In Nature Biotechnology Gao et al. (2016) describe a new gene editing system unrelated to Cas9 and based on a DNA-guided Argonaute protein with some interesting characteristics and the potential to become another useful tool.
The Cas9 gene editing system is a very facile but not without limitations and constraints. For example, the required protospacer-adjacent motif (PAM) constrains target selection when using Cas9-based editing tools, although clever variants with altered PAM requirements complement the original system so circumventing some of these PAM constraints is possible.
Gao et al. describe an Argonaute protein from Natronobacterium gregoryi (NgAgo), a haloalkaliphilic archaebacterium that uses a single stranded DNA molecule as a guide. The guide molecule is 24 bases in length and must be phosphorylated at the 5’ end.
NgAgo can only be loaded with ssDNA guides at 55 C and not at 37 C and once loaded the complex creates double stranded breaks in the target sequence. When NgAgo is being reloaded at 55 C the protein does not have DNA endonuclease activity and activity is only restored when the complex is returned to 37 C. NgAgo removes several nucleotides in the target region when it cuts DNA although it does not appear to have any exonuclease activity. Details of the endonuclease cleavage are not yet available.
The high temperature loading requirement might have some interesting implications. One being that in vivo expression of NgAgo and guideDNAs may not be an option in most organisms as is done in some applications of Cas9.
What about efficiency? Although not extensively tested in side by side comparisons, in the data presented NgAgo and a Cas9 directed to the same target had similar efficiencies.
After assessing the ability of some 47 guides targeting 8 genes in the human genome the authors report similar efficiencies among all guides tested – between 21% and 41%.
Mismatches between the guide and target reduced cleavage efficiency and a systematic analysis of single mismatches at all 24 positions showed that all positions were important with positions 8 -11 being particularly important. The system seems to be very sensitive to mismatches and this could make off target effects less of a concern. The authors found that 3 consecutive mismatches eliminated all activity.
The NgAgo system does not depend on anything resembling a protospacer-adjacent motif (PAM), nor does its efficacy decrease when targeting GC-rich regions, as appears to be the case with Cas9.
This is an interesting system and this paper is worth reading and considering. How this system develops and how much NgAgo might serve as an alternate to Cas9 remains to be seen but it is clear we have not seen the last of technology improvements and diversification in the area of gene editing.
NOTE: THIS PAPER HAS BEEN RETRACTED BECAUSE OF A LACK OF INDEPENDENT REPRODUCABILITY.