KnockoutbyTALENorCRISPRvs.knockdownby-GeneCopoeia.pdfVIP

TM TM GGeenneeCCooppooeeiiaa TECHNICAL NOTE Expressway to DiscoveryExpressway to Discovery Knockout by TALEN or CRISPR vs. Knockdown by shRNA or siRNA Ed Davis, Ph.D. Recent advances in technologies for genome editing-the use of TALEN or CRISPR to make targeted, permanent changes to genes-have revolutionized molecular genetics. They have also presented users with a choice between these relatively new technologies and that of the more established method of RNA interference (RNAi)-mediated knockdown using short hairpin RNA (shRNA) or short interfering RNA (siRNA). In this Technical Note, we explore the differences between the two methods for ablating gene function, and situations where one technology is more appropriate than the other. RNAi-mediated gene silencing In higher eukaryotes, RNAi-mediated knockdown is the most common strategy for depleting cells of a gene product of interest. However, RNAi usually does not completely shut off the gene. Essentially, short (approximately 20-25 nucleotides) double stranded RNA molecules are either generated from hairpin-forming precursors (shRNAs) or introduced exogenously (siRNAs). After processing by Dicer, a single stranded RNA base-pairs with a target mRNA (Ketting, 2013). Depending on the organism, RNAi- mediated gene silencing is carried out by Argonaute proteins via either mRNA degradation or translation inhibition (Figure 1). The end result is post transcriptional down-regulation of gene expression, without changing the genetic code (Mittal, 2004). Some functional RNA or protein remains and is translated at lower levels. So, the RNAi strategy for reducing gene function is termed a “knockdown”. G