Targeted chromosomal deletions in human cells using zinc finger nucleases.
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.
Lee
Hyung Joo
HJ
Department of Chemistry, Seoul National University, Seoul, South Korea.
Kim
Eunji
E
Kim
Jin-Soo
JS
D002460:Cell Line; D002872:Chromosome Deletion; D002877:Chromosomes, Human / Q000235:genetics*; D003001:Cloning, Molecular; D053903:DNA Breaks, Double-Stranded; D004720:Endonucleases / Q000378:metabolism*; D018390:Gene Targeting / Q000379:methods; D005818:Genetic Engineering; D005821:Genetic Techniques; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D054390:Receptors, CCR2 / Q000235:genetics; D019713:Receptors, CCR5 / Q000235:genetics; D017422:Sequence Analysis, DNA; D016335:Zinc Fingers
C515334:CCR2 protein, human; D054390:Receptors, CCR2; D019713:Receptors, CCR5; D004720:Endonucleases