting jackpot with the Natronobacterium gregoryi Argonaute – NgAgo.
- NgAgo uses 5’-phosphorylated ssDNA guides (~24 bp) to cleave supercoiled DNA at 37 C.
- 5’P-ssDNA guides must load soon after protein translation, and NgAgo locks once loaded – it does not exchange guides at 37 C.
- NgAgo will exchange guides at 55 C in vitro, but this high temperature harms the protein and reduces its later activity.
- NgAgo was about as effective as Cas9 for in vivo DNA cleavage in human cells, when an NgAgo-expressing plasmid was co-transfected with 5’P-ssDNA.
- Mammalian cells naturally have very low levels of 5’P-ssDNA, minimizing the risk of NgAgo finding an off-target guide.
Not only does this Argonaute usurper seem to work as well as Cas9, but it has several advantages:
- NgAgo is more specific than Cas9 – it completely loses of activity with only 3 mismatches. This reduces its off-target activity, an Achilles heel for CRISPR systems.
- At only 887 amino acids, NgAgo is slim and svelte for convenient vector packaging.
- No PAM is required at target sites.
- NgAgo is better at targeting (G+C)-rich sequences, since its ssDNA guides form less stable secondary structure than CRISPR’s RNA guides.
Will the N. gregoryi Argonaute overthrow the house of CRISPR, or will it become just one more player jostling for the gene editing throne? We’ll be watching intently, but in the meantime, you can catch this latest twist in the Game of Genomes at Nature Biotechnology, May 2016.