Trends in Biotechnology
ReviewZFN, TALEN, and CRISPR/Cas-based methods for genome engineering
Section snippets
Classical and contemporary approaches for establishing gene function
With the development of new and affordable methods for whole-genome sequencing, and the design and implementation of large genome annotation projects, scientists are poised to deliver upon the promises of the genomic revolution to transform basic science and personalized medicine. The resulting wealth of information presents researchers with a new primary challenge of converting this enormous amount of data into functionally and clinically relevant knowledge. Central to this problem is the need
Custom DNA-binding domains
The versatility of ZFNs and TALENs arises from the ability to customize the DNA-binding domain to recognize virtually any sequence. These DNA-binding modules can be combined with numerous effector domains to affect genomic structure and function (Box 1), including nucleases, transcriptional activators and repressors, recombinases, transposases, DNA and histone methyltransferases, and histone acetyltransferases. Thus, the ability to execute genetic alterations depends largely on the DNA-binding
Genome editing with site-specific nucleases
Historically, targeted gene inactivation, replacement, or addition has been achieved by homologous recombination; however, the low efficiency of homologous recombination in mammalian cells and model organisms dramatically limits the utility of this approach. Following the discovery that induction of a DSB increases the frequency of HDR by several orders of magnitude, targeted nucleases have emerged as the method of choice for improving the efficiency of HDR-mediated genetic alterations. By
Improving the performance of site-specific nucleases
In order for customizable nucleases to carry relevance for genetic analysis and clinical application, they must demonstrate strict specificity toward their intended DNA targets. Complex genomes, however, often contain multiple copies of sequences that are identical or highly homologous to the intended DNA target, leading to off-target activity and cellular toxicity. To address this problem, structure 52, 53 and selection-based 54, 55 approaches have been used to generate improved ZFN and TALEN
Site-specific nucleases in model organisms
Site-specific nucleases have enabled the introduction of targeted modifications in several model organisms common to biological research, including zebrafish 66, 67, 68, rats and mice 69, 70, Drosophila 71, 72, Caenorhabditis elegans [73], and many other species for various applications, including the monarch butterfly [74], frogs [75], and livestock 76, 77. ZFNs and TALENs have also allowed investigators to compare gene function across related species, such as C. elegans and Caenorhabditis
Therapeutic applications of site-specific nucleases
The use of site-specific nucleases for therapeutic purposes represents a paradigm shift in gene therapy. Unlike conventional methods, which either temporarily address disease symptoms or randomly integrate therapeutic factors in the genome, ZFNs and TALENs are capable of correcting the underlying cause of the disease, therefore permanently eliminating the symptoms with precise genome modifications. To date, ZFN-induced HDR has been used to directly correct the disease-causing mutations
Genome editing using programmable RNA-guided DNA endonucleases
Distinct from the site-specific nucleases described above, the CRISPR/Cas system has recently emerged as a potentially facile and efficient alternative to ZFNs and TALENs for inducing targeted genetic alterations. In bacteria, the CRISPR system provides acquired immunity against invading foreign DNA via RNA-guided DNA cleavage [98]. In the type II CRISPR/Cas system, short segments of foreign DNA, termed ‘spacers’ are integrated within the CRISPR genomic loci and transcribed and processed into
Concluding remarks and future directions
ZFNs, TALENs, and RNA-guided DNA endonucleases are transformative tools that have the potential to revolutionize biological research and affect personalized medicine. Indeed, these emerging technologies have dramatically expanded the ability to manipulate and study model organisms, and support the promise of correcting the genetic causes behind many diseases. However, in order to achieve the full potential of this technology, many important questions and challenges must be addressed (Box 3).
Acknowledgments
The authors are supported by the National Institutes of Health (Pioneer Award DP1CA174426 (CB) and DP2OD008586 (CG) and National Science Foundation (CBET-1151035). T.G. was supported by National Institute of General Medicine Sciences fellowship (T32GM080209). We apologize to those investigators whose important contributions may have been omitted due to space constraints.
Glossary
- CRISPR/Cas (CRISPR associated) systems
- clustered regulatory interspaced short palindromic repeats are loci that contain multiple short direct repeats, and provide acquired immunity to bacteria and archaea. CRISPR systems rely on crRNA and tracrRNA for sequence-specific silencing of invading foreign DNA. Three types of CRISPR/Cas systems exist: in type II systems, Cas9 serves as an RNA-guided DNA endonuclease that cleaves DNA upon crRNA–tracrRNA target recognition.
- crRNA
- CRISPR RNA base pairs with
References (119)
Structure of Aart, a designed six-finger zinc finger peptide, bound to DNA
J. Mol. Biol.
(2006)Effects of different zinc finger transcription factors on genomic targets
Biochem. Biophys. Res. Commun.
(2006)Rapid “open-source” engineering of customized zinc-finger nucleases for highly efficient gene modification
Mol. Cell
(2008)A TALEN genome-editing system for generating human stem cell-based disease models
Cell Stem Cell
(2013)Gene targeting of a disease-related gene in human induced pluripotent stem and embryonic stem cells
Cell Stem Cell
(2009)Directed evolution of an enhanced and highly efficient FokI cleavage domain for zinc finger nucleases
J. Mol. Biol.
(2010)Site-specific gene correction of a point mutation in human iPS cells derived from an adult patient with sickle cell disease
Blood
(2011)Generation of isogenic pluripotent stem cells differing exclusively at two early onset Parkinson point mutations
Cell
(2011)Generation of an HIV resistant T-cell line by targeted “stacking” of restriction factors
Mol. Ther.
(2013)A foundation for universal T-cell based immunotherapy: T cells engineered to express a CD19-specific chimeric-antigen-receptor and eliminate expression of endogenous TCR
Blood
(2012)
Gene targeting in mice: functional analysis of the mammalian genome for the twenty-first century
Nat. Rev. Genet.
Gene silencing in mammals by small interfering RNAs
Nat. Rev. Genet.
Genome editing with engineered zinc finger nucleases
Nat. Rev. Genet.
Genome engineering with zinc-finger nucleases
Genetics
DNA double-strand break repair: all's well that ends well
Annu. Rev. Genet.
Engineering polydactyl zinc-finger transcription factors
Nat. Biotechnol.
Design of polydactyl zinc-finger proteins for unique addressing within complex genomes
Proc. Natl. Acad. Sci. U.S.A.
Toward controlling gene expression at will: specific regulation of the erbB-2/HER-2 promoter by using polydactyl zinc finger proteins constructed from modular building blocks
Proc. Natl. Acad. Sci. U.S.A.
Positive and negative regulation of endogenous genes by designed transcription factors
Proc. Natl. Acad. Sci. U.S.A.
Getting a handhold on DNA: design of poly-zinc finger proteins with femtomolar dissociation constants
Proc. Natl. Acad. Sci. U.S.A.
Highly active zinc-finger nucleases by extended modular assembly
Genome Res.
Preassembled zinc-finger arrays for rapid construction of ZFNs
Nat. Methods
Modular system for the construction of zinc-finger libraries and proteins
Nat. Protoc.
Toward controlling gene expression at will: selection and design of zinc finger domains recognizing each of the 5′-GNN-3′ DNA target sequences
Proc. Natl. Acad. Sci. U.S.A.
Selection-free zinc-finger-nuclease engineering by context-dependent assembly (CoDA)
Nat. Methods
An optimized two-finger archive for ZFN-mediated gene targeting
Nat. Methods
Breaking the code of DNA binding specificity of TAL-type III effectors
Science
A simple cipher governs DNA recognition by TAL effectors
Science
The crystal structure of TAL effector PthXo1 bound to its DNA target
Science
Structural basis for sequence-specific recognition of DNA by TAL effectors
Science
Targeting DNA double-strand breaks with TAL effector nucleases
Genetics
A novel TALE nuclease scaffold enables high genome editing activity in combination with low toxicity
Nucleic Acids Res.
A TALE nuclease architecture for efficient genome editing
Nat. Biotechnol.
Efficient construction of sequence-specific TAL effectors for modulating mammalian transcription
Nat. Biotechnol.
Chimeric TALE recombinases with programmable DNA sequence specificity
Nucleic Acids Res.
Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting
Nucleic Acids Res.
FLASH assembly of TALENs for high-throughput genome editing
Nat. Biotechnol.
Iterative capped assembly: rapid and scalable synthesis of repeat-module DNA such as TAL effectors from individual monomers
Nucleic Acids Res.
A ligation-independent cloning technique for high-throughput assembly of transcription activator-like effector genes
Nat. Biotechnol.
A library of TAL effector nucleases spanning the human genome
Nat. Biotechnol.
Targeted gene addition into a specified location in the human genome using designed zinc finger nucleases
Proc. Natl. Acad. Sci. U.S.A.
Zinc-finger nuclease-driven targeted integration into mammalian genomes using donors with limited chromosomal homology
Nucleic Acids Res.
High-frequency genome editing using ssDNA oligonucleotides with zinc-finger nucleases
Nat. Methods
Targeted gene knockout in mammalian cells by using engineered zinc-finger nucleases
Proc. Natl. Acad. Sci. U.S.A.
Targeted chromosomal deletions in human cells using zinc finger nucleases
Genome Res.
Autonomous zinc-finger nuclease pairs for targeted chromosomal deletion
Nucleic Acids Res.
Targeted chromosomal duplications and inversions in the human genome using zinc finger nucleases
Genome Res.
Chromosomal translocations induced at specified loci in human stem cells
Proc. Natl. Acad. Sci. U.S.A.
In vivo cleavage of transgene donors promotes nuclease-mediated targeted integration
Biotechnol. Bioeng.
Obligate Ligation-Gated Recombination (ObLiGaRe): custom-designed nuclease-mediated targeted integration through nonhomologous end joining
Genome Res.
Cited by (2855)
Mitochondrial diseases and mtDNA editing
2024, Genes and DiseasesRecent advances in CRISPR-Cas9-based genome insertion technologies
2024, Molecular Therapy Nucleic Acids