When introducing DNA into cells there is a small chance that the introduced DNA will be inserted into the host cell DNA, resulting in gene editing. Introducing breaks into cellular DNA at the region where you want to edit increases the likelihood of gene editing taking place. A number of DNA cutting enzymes, called nucleases, have been engineered to cut at precise sequences of DNA but the older nucleases (meganucleases, Zinc Finger Nucleases and TALE Nucleases) were laborious to make, difficult to work with, expensive and inefficient. All of this changed with the discovery and development of Cas9 nuclease and the CRISPR-Cas9 system. As a result, gene editing is now something almost every molecular biology lab in the world is doing.
The CRISPR-Cas9 system was first identified in bacteria as part of its immune system helping prevent viral infection by cutting viral DNA. Scientists then discovered the Cas9 nuclease could be used to cut human DNA guided by a guide RNA (Jinek et al., 2012 and Cong et al., 2013). This system is reminiscent to RNA interference (RNAi) whose discovery earned Andrew Fire and Craig Mello a Nobel Prize in 2006.
RNAi utilises RNA-induced silencing compex (RISC), a multi-protein complex guided by micro or small interfering RNA to cleave messenger RNA thereby preventing gene expression. RNA cleavage is via a protein from the Argonaute protein family. Researchers recently made the startling discovery that Argonaute proteins from the heat-loving bacteria Thermus thermophilus and the heat-loving archaea Pyrococcus furiosus were able to introduce breaks in DNA using single stranded DNA (rather than RNA) as a guide (Swarts et al., 2014 and Swarts et al., 2015). However, these proteins only functioned at high temperatures (65oC) precluding their use in human cells which work at 37oC.
A team of scientists in China searched the US National Center for Biotechnology Information databases for proteins with sequences similar to those of the DNA cutting Argonautes from the heat-loving organisms and found the Natronobacterium gregoryi bacteria has an Argonaute protein (NgAgo) that cleaves DNA at 37oC, as Cas9 does, indicating a potential to utilise this protein for gene editing of human cells. The group showed that NgAgo was equally as efficient at inducing breaks in DNA as Cas9. They also found that whilst Cas9 utilises an RNA guide, NgAgo requires single stranded DNA with a length of 24 base pairs. Gao et al., (2016) demonstrate that NgAgo does not switch guide DNA once the single stranded DNA is loaded and the co-ordinates set.
Whilst Cas9 based gene editing requires DNA ending in “GG” nucleotides, the researchers demonstrate no such limitation for NgAgo based strategies, which may enable larger portion of the genome to become amenable to genetic manipulation.
The human genome has many stretches of DNA with sequences rich in “GC” bases which are difficult to target with conventional Cas9 as RNA with a high number of “GC” bases tend to form secondary structures making them unsuitable for loading into Cas9. DNA is less prone to forming secondary structures and as NgAgo utilises DNA as its guide this opens up the possibility of targeting “GC” rich regions of the genome– a finding confirmed by Gao et al. (2016).
A major concern for all nucleases used for gene editing is potential “off-target” effects, where a nuclease cuts at a position where it shouldn’t. Gao et al., (2016) found that introducing just a single change in the guide DNA of NgAgo reduced its cutting efficiency by 73-100% and three changes prevented any cutting at the target gene- compared to Cas9 which can tolerate up to five mismatches in the guide RNA sequence. Whilst these data show a very low tolerance for guide-target mismatches with NgAgo further studies will be needed to properly look for “off-target” effects with the gold-standard being deep sequencing to identify any “off target” changes in the genome.
In summary, NgAgo has a number of benefits of Cas9 including;
• Low tolerance of mismatches between guide DNA sequence and target DNA sequence
• No switching of guide DNA sequences
• Ability to target GC-rich DNA sequences
Further research will be required to determine the propensity for off-target cleavage with NgAgo.
No doubt NgAgo will not render Cas9 obsolete given that CRISPR-Cas9 is now a staple in any molecular biology lab. However the unique advantages will ensure this is not the last you will hear about the NgAgo nuclease.
Cong, L., Ran, F. A., et al. (2013). Multiplex Genome Engineering Using CRISPR/Cas Systems. Science. 339, 819-823.
Jinek, M., Chylinski, K., et al. (2012). A Programmable Dual-RNA-Guided DNA Endonuclease in Adaptive Bacterial Immunity. Science. 337, 816-821.
Swarts, D. C., Hegge, J. W., et al. (2015). Argonaute of the archaeon Pyrococcus furiosus is a DNA-guided nuclease that targets cognate DNA. Nucleic Acids Res. 43, 5120-5129.
Swarts, D. C., Jore, M. M., et al. (2014). DNA-guided DNA interference by a prokaryotic Argonaute. Nature. 507, 258-261.