A recent report of genome editing using Natronobacterium gregoryi Argonaute (NgAgo) with guide DNA (gDNA) in human cells1 prompted us to explore the utility of this protein for in vivo genetic manipulation in zebrafish (Danio rerio). Zebrafish is a model organism that offers several distinct advantages for studying genetics, developmental biology, vascular biology and disease modeling. In the last several years, loss-of-function genomic editing techniques, including zinc-finger nucleases (ZFNs), artificial transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 system (CRISPR/Cas9), have been adopted for zebrafish. Recently the gDNA/NgAgo system has elicited much interest because of some unique advantages: low tolerance to guide-target mismatch, minimum off-target effects, and easy to design. The scientists from Nantong University and Fudan University investigate whether the gDNA/NgAgo system can be used to manipulate zebrafish genes in vivo using fabp11a as a test case.

Their study shows that gene knockdown is the main mechanism by which gDNA/NgAgo affects gene function in zebrafish. This is supported by results of the experiments using presumably catalytically dead NgAgo. In addition, they have failed to detect any mutation in all the embryos we examined. Since the catalytically inactive (dead) Cas9 (dCas9):sgRNA complex could efficiently inhibit gene expression through binding to the coding sequence, they hypothesize that gDNA/NgAgo may bind to a target gene to block its transcription. Overall, they suggest that the gDNA/NgAgo system provides an alternative strategy for gene knockdown in zebrafish.

(SOURCE: Cell Research)