Abstract
Chimeric restriction enzymes are a novel class of engineered nucleases in which the non-specific DNA cleavage domain of FokI (a type IIS restriction endonuclease) is fused to other DNA-binding motifs. The latter include the three common eukaryotic DNA-binding motifs, namely the helix-turn-helix motif, the zinc finger motif and the basic helix-loop-helix protein containing a leucine zipper motif. Such chimeric nucleases have been shown to make specific cuts in vitro very close to the expected recognition sequences. The most important chimeric nucleases are those based on zinc finger DNA-binding proteins because of their modular structure. Recently, one such chimeric nuclease, Zif-QQR-F(N) was shown to find and cleave its target in vivo. This was tested by microinjection of DNA substrates and the enzyme into frog oocytes. The injected enzyme made site-specific double-strand breaks in the targets even after assembly of the DNA into chromatin. In addition, this cleavage activated the target molecules for efficient homologous recombination. Since the recognition specificity of zinc fingers can be manipulated experimentally, chimeric nucleases could be engineered so as to target a specific site within a genome. The availability of such engineered chimeric restriction enzymes should make it feasible to do genome engineering, also commonly referred to as gene therapy.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 841-848 |
| Number of pages | 8 |
| Journal | Biological Chemistry |
| Volume | 380 |
| Issue number | 7-8 |
| DOIs | |
| State | Published - Jul 1999 |
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Keywords
- Chimeric restriction endonuclease
- Flavobacterium okeanokoites
- Gene therapy
- Genome engineering
- Hybrid restrictions enzymes
- Recognition and cleavage domains
ASJC Scopus subject areas
- Biochemistry
Cite this
Chimeric restriction enzymes : What is next? / Chandrasegaran, Srinivasan; Smith, Jeff.
In: Biological Chemistry, Vol. 380, No. 7-8, 07.1999, p. 841-848.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Chimeric restriction enzymes
T2 - What is next?
AU - Chandrasegaran, Srinivasan
AU - Smith, Jeff
PY - 1999/7
Y1 - 1999/7
N2 - Chimeric restriction enzymes are a novel class of engineered nucleases in which the non-specific DNA cleavage domain of FokI (a type IIS restriction endonuclease) is fused to other DNA-binding motifs. The latter include the three common eukaryotic DNA-binding motifs, namely the helix-turn-helix motif, the zinc finger motif and the basic helix-loop-helix protein containing a leucine zipper motif. Such chimeric nucleases have been shown to make specific cuts in vitro very close to the expected recognition sequences. The most important chimeric nucleases are those based on zinc finger DNA-binding proteins because of their modular structure. Recently, one such chimeric nuclease, Zif-QQR-F(N) was shown to find and cleave its target in vivo. This was tested by microinjection of DNA substrates and the enzyme into frog oocytes. The injected enzyme made site-specific double-strand breaks in the targets even after assembly of the DNA into chromatin. In addition, this cleavage activated the target molecules for efficient homologous recombination. Since the recognition specificity of zinc fingers can be manipulated experimentally, chimeric nucleases could be engineered so as to target a specific site within a genome. The availability of such engineered chimeric restriction enzymes should make it feasible to do genome engineering, also commonly referred to as gene therapy.
AB - Chimeric restriction enzymes are a novel class of engineered nucleases in which the non-specific DNA cleavage domain of FokI (a type IIS restriction endonuclease) is fused to other DNA-binding motifs. The latter include the three common eukaryotic DNA-binding motifs, namely the helix-turn-helix motif, the zinc finger motif and the basic helix-loop-helix protein containing a leucine zipper motif. Such chimeric nucleases have been shown to make specific cuts in vitro very close to the expected recognition sequences. The most important chimeric nucleases are those based on zinc finger DNA-binding proteins because of their modular structure. Recently, one such chimeric nuclease, Zif-QQR-F(N) was shown to find and cleave its target in vivo. This was tested by microinjection of DNA substrates and the enzyme into frog oocytes. The injected enzyme made site-specific double-strand breaks in the targets even after assembly of the DNA into chromatin. In addition, this cleavage activated the target molecules for efficient homologous recombination. Since the recognition specificity of zinc fingers can be manipulated experimentally, chimeric nucleases could be engineered so as to target a specific site within a genome. The availability of such engineered chimeric restriction enzymes should make it feasible to do genome engineering, also commonly referred to as gene therapy.
KW - Chimeric restriction endonuclease
KW - Flavobacterium okeanokoites
KW - Gene therapy
KW - Genome engineering
KW - Hybrid restrictions enzymes
KW - Recognition and cleavage domains
UR - http://www.scopus.com/inward/record.url?scp=0032876974&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0032876974&partnerID=8YFLogxK
U2 - 10.1515/BC.1999.103
DO - 10.1515/BC.1999.103
M3 - Article
C2 - 10494832
AN - SCOPUS:0032876974
VL - 380
SP - 841
EP - 848
JO - Biological Chemistry
JF - Biological Chemistry
SN - 1431-6730
IS - 7-8
ER -