A guide to genome engineering with programmable nucleases

Journal name:
Nature Reviews Genetics
Year published:
Published online


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.

At a glance


  1. Outcome of genome editing using programmable nucleases.
    Figure 1: Outcome of genome editing using programmable nucleases.

    a | Nuclease-induced double-strand breaks (DSBs) can lead to sequence insertion, nucleotide correction or change (red box) through homology-directed repair (HDR) in the presence of a donor DNA or a single-strand oligodeoxynucleotide (ssODN), both of which contain homology arms. DSBs can also be repaired through error-prone non-homologous end-joining (NHEJ), which does not require donor DNA or ssODN and consequently often leads to small insertions and deletions (indels). Typical indel sequences and the number of inserted (+3 and +1) or deleted (−2, −4 and −10) bases are shown. b | When two DSBs are generated in cis on a single chromosome by programmable nucleases, the flanking region can be deleted or inverted. c | When two DSBs are generated on two different chromosomes, chromosomal translocations can be induced.

  2. Structure of ZFNs.
    Figure 2: Structure of ZFNs.

    a | A schematic representation of a zinc-finger nuclease (ZFN) pair is shown. Each ZFN is composed of a zinc-finger protein (ZFP) at the amino terminus and the FokI nuclease domain at the carboxyl terminus. In the zinc-finger motif consensus, X represents any amino acid. Target sequences of ZFN pairs are typically 18–36 bp in length, excluding spacers. b | A computer model structure of a ZFN pair bound to DNA is shown. Each zinc-finger is shown in shades of pink in ribbon (left) and space-filling (right) representations. The grey region represents the linker between the DNA-binding and catalytic domains. The FokI catalytic domains are shown in blue and purple at the centre using space-filling representations. Part b is modified, with permission, from Ref. 191 © (2011) Genetics Society of America.

  3. Structure of TALENs.
    Figure 3: Structure of TALENs.

    a | A schematic representation of a transcription activator-like effector nuclease (TALEN) pair is shown. Each TALEN is composed of transcription activator-like effectors (TALEs) at the amino terminus and the FokI nuclease domain at the carboxyl terminus. Each TALE repeat is comprised of 33–35 amino acids and recognizes a single base pair through the amino acids at positions 12 and 13, which is called the repeat variable diresidue (RVD; shown in red). Target sequences of TALEN pairs are typically 30–40 bp in length, excluding spacers. b | In the TALE–DNA co-crystal structure, the RVDs in TALE interact with DNA in the major groove. The amino-terminal repeats (designated as 0 and −1 in the box) contact 5′ thymine. Part b is modified, with permission, from Ref. 73 © (2012) American Association for the Advancement of Science.

  4. Structure of RGENs.
    Figure 4: Structure of RGENs.

    Schematic representations of RNA-guided engineered nucleases (RGENs) are shown. a | An RGEN is comprised of CRISPR (clustered regularly interspaced short palindromic repeat)-associated protein 9 (Cas9), a CRISPR RNA (crRNA) and a trans-activating crRNA (tracrRNA), which form the dualRNA–Cas9. b | Alternatively, an RGEN can contain Cas9 and a single-chain guide RNA (sgRNA). The guide sequence in the crRNA (part a) or sgRNA (part b) is complementary to a 20-bp target DNA sequence known as protospacer, which is next to the 5′-NGG-3′ (where N represents any nucleotide) sequence known as protospacer adjacent motif (PAM). Grey dots indicate weak bonding. c | Target DNA cleaved by an RGEN yielding blunt ends is shown. d | A three-dimensional model of Cas9 complexed with DNA is shown. Part d courtesy of D. W. Taylor (University of California, Berkeley, USA), J. A. Doudna (University of California, Berkeley, USA) and M. Jinek (University of Zurich, Switzerland).


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  1. Graduate School of Biomedical Science and Engineering, and College of Medicine, Hanyang University, Wangsimni-ro 222, Sungdong-gu, Seoul 133-791, South Korea.

    • Hyongbum Kim
  2. Center for Genome Engineering, Institute for Basic Science, Gwanak-ro 1, Gwanak-gu, Seoul 151-747, South Korea.

    • Jin-Soo Kim
  3. Department of Chemistry, Seoul National University, Gwanak-ro 1, Gwanak-gu, Seoul 151-747, South Korea.

    • Jin-Soo Kim

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The authors hold stocks in ToolGen, Inc. mentioned in this article.

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  • Hyongbum Kim

    Hyongbum Kim is an assistant professor in the Graduate School of Biomedical Science and Engineering, and College of Medicine at Hanyang University, Seoul, South Korea. He received his M.D. in 2001 and Ph.D. in 2006 from Yonsei University, Seoul. During his Ph.D. programme, he studied tissue engineering using mesenchymal stem cells and biomaterials. After postdoctoral training at Emory University, Atlanta, Georgia, USA, in the field of stem cell biology, he became an independent researcher in 2010. His laboratory is interested in genome engineering in several types of cultured cells (including stem cells) and in mammals. Hyongbum Kim's homepage.

  • Jin-Soo Kim

    Jin-Soo Kim is an entrepreneur and a chemist-turned-biologist. He graduated from Seoul National University, South Korea, in 1987 with a major in chemistry. He obtained a master degree in chemistry from Seoul National University in 1989 and a Ph.D. in biochemistry from the University of Wisconsin–Madison, USA, in 1994. After postdoctoral training at Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, USA, he returned to Seoul in 1997 as a principal investigator at Samsung Biomedical Research Institute. He co-founded a biotechnology company, ToolGen, Inc., in 1999, and was chief executive officer and chief strategy officer until he joined the faculty of the Department of Chemistry at Seoul National University in 2005. He now serves as Director of the Center for Genome Engineering at the Institute for Basic Science, Seoul.

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