Choosing the Right Tool for the Job: RNAi, TALEN, or CRISPR

Abstract

The most widely used approach for defining a genes’ function is to reduce or completely disrupt its normal expression. For over a decade, RNAi has ruled the lab, offering a magic bullet to disrupt gene expression in many organisms. However, new biotechnological tools - specifically CRISPR-based technologies - have become available and are squeezing out RNAi dominance in mammalian cell studies. These seemingly competing technologies leave research investigators with the question: ‘Which technology should I use in my experiment?’ This review offers a practical resource to compare and contrast these technologies, guiding the investigator when and where to use this fantastic array of powerful tools.

Dynamics of CRISPR/Cas9-mediated genomic editing of the AXL locus in hepatocellular carcinoma cells

“…In particular, all functional copies of the target gene must be edited in cancer cell lines to accomplish a complete knockout situation (24). As NHEJ works in a random fashion, there may arise altered structures without gene inactivation along NHEJ repair events.…”

“…As NHEJ works in a random fashion, there may arise altered structures without gene inactivation along NHEJ repair events. These insufficient knockout events, often combined with cellular heterogeneity, enhance the probability to generate partial knockouts that still harbor alleles coding for functional gene products or gene products with altered functionality (24). Hence, the determination of target gene copy number and cellular heterogeneity is essential in cancer cell populations to allow generation of solid CRISPR/Cas9-mediated knockouts and to correctly interpret the subsequent confirmation of knockout events.…”

Genome Editing with Engineered Nucleases in Economically Important Animals and Plants: State of the Art in the Research Pipeline

“…Cas9, which is utilised by bacteria such as Streptococcus pyogenes in their adaptive immunity systems to recognize and cleave a specific site in the target DNA (Puchta and Fauser, 2014;Rinaldo and Ayliffe, 2015). The CRISPR/Cas9 system is the most recent of the SDNs (Boettcher and McManus, 2015); the d e s i g n i s s t r a i g h t f o r w a r d a n d c a n b e accomplished quickly as the only part that needs redesign is the guide RNA (Puchta and Fauser, 2014). The CRISPR/Cas9 system also allows multiplexing to generate organisms with multiple mutations or large chromosomal deletions (Belhaj et al, 2015).…”

“…The most common and simple DNA mutation is the gene knock-out via NHEJ. The function of a gene is disrupted by introducing an error in the gene sequence most often within the open reading frame of the target gene creating early frame shift mutations or premature stop codons (Boettcher and McManus, 2015;Bortesi and Fischer, 2015). It is possible to designate the target location but the random nature of NHEJ prevents, in most cases, any control over the nature of the mutation (Chen and Gao, 2013;Voytas, 2013;Rinaldo and Ayliffe, 2015).…”

Regulators of G-Protein Signaling 10 and Heart Failure

“…4 Generation of RGS10KO mice by Miao et al was accomplished by CRISPRCas9 technology, a precise and efficient genome editing tool that identifies any DNA sequence by a programmable single guide RNA, which has become a popular approach in genetic engineering. 4,12 As used in this study, this technology created a basic germline knockout of RGS10, meaning that RGS10…”