Enhancing Gene Targeting with Designed Zinc Finger Nucleases

Bibikova, Marina; Beumer, Kelly J.; Trautman, Jonathan K.; Carroll, Dana

doi:10.1126/science.1079512

Cited by 742 publications

(506 citation statements)

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“…R GENs are derived from the adaptive immune system in bacteria and archaea known as clustered, regularly interspaced, short palindromic repeats (CRISPR)/CRISPRassociated protein (Cas) 1 ; they now belong to a family of genome-editing engineered nucleases that induce site-specific DNA cleavages in cells and organisms, whose repair via endogenous DNA double-strand break (DSB) repair systems gives rise to targeted mutagenesis 2,3 and chromosomal rearrangements 4,5 . Unlike zinc-finger nucleases (ZFNs) or transcription activator-like effector (TALE) nucleases (TALENs), whose DNA sequence specificities are determined by protein moieties, RGENs recognize and cleave DNA in a targeted manner using two separate components, that is, a small RNA component termed CRISPR RNA (crRNA) or single-chain guide RNA (sgRNA) that hybridizes with a 20-base pair (bp) long target DNA sequence and the Cas9 protein, derived from Streptococcus pyogenes, which recognizes the NGG (or, to a lesser extent, NAG 6 ) trinucleotide sequence known as the protospacer-adjacent motif (PAM).…”

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Genotyping with CRISPR-Cas-derived RNA-guided endonucleases

et al. 2014

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Restriction fragment length polymorphism (RFLP) analysis is one of the oldest, most convenient and least expensive methods of genotyping, but is limited by the availability of restriction endonuclease sites. Here we present a novel method of employing CRISPR/ Cas-derived RNA-guided engineered nucleases (RGENs) in RFLP analysis. We prepare RGENs by complexing recombinant Cas9 protein derived from Streptococcus pyogenes with in vitro transcribed guide RNAs that are complementary to the DNA sequences of interest. Then, we genotype recurrent mutations found in cancer and small insertions or deletions (indels) induced in cultured cells and animals by RGENs and other engineered nucleases such as transcription activator-like effector nucleases (TALENs). Unlike T7 endonuclease I or Surveyor assays that are widely used for genotyping engineered nuclease-induced mutations, RGEN-mediated RFLP analysis can detect homozygous mutant clones that contain identical biallelic indel sequences and is not limited by sequence polymorphisms near the nuclease target sites.

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confidence: 99%

Genotyping with CRISPR-Cas-derived RNA-guided endonucleases

et al. 2014

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“…Meganucleases such as I-SceI are valuable cleavage reagents because of their long recognition sites (19-21), but their sites must be introduced before they can be attacked. Because zinc fingers exist for many of the DNA triplets, nucleases of this type can theoretically be created to target almost any preexisting locus.Studies performed in Drosophila and in human cells have shown that ZFNs can be designed to effectively cleave specific sequences (22)(23)(24)(25)(26)(27)(28). Additional studies with synthetic targets have demonstrated the efficiency of ZFN-directed cleavage in Xenopus, in mammalian cells and in plants (16,(29)(30)(31).…”

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confidence: 99%

Induction and repair of zinc-finger nuclease-targeted double-strand breaks in Caenorhabditis elegans somatic cells

Morton

Davis

Jørgensen

et al. 2006

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

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Zinc-finger nucleases are chimeric proteins consisting of engineered zinc-finger DNA-binding motifs attached to an endonuclease domain. These proteins can induce site-specific DNA double-strand breaks in genomic DNA, which are then substrates for cellular repair mechanisms. Here, we demonstrate that engineered zinc-finger nucleases function effectively in somatic cells of the nematode Caenorhabditis elegans. Although gene-conversion events were indistinguishable from uncut DNA in our assay, nonhomologous end joining resulted in mutations at the target site. A synthetic target on an extrachromosomal array was targeted with a previously characterized nuclease, and an endogenous genomic sequence was targeted with a pair of specifically designed nucleases. In both cases, Ϸ20% of the target sites were mutated after induction of the corresponding nucleases. Alterations in the extrachromosomal targets were largely products of end-filling and blunt ligation. By contrast, alterations in the chromosomal target were mostly deletions. We interpret these differences to reflect the abundance of homologous templates present in the extrachromosomal arrays versus the paucity of such templates for repair of chromosomal breaks. In addition, we find evidence for the involvement of error-prone DNA synthesis in both homologous and nonhomologous pathways of repair. DNA ligase IV is required for efficient end joining, particularly of blunt ends. In its absence, a secondary end-joining pathway relies more heavily on microhomologies in producing deletions.DNA repair ͉ gene targeting ͉ nematodes ͉ nonhomologous end joining T he ability to make targeted double-strand breaks in chromosomal DNA has several important uses. It allows the detailed study of DNA repair mechanisms; it leads to localized mutagenesis at the break site; and it enhances the efficiency of targeted gene replacement through homologous recombination. We have been exploring the capabilities of one class of targetable cleavage reagents, the zinc-finger nucleases (ZFNs).ZFNs are chimeric proteins composed of DNA-binding Cys 2 His 2 zinc fingers fused to the nonspecific nuclease domain of the restriction enzyme FokI (1). Each finger makes contact primarily with a separate DNA triplet (2, 3). Natural and artificial zinc fingers have been characterized that bind to all 5Ј-GNN-3Ј, many ANN and CNN, and some TNN triplets (4-7). Furthermore, the modular nature of the zinc fingers allows them to be joined in essentially arbitrary combinations. Typically, three zinc fingers are combined to bind to a specific 9-bp DNA sequence with the nanomolar affinity required to be biologically useful, but additional fingers can be incorporated to confer increased specificity (8-11). Zinc-finger fusions to various functional domains have been used to create artificial transcription factors and DNA-modifying proteins (12,13).When attached to the FokI nuclease domain, zinc fingers can direct cleavage to specific DNA sequences. The nuclease domain must dimerize to cleave DNA (14), and because the di...

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“…It is also possible to hijack the repair of the DSB induced by a ZFN or TALEN to introduce alternations (point mutations, deletions) at the cut site [61]. From their beginnings in Drosophila [62], ZFNs have recently been used to perform targeted mutagenesis in mouse zygotes (one-cell embryos) ( Figure 1C) [63,64]. Modification of somatic cells in vivo and gene correction at the haemophilia B locus have also been achieved using ZFNs [65].…”

Section: Engineered Nucleases and Tools For Genome Editingmentioning

confidence: 99%