We present a novel approach for generating targeted deletions of genomic segments in human and other eukaryotic cells using engineered zinc finger nucleases (ZFNs). We found that ZFNs designed to target two different sites in a human chromosome could introduce two concurrent DNA double-strand breaks (DSBs) in the chromosome and give rise to targeted deletions of the genomic segment between the two sites. Using this method in human cells, we were able to delete predetermined genomic DNA segments in the range of several-hundred base pairs (bp) to 15 mega-bp at frequencies of 10(-3) to 10(-1). These high frequencies allowed us to isolate clonal populations of cells, in which the target chromosomal segments were deleted, by limiting dilution. Sequence analysis revealed that many of the deletion junctions contained small insertions or deletions and microhomologies, indicative of DNA repair via nonhomologous end-joining. Unlike other genome engineering tools such as recombinases and meganucleases, ZFNs do not require preinsertion of target sites into the genome and allow precise manipulation of endogenous genomic scripts in animal and plant cells. Thus, ZFN-induced genomic deletions should be broadly useful as a novel method in biomedical research, biotechnology, and gene therapy.
Targeted chromosomal deletions in human cells using zinc finger nucleases.
Published 2010 in Genome Research
ABSTRACT
PUBLICATION RECORD
- Publication year
2010
- Venue
Genome Research
- Publication date
Unknown publication date
- Fields of study
Biology, Medicine
- Identifiers
- External record
- Source metadata
Semantic Scholar, PubMed
CITATION MAP
EXTRACTION MAP
CLAIMS
CONCEPTS
- clonal populations
Cell populations derived from a single progenitor cell and therefore genetically uniform for the edited event of interest.
Aliases: clones
- engineered zinc finger nucleases
Engineered DNA-cutting proteins built from zinc finger DNA-binding modules and a nuclease domain to target chosen genomic sequences.
Aliases: ZFNs
- human cells
Cultured human cells used as the experimental system for inducing chromosomal deletions.
- limiting dilution
A cell-culture isolation method that dilutes cells to low density so clonal descendants can be recovered.
- microhomology
Short shared DNA sequence overlap present at some deletion junctions.
- nonhomologous end-joining
An error-prone DNA repair pathway that rejoins broken DNA ends without requiring a homologous template.
Aliases: NHEJ
- targeted chromosomal deletions
Removal of the chromosomal DNA segment lying between two nuclease target sites.
Aliases: genomic deletions
REFERENCES
Showing 1-41 of 41 references · Page 1 of 1