Gene Inactivation Strategies: An Update

Abstract

The ability to manipulate gene expression levels has been essential to the study of gene function and biological processes. Classically, whole body deletions of genes were generated via homologous recombination. Over the past two decades, the development of Cre‐Lox and flippase/flippase recombinase target based gene inactivation technologies allowed researchers to closely regulate the location and timing of gene expression. However, these methods are costly and time consuming. The last few years have seen a revolution in the approaches scientists use to inactivate gene expression, such as the development of highly efficient ribonucleic acid interference (RNAi) delivery systems and the groundbreaking genome editing technologies. Here, the authors describe the recent updates on small interfering RNA (siRNA)in vivo delivery as well as the methodologies and applications of zinc‐finger nucleases, transcription activator‐like effector nucleases and clustered regularly interspaced short palindromic repeats. Equipped with these powerful tools, scientists can now rapidly modulate the expression of genes of interest and precisely modify the genomic sequences in virtually any organisms.

Key Concepts:

  • RNA interference (RNAi) is an endogenous post‐transcriptional gene‐regulatory mechanism mediated by noncoding RNA molecules to regulate gene expression.

  • Chemical modification on siRNA reduces off‐target effect, improves RNAi efficiency and enhances the stability of siRNA for in vivo applications.

  • Genome editing is a genetic approach used to directly manipulate an organism's genome by inserting, replacing, or removing DNA sequences.

  • Engineered nucleases, such as zinc‐finger nucleases (ZFNs), transcription activator‐like effector nucleases (TALENs) and the bacterial clustered regularly interspaced short palindromic repeat (CRISPR)/Cas (CRISPR‐associated) system enable targeted genetic modifications in cells, tissues and organisms.

Keywords: gene inactivation; RNAi; siRNA delivery; genome editing; programmable nucleases; ZFN; TALEN; CRISPR

Figure 1.

Chemically modified siRNA enhances RNA stability and decreases its vulnerability to nucleases and susceptibility to innate immune responses. Common chemical modifications of siRNA include nucleobase, internucleotide phosphate linkage and 2′‐hydroxyl modifications.

Figure 2.

Structure of zinc‐finger nucleases (ZFNs) and transcription activator‐like effector nucleases (TALENs). (a) ZFNs utilise FokI nucleases as the DNA‐cleavage domain and bind DNA by engineered Cys2–His2 zinc fingers which recognise different nucleotide triplets and dimerise the FokI nuclease. The activated nuclease introduces a double‐stranded break between the two distinct zinc‐finger binding sites. (b) TALEN systems are a fusion of TALEs derived from the Xanthomonas spp. to endonuclease FokI. Individual TALE repeats contain 33–35 amino acids that recognise a single base pair via two hypervariable residues (NN, NI, HD and NG for the recognition of G,A,C and T nucleotide, respectively).

Figure 3.

CRISPR/Cas9 is an RNA‐guided genome‐editing platform. The system contains two critical components: (1) a guide RNA and (2) Cas9 nuclease. The guide RNA is a combination of the crRNA and tracrRNA into a single chimeric guide RNA (gRNA) transcript. The gRNA combines the targeting specificity of the crRNA with the scaffolding properties of the tracrRNA into a single transcript. The binding of the gRNA/Cas9 complex localises the Cas9 to the genomic target sequence so that the wild‐type Cas9 can cut both strands of DNA causing a double‐strand break (DSB). For successful binding of Cas9, the genomic target sequence must also contain the correct protospacer adjacent motiff (PAM) sequence immediately following the target sequence.

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Guo, Chang‐An, O'Neill, Lucas M, and Ntambi, James M(Oct 2014) Gene Inactivation Strategies: An Update. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0021020.pub2]