Matthew Wilson
Last active: 3/24/2020

Comparative analysis of chimeric ZFP-, TALE- and Cas9-piggyBac transposases for integration into a single locus in human cells.

Luo W, Galvan DL, Woodard LE, Dorset D, Levy S, Wilson MH
Nucleic Acids Res. 2017 45 (14): 8411-8422

PMID: 28666380 · PMCID: PMC5737283 · DOI:10.1093/nar/gkx572

Integrating DNA delivery systems hold promise for many applications including treatment of diseases; however, targeted integration is needed for improved safety. The piggyBac (PB) transposon system is a highly active non-viral gene delivery system capable of integrating defined DNA segments into host chromosomes without requiring homologous recombination. We systematically compared four different engineered zinc finger proteins (ZFP), four transcription activator-like effector proteins (TALE), CRISPR associated protein 9 (SpCas9) and the catalytically inactive dSpCas9 protein fused to the amino-terminus of the transposase enzyme designed to target the hypoxanthine phosphoribosyltransferase (HPRT) gene located on human chromosome X. Chimeric transposases were evaluated for expression, transposition activity, chromatin immunoprecipitation at the target loci, and targeted knockout of the HPRT gene in human cells. One ZFP-PB and one TALE-PB chimera demonstrated notable HPRT gene targeting. In contrast, Cas9/dCas9-PB chimeras did not result in gene targeting. Instead, the HPRT locus appeared to be protected from transposon integration. Supplied separately, PB permitted highly efficient isolation of Cas9-mediated knockout of HPRT, with zero transposon integrations in HPRT by deep sequencing. In summary, these tools may allow isolation of 'targeted-only' cells, be utilized to protect a genomic locus from transposon integration, and enrich for Cas9-mutated cells.

Published by Oxford University Press on behalf of Nucleic Acids Research 2017.

MeSH Terms (18)

Bacterial Proteins Cell Line, Tumor CRISPR-Associated Protein 9 CRISPR-Cas Systems DNA Transposable Elements Endonucleases Gene Knockout Techniques Gene Targeting Gene Transfer Techniques Humans Hypoxanthine Phosphoribosyltransferase Mutagenesis, Insertional Recombinant Fusion Proteins Reproducibility of Results Transcription Activator-Like Effector Nucleases Transcription Activator-Like Effectors Transposases Zinc Fingers

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