Targeting individual subunits of the FokI restriction endonuclease to specific DNA strands

Sanders, Kelly; Catto, Lucy E.; Bellamy, Stuart R.W.; Halford, Stephen E.

doi:10.1093/nar/gkp046

Cited by 48 publications

(50 citation statements)

References 60 publications

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“…1A) necessary for dimerization and subsequent DNA cleavage (Bitinaite et al 1998). Specifically, we focused on amino acids D450 and D467, both previously shown to result in catalytically inactive FokI variants when mutated (Waugh and Sauer 1993;Sanders et al 2009). …”

Section: Resultsmentioning

confidence: 99%

Targeted gene addition to a predetermined site in the human genome using a ZFN-based nicking enzyme

Wang¹,

Friedman²,

Doyon³

et al. 2012

Genome Res.

124

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Zinc-finger nucleases (ZFNs) drive highly efficient genome editing by generating a site-specific DNA double-strand break (DSB) at a predetermined site in the genome. Subsequent repair of this break via the nonhomologous end-joining (NHEJ) or homology-directed repair (HDR) pathways results in targeted gene disruption or gene addition, respectively. Here, we report that ZFNs can be engineered to induce a site-specific DNA single-strand break (SSB) or nick. Using the CCR5-specific ZFNs as a model system, we show that introduction of a nick at this target site stimulates gene addition using a homologous donor template but fails to induce significant levels of the small insertions and deletions (indels) characteristic of repair via NHEJ. Gene addition by these CCR5-targeted zinc finger nickases (ZFNickases) occurs in both transformed and primary human cells at efficiencies of up to ∼1%–8%. Interestingly, ZFNickases targeting the AAVS1 “safe harbor” locus revealed similar in vitro nicking activity, a marked reduction of indels characteristic of NHEJ, but stimulated far lower levels of gene addition—suggesting that other, yet to be identified mediators of nick-induced gene targeting exist. Introduction of site-specific nicks at distinct endogenous loci provide an important tool for the study of DNA repair. Moreover, the potential for a SSB to direct repair pathway choice (i.e., HDR but not NHEJ) may prove advantageous for certain therapeutic applications such as the targeted correction of human disease-causing mutations.

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Section: Resultsmentioning

confidence: 99%

Targeted gene addition to a predetermined site in the human genome using a ZFN-based nicking enzyme

Wang¹,

Friedman²,

Doyon³

et al. 2012

Genome Res.

124

View full text Add to dashboard Cite

show abstract

“…It has been shown that DNA-nicking enzymes allow homology-directed gene targeting in mammalian cells (Lee et al 2004;van Nierop et al 2009) and that nickases can be created by engineering naturally occurring restriction enzymes (Sanders et al 2009) or meganucleases (McConnell Smith et al 2009). However, these enzymes are not readily reprogrammed to target any predetermined DNA sequence and thus cannot be used to modify DNA sequences at user-defined genomic sites.…”

Section: Discussionmentioning

confidence: 99%

“…(Hereinafter, L and R refer to zinc finger proteins that bind to the left half-site and the right half-site, respectively, and EL and KK refer to two forms of obligatory heterodimeric FokI domains.) We introduced a mutation (Asp450 to Ala) at the active site of the FokI domain in one subunit to make a catalytically inert monomer (Sanders et al 2009), which can be paired with a wild-type monomer to yield a nickase. A wild-type monomer alone cannot induce a SSB because two FokI nuclease domains must dimerize to cleave a phosphodiester bond.…”

Section: Redesign Of a Zfn Pair To Make Nickasesmentioning

confidence: 99%

Precision genome engineering with programmable DNA-nicking enzymes

Kim¹,

Kim²,

Kim³

et al. 2012

Genome Res.

130

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Zinc finger nucleases (ZFNs) are powerful tools of genome engineering but are limited by their inevitable reliance on error-prone nonhomologous end-joining (NHEJ) repair of DNA double-strand breaks (DSBs), which gives rise to randomly generated, unwanted small insertions or deletions (indels) at both on-target and off-target sites. Here, we present programmable DNA-nicking enzymes (nickases) that produce single-strand breaks (SSBs) or nicks, instead of DSBs, which are repaired by error-free homologous recombination (HR) rather than mutagenic NHEJ. Unlike their corresponding nucleases, zinc finger nickases allow site-specific genome modifications only at the on-target site, without the induction of unwanted indels. We propose that programmable nickases will be of broad utility in research, medicine, and biotechnology, enabling precision genome engineering in any cell or organism.

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“…1F). A targeted single-strand break (SSB) has the potential to restrict repair to the homology-directed repair (HDR) pathway (28,29). Zinc-finger nickases (ZFNs) are well established for generating SSBs; more recently, this strategy even has been reported in TALENs (5,(29)(30)(31)(32).…”

Section: Resultsmentioning

confidence: 99%

“…Zinc-finger nickases (ZFNs) are well established for generating SSBs; more recently, this strategy even has been reported in TALENs (5,(29)(30)(31)(32). Therefore, a mutation at the active site (D450A) that abolishes catalytic activity without affecting protein dimerization or DNA recognition was introduced to FokI of the right hand of TALENs (28).…”

Section: Resultsmentioning

confidence: 99%

TALE nickase-mediatedSP110knockin endows cattle with increased resistance to tuberculosis

Wang

Zhang

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

161

112

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Transcription activator-like effector nuclease (TALEN)-mediated genome modification has been applied successfully to create transgenic animals in various species, such as mouse, pig, and even monkey. However, transgenic cattle with gene knockin have yet to be created using TALENs. Here, we report site-specific knockin of the transcription activator-like effector (TALE) nickase-mediated SP110 nuclear body protein gene (SP110) via homologous recombination to produce tuberculosis-resistant cattle. In vitro and in vivo challenge and transmission experiments proved that the transgenic cattle are able to control the growth and multiplication of Mycobacterium bovis, turn on the apoptotic pathway of cell death instead of necrosis after infection, and efficiently resist the low dose of M. bovis transmitted from tuberculous cattle in nature. In this study, we developed TALE nickases to modify the genome of Holstein–Friesian cattle, thereby engineering a heritable genome modification that facilitates resistance to tuberculosis.

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Targeting individual subunits of the FokI restriction endonuclease to specific DNA strands

Cited by 48 publications

References 60 publications

Targeted gene addition to a predetermined site in the human genome using a ZFN-based nicking enzyme

Targeted gene addition to a predetermined site in the human genome using a ZFN-based nicking enzyme

Precision genome engineering with programmable DNA-nicking enzymes

TALE nickase-mediatedSP110knockin endows cattle with increased resistance to tuberculosis

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