Abstract
The field of genome editing started with the discovery of meganucleases (e.g., the LAGLIDADG family of homing endonucleases) in yeast. After the discovery of transcription activator-like effector nucleases and zinc finger nucleases, the recently discovered clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated proteins (Cas) system has opened a new window of applications in the field of gene editing. Here, we review different Cas proteins and their corresponding features including advantages and disadvantages, and we provide an overview of the different endonuclease-deficient Cas protein (dCas) derivatives. These dCas derivatives consist of an endonuclease-deficient Cas9 which can be fused to different effector domains to perform distinct in vitro applications such as tracking, transcriptional activation and repression, as well as base editing. Finally, we review the in vivo applications of these dCas derivatives and discuss their potential to perform gene activation and repression in vivo, as well as their potential future use in human therapy.
Highlights
In the last ten years, major breakthroughs have been made in the field of gene editing, which is the process where DNA is modified, deleted, inserted or replaced
We have demonstrated that dCas9-TET3CD induces gene-specific re-activation and demethylation, which results in the amelioration of kidney fibrosis in vivo via lentiviral vectors [44]
The discovery of endonuclease-deficient Cas9 has led to the development of different clustered regularly interspaced short palindromic repeats (CRISPR)/dCas9 derivatives, which can be used to perform distinct functions such as tracking, transcriptional activation and repression, and base editing
Summary
In the last ten years, major breakthroughs have been made in the field of gene editing, which is the process where DNA is modified, deleted, inserted or replaced. The sequence in between the two ZFN binding sites is the so-called spacer region, consisting of 5–7 nucleotides where Fokl monomers form a catalytically active dimer, which cleaves the DNA, resulting in double-strand breaks [6]. The second TALEN monomer is designed for the complementary strand These Fokl nuclease monomers will again form a catalytically active dimer in the spacer region and cleave the DNA. These repeat sequences are subsequently transcribed into so-called CRISPR RNAs (crRNAs) which recognize foreign DNA and recruit a Cas protein to cleave the foreign DNA This system can be adapted by the design of a so-called single guide RNA (sgRNA), which is a custom-designed short crRNAs sequence fused to the scaffold trans-activating crRNA sequence (tracrRNA) and guides the Cas nuclease to a specific DNA region of interest. Colors indicate different scores: green (high), blue (middle) and peachpuff (low)
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