Standardized reagents and protocols for engineering zinc finger nucleases by modular assembly

Abstract

Engineered zinc finger nucleases can stimulate gene targeting at specific genomic loci in insect, plant and human cells. Although several platforms for constructing artificial zinc finger arrays using "modular assembly" have been described, standardized reagents and protocols that permit rapid, cross-platform "mixing-and-matching" of the various zinc finger modules are not available. Here we describe a comprehensive, publicly available archive of plasmids encoding more than 140 well-characterized zinc finger modules together with complementary web-based software (termed ZiFiT) for identifying potential zinc finger target sites in a gene of interest. Our reagents have been standardized on a single platform, enabling facile mixing-and-matching of modules and transfer of assembled arrays to expression vectors without the need for specialized knowledge of zinc finger sequences or complicated oligonucleotide design. We also describe a bacterial cell-based reporter assay for rapidly screening the DNA-binding activities of assembled multi-finger arrays. This protocol can be completed in approximately 24-26 d.

Site-directed mutagenesis in Arabidopsis using custom-designed zinc finger nucleases

“…Amino acid sequences of helix motif in ZF are used as follows: QRAHLER for finger 1, QSGHLQR for finger 2, and QRAHLER for finger 3 in ZF_AAA; and RSDALTR for finger 1, RSDDLQR for finger2, RSDDLQR for finger 3 in ZF_TCC. The coding sequences of ZF proteins were cloned between the Xba I and Bam HI sites of the pGB-FB vector (41).…”

“…B2H assays were performed according to the protocol of Wright et al (41). Plasmid DNA and bacterial strains for B2H were obtained from Addgene.…”

Zinc-finger directed double-strand breaks within CAG repeat tracts promote repeat instability in human cells

“…Individual zinc fingers have now been engineered in vitro to recognize many DNA triplets (3)(4)(5)(6). Zinc fingers are modular, enabling their assembly into linear arrays that can be designed to bind a variety of genome sequences (7)(8)(9). These custom zinc finger proteins can be attached to other protein domains to direct various functions to specific locations in the genomes of cells and organisms (10).…”

Method of Detecting and Targeting Mutations in Cancer

“…These proteins have been fused to various other protein moieties, producing for example artificial transcription factors and site-specific nucleases [5][6][7][8][9][10][11]. Additionally, ZF proteins fused to reporter proteins have been used to detect specific DNA sequences both in vitro [12][13][14] and in plants and mammalian cell culture [15].…”