“New Tool Changes Biology Forever.”
“Magic Biological Wand Takes Over Labs Across the Country.”
“Welcome to the Modern Era of Molecular Biology.”
Such headlines dominated the front pages of newspapers and scientific magazines when the genetic engineering tool CRISPR-Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR associated protein 9) was first introduced. The advent of CRISPR-Cas9 enabled scientists to edit genes with unparalleled efficiency and versatility. Derived from the natural defense system of bacteria, CRISPR-Cas9 employs a single guide RNA to direct the enzyme Cas9 to cut a specific target gene sequence. This revolutionary genetic engineering tool has significantly impacted a wide variety of fields, from medicine to agriculture to even paleontology. Nowadays, CRISPR has become so influential that nearly all molecular biology laboratories employ this tool — it has become as common as PCR machines. Even with the widespread use of this game-changing tool, research scientists have begun to think towards the future about ways to further improve genetic engineering. The announcement of another genome editing tool has garnered as much debate and criticism as it has elicited laudatory remarks.
Enter NgAgo. On March 21, 2016, Chinese scientists from the Hebei University of Science and Technology announced the breakthrough of their DNA-guided genome editing tool using the Argonaute protein derived from Natronobacterium gregoryi. The scientists claim that the new gene editing protein has multiple advantages over CRISPR-Cas9, especially in regards to flexibility and efficiency in gene editing. First of all, while the enzyme Cas9 utilizes RNA as a guide to locate the site of gene editing, the Argonaute enzyme employs a single-stranded DNA guide (gDNA) of roughly 24 nucleotides that can efficiently induce site-specific double-stranded breaks in DNA. Argonaute’s DNA guides are advantageous since the shorter, 24-base DNA guides can be directly purchased, thus eliminating the need for scientists to undergo the arduous process of constructing the single-guide RNA required in CRISPR gene editing.
Another advantage of NgAgo is its greater targeting flexibility. For Cas9 to cut genes, the target DNA sequence of the guide RNA must be next to a specific sequence called a PAM (Protospacer Adjacent Motif). In contrast to CRISPR/Cas9, NgAgo is not limited to editing in regions adjacent to the PAM sequence, which gives it more flexibility in editing DNA wherever scientists decide. In addition, NgAgo has shown higher efficiency in editing DNA regions. This greater editing efficiency is promising, especially since CRISPR-mediated editing often leads to undesirable off-target effects in which the guide RNA may accidentally direct the Cas9 to cut the DNA at the wrong sequence, leading to disastrous mutations. While these numerous advantages of NgAgo over CRISPR-Cas9 might make one wonder whether CRISPR is phasing out of popularity and NgAgo is now seizing the spotlight, the viral debate over the efficacy of NgAgo reminds us never to be too hasty when it comes to scientific breakthroughs.
“Biotech journal to check DNA claims”
While the announcement of NgAgo has captivated many researchers, ensuing concerns over the reproducibility of the experiments caution us against drawing such bold claims of a scientific breakthrough from a single paper. The original paper stemmed from Han Chunyu, a biologist at Hebei University of Science and Technology in Shijiazhuang. After the publication of his paper in Nature Biotechnology, many scientists criticized that they were unable to replicate the results of Han’s experiment. Since the introduction of CRISPR-Cas9 greatly increased the potency of scientists’ laboratory tool kits, many scientists were understandably eager to see the success of NgAgo. Geneticist George Church at Harvard Medical School summarized one side of the debate in support of NgAgo, saying, “A lot of us are really cheerleading and hoping that it works.” Yet the scientific community has been divided by the uncertainty about the reproducibility of the results. For example, geneticist Gaetan Burgio of the Australian National University reported his failure to replicate Han’s experiments. Other researchers have likewise warned not to waste time and money on using NgAgo.
This ongoing debate highlights an important lesson of scientific inquiry. Research is an ongoing process with every new experiment testing the efficacy of previous experiments and adding new knowledge to the field. While NgAgo seems to be teetering on a balance beam of long-lasting success and ephemeral fluke, Han’s paper and the ensuing debate give us hope.
UPDATE: Since the initial publication of Gao’s paper in Nature Biotechnology, several reports have indicated that NgAgo is not a DNA-guided endonuclease and does not create double-stranded breaks in DNA, but rather single-stranded breaks in RNA. These recent studies definitively disprove the earlier suggestions that NgAgo is a genome editing tool. This article was written before these new results were announced.