The CRISPR-Cas9 system renews the gene editing approach into a more convenient and efficient way. By rapidly introducing genetic modifications in cell lines, organs and animals, CRISPR-Cas9 system extends the gene editing into whole genome screening, both in loss-of-function and gain-of-function manners. Furthermore, CRISPR-Cas9 system is modified into diverse innovative tools for observing the dynamic bioprocesses in cancer studies, such as image tracing for targeted DNA, regulation of transcription activation or repression. As scientists continually strive to improve the system, CRISPR-Cas9 system exhibits little side effects and excellent efficiency in gene editing, and extend their applications to gene therapy and functional studies besides cancer research, etc.
The CRISPR-Cas9 system is a versatile tool for gene editing that uses a single guide RNA (sgRNA) to target Cas9 to a specific sequence. This simple RNA-guided genome-editing technology has become a revolutionary tool in biology and has many innovative applications in different fields. Originally discovered as an adaptive prokaryotic immune system, CRISPR-Cas9 holds the promise of achieving precise modiﬁcations in the genome with a simplicity and versatility that surpasses previous genome editing methods. It has been repurposed for gene editing in a broad range of model organisms, from yeast to mammalian cells, and including protist parasites. cancer characterization and modeling have beneﬁtted greatly from the genome editing capabilities of CRISPR-Cas9.
Gene editing is a strategy to modify genomic DNA and change the genetic information artificially, including gain-of-function (gene knock-in, gene mutation, gene labeling and gene activation) and loss-of-function (gene knock-out, and gene mutation). CRISPR-Cas9, a type II CRISPR system from Streptococcus pyogenes, has rapidly become the most promising gene editing tool with great potential to revolutionize medicine. Through guidance of a 20 nucleotide RNA (gRNA), CRISPR-Cas9 finds and cuts target protospacer DNA precisely 3 base pairs upstream of a PAM, producing double strand breaks (DSBs).
The DSBs are usually repaired by either nonhomologous end-joining (NHEJ) resulting in small indels, or by homology-directed repair (HDR) for precise gene or nucleotide replacement. Theoretically, CRISPR-Cas9 could be used to modify any genomic sequences, thereby providing a simple, easy, and cost effective means of genome wide gene editing. HDR works when the impaired sites have homologue DNA sequences in the nucleus. This mechanism protects genetic information because homologous DNA will be used as templates for the repair. As a result, the sequence of donor fragment is integrated into the genome at the DSBs site.
If homologous DNA is absent, NHEJ which can introduce a small insertion or deletion at the site, thus knocking out the gene, works to repair the DSBs. Lacking of templates, NHEJ easily loses genetic information and introduces insertions or deletions into damaged sites. When site-specially inducing DSBs, the NHEJ could be used to introduce gene alteration in cell lines or animal organs. The DSBs repair system (NHEJ and HDR) is a ubiquitous component of all living cells; therefore, an artificial nuclease whose recognition site is reprogrammable is the most critical part of gene editing.
Nowadays, three programmable nucleases have been developed for gene editing, including zinc finger nuclease (ZFN), transcription activator-like eﬀector nuclease (TALEN), and CRISPR. Among these nucleases, the Cas9 nuclease, which recognizes target DNA according to Watson-Crick base pairing between its guide RNA(s) and DNA, shows the highest specificity and efficiency and is the simplest one to implement and quickly became the most popular and powerful tool for genome engineering. This two-component system (Cas9-sgRNA) has been easily adapted to many biological models. And it not only provides a molecular tool for investigating biological questions in depth, but also enables the development of innovative and practical applications of biology.
Fig 1. The comparison of working mechanism among ZFN, TALEN and CRISPR
ZFN consists of a zinc finger DNA-binding domain and DNA cleavage domain of the FokI type IIS restriction endonuclease. In 1990s, ZFN was applied to site-specific gene editing. Since this editing system needs to be synthesized commercially and is difficult to use, ZFN was gradually replaced by other systems. TALEN is another engineered nuclease, which shows better specificity and efficiency than ZFN. Similar to ZFN, TALEN consists of DNA-binding domain and DNA cleavage domain. However, the major challenge for TALEN is to clone the large modules in series, joint these modules in designed order by ligase in an efficient way. And the other technique barrier is low screening efficiency for successful targeted cells. These three systems can all create DSBs near the targeted DNA locus and initiate the DNA repair procedures.
However, Cas9 can tolerate, to a certain extent, mismatches between the sgRNA and the target sequence in the genome, resulting in off-target effects, as some previous studies have shown. These undesirable effects of the CRISPR-Cas9 system might impede the use of this gene editing technology for clinical applications; therefore, a great deal of effort has been made to improve the efﬁciency and speciﬁcity of CRISPR-Cas9. Shortly after CRISPR-Cas9 system was applied to mammalian genome editing, scientists continually modified the system to increase its efficiency, reduce the off-target effect and simplify its delivery method. The system is also modified for new applications besides gene editing, such as imaging targeted DNA, and regulating transcription activation or repression, etc.
To date, targeted gene knock-out using the CRISPR-Cas9 system has been established in many plant species, and the targeting efficiency and capacity of Cas9 has been improved by optimizing its expression and that of its gRNA. The CRISPR-Cas9 system can also be used for sequence-specific mutagenesis / integration and transcriptional control of target genes. Cancer research has and will continue to beneﬁt from this new genome editing technology. In particular, modeling oncogenesis in mice using CRISPR-Cas9 surpassed previous genome editing tools in terms of time length and cost effectiveness without the need of multiplying colonies of mice.
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