Programmable nucleases — including zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and CRISPR (clustered regularly interspaced short palindromic repeat)-associated protein 9 (Cas9) RNA-guided engineered nucleases (RGENs) — enable genome editing in cultured cells, as well as in whole animals and plants. These nucleases are useful for a broad range of applications in biomedical research, medicine and biotechnology.
ZFNs and TALENs are composed of DNA-binding proteins and the FokI nuclease domain. RGENs are derived from the type II CRISPR–Cas adaptive immune system in bacteria and are composed of guide RNAs and the Cas9 protein.
DNA double-strand breaks (DSBs) introduced by programmable nucleases can be repaired by homology-directed repair, which leads to gene insertion, correction and point mutagenesis, or by erroneous non-homologous end-joining, which results in gene disruptions. The repair of two concurrent DSBs can give rise to chromosomal rearrangements such as deletions, inversions and translocations.
Programmable nucleases induce off-target mutations at sites that are highly homologous to their target sites. Measuring and reducing off-target effects of engineered nucleases is of great importance in research, biotechnology and medicine.
Programmable nickases derived from nucleases induce single-strand breaks (that is, 'nicks'), the repair of which can lead to precise genome editing. Paired nickases can induce genome editing as efficiently as nucleases but with a much higher specificity.
With programmable nucleases, artificial selection can be driven by desired genotypes a priori rather than by unpredictable phenotypes a posteriori.
Programmable nucleases — including zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and RNA-guided engineered nucleases (RGENs) derived from the bacterial clustered regularly interspaced short palindromic repeat (CRISPR)–Cas (CRISPR-associated) system — enable targeted genetic modifications in cultured cells, as well as in whole animals and plants. The value of these enzymes in research, medicine and biotechnology arises from their ability to induce site-specific DNA cleavage in the genome, the repair (through endogenous mechanisms) of which allows high-precision genome editing. However, these nucleases differ in several respects, including their composition, targetable sites, specificities and mutation signatures, among other characteristics. Knowledge of nuclease-specific features, as well as of their pros and cons, is essential for researchers to choose the most appropriate tool for a range of applications.
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This work was supported by the National Research Foundation of Korea (2013-000718 to J.S.K. and 2011-0013568 to H.K.), Health Technology Research and Development Project by the Korean Ministry of Health and Welfare (H10C1740 to H.K.), and Converging Research Center Program funded by the Korean Ministry of Science, ICT and Future Planning (2013K000275 to H.K.). The authors thank three anonymous referees for comments and all the researchers, including our collaborators and competitors, who have contributed to the development of programmable nucleases in the past 20 years and regret the need to omit many relevant papers owing to the page limitation.
The authors hold stocks in ToolGen, Inc. mentioned in this article.
- Homologous recombination
A genetic recombination process in which two similar DNA strands exchange nucleotide sequences.
- Non-homologous end-joining
(NHEJ). A repair pathway for DNA double-strand breaks (DSBs) through direct ligation of the break ends without using a homologous template. This error-prone process often causes small insertions and deletions at the DSB site.
- Zinc-finger nucleases
(ZFNs). Programmable nucleases composed of the FokI catalytic domain and zinc-finger DNA-binding domains.
- Transcription activator-like effector nucleases
(TALENs). Programmable nucleases composed of the FokI catalytic domain and TALE proteins.
- RNA-guided engineered nucleases
(RGENs). Programmable nucleases composed of the Cas9 protein and a guide RNA.
- Clustered regularly interspaced short palindromic repeat
(CRISPR). A genomic locus in bacteria or archaea where protospacers and direct repeat sequences are arrayed in tandem. It is associated with adaptive immunity against invading phages and plasmids.
- Homology-directed repair
(HDR). Template-dependent repair of DNA strand breaks using homologous DNA sequences such as double-strand donor DNA or single-strand oligonucleotides. HDR of programmable nuclease-induced strand breaks leads to precise genome editing, including targeted gene insertion, correction and point mutagenesis.
- Insertions and deletions
(Indels). Small insertions or deletions of DNA sequences relative to a reference sequence.
A type IIS restriction enzyme found in Flavobacterium okeanokoites that is composed of a separable DNA-binding domain and a nuclease domain that is used to construct ZFNs and TALENs.
- Transcription activator-like effectors
(TALEs). DNA-binding proteins with a modular structure derived from Xanthomonas spp. (a plant pathogen). Each module is composed of ~34 amino acids and recognizes a single nucleotide. The base specificity is determined by the amino acids at positions 12 and 13 (known as repeat variable diresidues) in each module.
DNA sequences of 26–72 bp that are initially derived from invading phages and plasmids and that are embedded in the CRISPR loci in bacteria or archaea.
- CRISPR RNA
(crRNA). A small RNA transcribed from the CRISPR loci that determines the target-sequence specificity of Cas9 RNA-guided endonulceases.
- CRISPR-associated protein 9
(Cas9). A protein derived from bacteria such as Streptococcus pyogenes. The Cas9 protein forms an active DNA endonuclease when complexed with guide RNAs.
- Protospacer adjacent motif
(PAM). A short (2–5-bp) nucleotide motif adjacent to protospacers that is recognized by Cas9.
- Single-chain guide RNA
(sgRNA). A small, single-chain guide RNA that is created by the fusion of CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA).
Enzymes that generate DNA single-strand breaks (that is, 'nicks').
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Kim, H., Kim, JS. A guide to genome engineering with programmable nucleases. Nat Rev Genet 15, 321–334 (2014). https://doi.org/10.1038/nrg3686
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