Abstract
The discovery of clustered, regularly interspaced short palindromic repeats (CRISPR) and their cooperation with CRISPR-associated (Cas) genes is one of the greatest advances of the century and has marked their application as a powerful genome engineering tool. The CRISPR–Cas system was discovered as a part of the adaptive immune system in bacteria and archaea to defend from plasmids and phages. CRISPR has been found to be an advanced alternative to zinc-finger nucleases (ZFN) and transcription activator-like effector nucleases (TALEN) for gene editing and regulation, as the CRISPR–Cas9 protein remains the same for various gene targets and just a short guide RNA sequence needs to be altered to redirect the site-specific cleavage. Due to its high efficiency and precision, the Cas9 protein derived from the type II CRISPR system has been found to have applications in many fields of science. Although CRISPR–Cas9 allows easy genome editing and has a number of benefits, we should not ignore the important ethical and biosafety issues. Moreover, any tool that has great potential and offers significant capabilities carries a level of risk of being used for non-legal purposes. In this review, we present a brief history and mechanism of the CRISPR–Cas9 system. We also describe on the applications of this technology in gene regulation and genome editing; the treatment of cancer and other diseases; and limitations and concerns of the use of CRISPR–Cas9.
Highlights
Precise and efficient genome modification is significant for genetic engineering
clustered regularly-interspaced short palindromic repeats (CRISPR) were identified in the Escherichia coli genome in 1987, and they were characterized as extraordinary sequence elements, which consisted of a series of 29 nucleotide repeats separated by 32 nucleotide “spacer” sequences, which appeared whenever bacteria came in contact with phage DNA [5]
Most of the current human immunodeficiency virus (HIV), human papillomavirus (HPV), hepatitis B virus (HBV) and herpesvirus antiviral therapies do not provide a clinical cure—mainly due to the inability to remove the viral genome from the infected host cell because of a latent state, in which the viruses minimize their activity inside the host cell in order to avoid host immune surveillance
Summary
Precise and efficient genome modification is significant for genetic engineering. The progressive development of technology enables the use of techniques that allow for changes in genomes. The discovery of clustered regularly-interspaced short palindromic repeats (CRISPR); their description as an adaptative prokaryotic immune system (CRISPR–Cas), providing specific and acquired immunity against mobile and exogenic genetic elements; and the following development into a precision genomic editing tool has changed the field of molecular biology [3,4]. Subsequent comparative genomic analysis indicated that CRISPR and Cas proteins cooperate and provide an acquired immune system to protect prokaryotic cells from invading genetic elements, such as viruses and plasmids, analogous to the eukaryotic RNA interference (RNAi) system. This assumption was experimentally proven in 2007, using the lactic acid bacteria Streptococcus thermophilus [3]. It was demonstrated that CRISPRs are transcribed into RNA, which is cleaved and loaded into CRISPR–Cas proteins, and the RNA–protein complex is sufficient for RNA-guided dsDNA endonuclease activity [14,15]
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