ReviewTALEN or Cas9 – Rapid, Efficient and Specific Choices for Genome Modifications
Introduction
Modifications of genomes have laid the foundation of functional studies in modern biology and have led to significant discoveries (Esvelt and Wang, 2013). Since the time of Thomas Morgan, scientists, particularly geneticists, have been seeking methods to manipulate genetic materials in different organisms. For a long time, genome editing has largely relied on traditional forward genetic screens, such as chemical mutagenesis (Eeken and Sobels, 1983; Solnica-Krezel et al., 1994) and transposon-mediated mutagenesis (Marx, 1982; Rubin and Spradling, 1982). These screens are intrinsically limited, because (1) it is ineffective to map the mutations to a single gene due to the existence of functional redundancy of different genes; (2) not every mutation produces measurable phenotypes; (3) the biggest constraint is the inability to make specific targeted mutations. The completion of several model organisms' genome sequence has greatly facilitated functional studies of specific genes and opened the era of reverse genetics. Therefore, scientists developed in the past decades reverse genetic technologies that can be used to make precise genetic manipulations, including homologous recombination-based gene targeting (Thomas and Capecchi, 1987; Xu and Rubin, 1993; Melton, 1994; Golic and Golic, 1996; Xu et al., 2009; Chen et al., 2010; Du et al., 2010; Yu and Jiao, 2010; Huang et al., 2011a; Liu et al., 2011; Xie et al., 2012; Dui et al., 2012), ΦC31-mediated integration system (Groth et al., 2004) and zinc finger nucleases (ZFNs)-mediated genomic edition (Bibikova et al., 2002; Bibikova et al., 2003). However, these techniques are often inefficient, time consuming, laborious and expensive, which have been pushing the demand of developing new simpler, more rapid, more efficient and less expensive genome editing techniques to meet the new era of biomedical research.
The principle of genome editing relies on DNA repair system that works when DNA double strand breaks (DSBs) occur. In eukaryotic cells, there are two main types of DNA double strand breaks repair mechanisms, non-homologous end-joining (NHEJ) (Barnes, 2001; Lieber, 2010) and homologous recombinational (HR) repair (van den Bosch et al., 2002). NHEJ rejoins the broken ends and is often accompanied by loss/gain of some nucleotides, thus the outcome of NHEJ is variable: nucleotide insertions, deletions, or nucleotide substitutions in the broken region. HR uses homologous DNA as a template to restore the DSBs, and the outcome of this kind of repair is precise and controllable. For example, through HR repair an exogenous DNA sequence can be added at the break site in the genome. Scientists have been seeking to develop better genetic tools to manipulate the genome by creating a DNA binding domain that can recognize a specific DNA sequence and fusing it with a protein that can offer a nuclease activity. The discovery and application of zinc finger proteins made a revolutionary contribution to genomic editing toolbox. Based on the feature that different zinc fingers recognize different sets of nucleotide triplets, an hybrid protein containing specific zinc finger DNA binding domains and the endonuclease Fok I (ZFN) was generated to target specific DNA sequences (Kim et al., 1996; Urnov et al., 2010). Although considerable progress has been achieved, the use of ZFNs has not been picked up as widely as anticipated mainly due to: (1) there exist context effects on the specificities of individual finger in an array; (2) not all nucleotide triplets have got their corresponding zinc fingers discovered; (3) production of the ZFN proteins with high selectivity is costly, laborious, and time consuming (Bibikova et al., 2002; Bibikova et al., 2003; Urnov et al., 2010; Cradick et al., 2011).
In contrast, TALEN (transcription activator-like effector nuclease)-mediated specific genome editing has much more attractive advantages than ZFNs since its birth about 2–3 years ago (Morbitzer et al., 2010; Hockemeyer et al., 2011; Huang et al., 2011b; Tesson et al., 2011). It has been rapidly and widely used to perform precise genome editing in a wide range of organisms and cell types, including plants (Christian et al., 2010, Morbitzer et al., 2010, Li et al., 2012), frogs (Lei et al., 2012), fish (Huang et al., 2011b, 2012; Shen et al., 2013a; Zu et al., 2013), flies (Liu et al., 2012), worms (Wood et al., 2011), rats (Tesson et al., 2011; Tong et al., 2012), mice (Sung et al., 2013), livestock (Carlson et al., 2012), human somatic cells (Cermak et al., 2011) and human pluripotent stem cells (Hockemeyer et al., 2011). Interestingly, a very recent burst of publications (in the last 2–3 months) indicate that another new site-specific genomic editing tool is being developed, which borrows the CRISPR (clusters of regularly interspaced short palindromic repeats) system and the Cas9 endonuclease (Ishino et al., 1987; Hale et al., 2009; Jore et al., 2011; Carroll, 2012; Jinek et al., 2012). Unlike ZFN or TALEN, CRISPR/Cas9-mediated genome editing system adopts the Watson–Crick complementary rule to recognize and cleave target DNA sequence via a short RNA molecule and the endonuclease Cas9, respectively. It has appeared to be a very effective and promising genome editing tool in mammalian cells (Cho et al., 2013; Cong et al., 2013; Jiang et al., 2013; Jinek et al., 2013) and zebrafish somatic cells at the organismal level (Hwang et al., 2013). However, no success of inheritable Cas9-mediated genome modifications has been reported yet thus far although it is expected to come soon. Up to date, in the family of genomic editing toolbox, TALEN has shown to be an efficient, rapid, specific and economic method with a wide range of applications, and CRISPR/Cas9 system is emerging to be a new choice.
Section snippets
TALEN – an established genomic editing tool
TAL effectors (TALEs), originally discovered in the plant pathogen Xanthomonas sp., act as the bacteria invasion strategies to infect plant (Bonas et al., 1989). These effectors are injected into plant cells via the bacterial type III secretion system, imported into the plant cell nuclei, targeting effector-specific gene promoters to activate gene transcription (Kay et al., 2007; Romer et al., 2007), which may contribute to bacterial colonization. TALEs consist of a group of special effector
Perspectives
The availability of easily customizable DNA binding factors has offered scientists versatile tools to target specific genomic loci. The elegant TALE code for DNA recognition has been well exploited to artificially design TALEN proteins to make genome-wide targeted genetic modifications. The emerging CRISPR/Cas9 system may become (has the potential to be) more effective in artificial genomic editing than TALEN. Unlike the TALE code, the specificity of CRISPR/Cas9-mediated genome editing relies
Acknowledgements
This work has been supported financially by the National Basic Research Program of China (973 Program) (Nos. 2009CB918702 and 2012CB825504), the National Natural Science Foundation of China (Nos. 31201007, 31271573 and 31071087).
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