Review
TALEN or Cas9 – Rapid, Efficient and Specific Choices for Genome Modifications

https://doi.org/10.1016/j.jgg.2013.03.013Get rights and content

Abstract

Precise modifications of complex genomes at the single nucleotide level have been one of the big goals for scientists working in basic and applied genetics, including biotechnology, drug development, gene therapy and synthetic biology. However, the relevant techniques for making these manipulations in model organisms and human cells have been lagging behind the rapid high throughput studies in the post-genomic era with a bottleneck of low efficiency, time consuming and laborious manipulation, and off-targeting problems. Recent discoveries of TALEs (transcription activator-like effectors) coding system and CRISPR (clusters of regularly interspaced short palindromic repeats) immune system in bacteria have enabled the development of customized TALENs (transcription activator-like effector nucleases) and CRISPR/Cas9 to rapidly edit genomic DNA in a variety of cell types, including human cells, and different model organisms at a very high efficiency and specificity. In this review, we first briefly summarize the development and applications of TALENs and CRISPR/Cas9-mediated genome editing technologies; compare the advantages and constraints of each method; particularly, discuss the expected applications of both techniques in the field of site-specific genome modification and stem cell based gene therapy; finally, propose the future directions and perspectives for readers to make the choices.

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).

References (91)

  • L.S. Qi et al.

    Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression

    Cell

    (2013)
  • Y. Song et al.

    CAF-1 is essential for Drosophila development and involved in the maintenance of epigenetic memory

    Dev. Biol.

    (2007)
  • K.R. Thomas et al.

    Site-directed mutagenesis by gene targeting in mouse embryo-derived stem cells

    Cell

    (1987)
  • C. Tong et al.

    Rapid and cost-effective gene targeting in rat embryonic stem cells by TALENs

    J. Genet. Genomics

    (2012)
  • V.M. Bedell et al.

    In vivo genome editing using a high-efficiency TALEN system

    Nature

    (2012)
  • M. Bibikova et al.

    Targeted chromosomal cleavage and mutagenesis in Drosophila using zinc-finger nucleases

    Genetics

    (2002)
  • M. Bibikova et al.

    Enhancing gene targeting with designed zinc finger nucleases

    Science

    (2003)
  • J. Boch et al.

    Xanthomonas AvrBs3 family-type III effectors: discovery and function

    Annu. Rev. Phytopathol.

    (2010)
  • J. Boch et al.

    Breaking the code of DNA binding specificity of TAL-type III effectors

    Science

    (2009)
  • A.J. Bogdanove et al.

    TAL effectors: customizable proteins for DNA targeting

    Science

    (2011)
  • A. Bolotin et al.

    Clustered regularly interspaced short palindrome repeats (CRISPRs) have spacers of extrachromosomal origin

    Microbiology

    (2005)
  • U. Bonas et al.

    Genetic and structural characterization of the avirulence gene avrBs3 from Xanthomonas campestris pv. vesicatoria

    Mol. Gen. Genet.

    (1989)
  • A.W. Briggs et al.

    Iterative capped assembly: rapid and scalable synthesis of repeat-module DNA such as TAL effectors from individual monomers

    Nucleic Acids Res.

    (2012)
  • D.F. Carlson et al.

    Efficient TALEN-mediated gene knockout in livestock

    Proc. Natl. Acad. Sci. USA

    (2012)
  • T. Cermak et al.

    Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting

    Nucleic Acids Res.

    (2011)
  • F. Chen et al.

    High-frequency genome editing using ssDNA oligonucleotides with zinc-finger nucleases

    Nat. Methods

    (2011)
  • Y. Chen et al.

    Drosophila RecQ5 is required for efficient SSA repair and suppression of LOH in vivo

    Protein Cell

    (2010)
  • S.W. Cho et al.

    Targeted genome engineering in human cells with the Cas9 RNA-guided endonuclease

    Nat. Biotechnol.

    (2013)
  • S.M. Choi et al.

    Efficient drug screening and gene correction for treating liver disease using patient-specific stem cells

    Hepatology

    (2013)
  • M. Christian et al.

    Targeting DNA double-strand breaks with TAL effector nucleases

    Genetics

    (2010)
  • L. Cong et al.

    Comprehensive interrogation of natural TALE DNA-binding modules and transcriptional repressor domains

    Nat. Commun.

    (2012)
  • L. Cong et al.

    Multiplex genome engineering using CRISPR/Cas systems

    Science

    (2013)
  • T.J. Cradick et al.

    ZFN-site searches genomes for zinc finger nuclease target sites and off-target sites

    BMC Bioinformatics

    (2011)
  • D. Deng et al.

    Structural basis for sequence-specific recognition of DNA by TAL effectors

    Science

    (2012)
  • G. Du et al.

    Drosophila histone deacetylase 6 protects dopaminergic neurons against {alpha}-synuclein toxicity by promoting inclusion formation

    Mol. Biol. Cell

    (2010)
  • K.M. Esvelt et al.

    Genome-scale engineering for systems and synthetic biology

    Mol. Syst. Biol.

    (2013)
  • A. Garg et al.

    Engineering synthetic TAL effectors with orthogonal target sites

    Nucleic Acids Res.

    (2012)
  • J.E. Garneau et al.

    The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA

    Nature

    (2010)
  • G. Gasiunas et al.

    Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria

    Proc. Natl. Acad. Sci. USA

    (2012)
  • K.G. Golic et al.

    Engineering the Drosophila genome: chromosome rearrangements by design

    Genetics

    (1996)
  • A.C. Groth et al.

    Construction of transgenic Drosophila by using the site-specific integrase from phage phiC31

    Genetics

    (2004)
  • D. Hockemeyer et al.

    Genetic engineering of human pluripotent cells using TALE nucleases

    Nat. Biotechnol.

    (2011)
  • P. Horvath et al.

    CRISPR/Cas, the immune system of bacteria and archaea

    Science

    (2010)
  • H. Huang et al.

    Roles of chromatin assembly factor 1 in the epigenetic control of chromatin plasticity

    Sci. China Life. Sci.

    (2012)
  • H. Huang et al.

    Drosophila CAF-1 regulates HP1-mediated epigenetic silencing and pericentric heterochromatin stability

    J. Cell Sci.

    (2010)
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