Histone deacetylase inhibition rescues gene knockout levels achieved with integrase-defective lentiviral vectors encoding zinc-finger nucleases.
Zinc-finger nucleases (ZFNs) work as dimers to induce double-stranded DNA breaks (DSBs) at predefined chromosomal positions. In doing so, they constitute powerful triggers to edit and to interrogate the function of genomic sequences in higher eukaryotes. A preferred route to introduce ZFNs into somatic cells relies on their cotransduction with two integrase-defective lentiviral vectors (IDLVs) each encoding a monomer of a functional heterodimeric pair. The episomal nature of IDLVs diminishes the risk of genotoxicity and ensures the strict transient expression profile necessary to minimize deleterious effects associated with long-term ZFN activity. However, by deploying IDLVs and conventional lentiviral vectors encoding HPRT1- or eGFP-specific ZFNs, we report that DSB formation at target alleles is limited after IDLV-mediated ZFN transfer. This IDLV-specific underperformance stems, to a great extent, from the activity of chromatin-remodeling histone deacetylases (HDACs). Importantly, the prototypic and U.S. Food and Drug Administration-approved inhibitors of metal-dependent HDACs, trichostatin A and vorinostat, respectively, did not hinder illegitimate recombination-mediated repair of targeted chromosomal DSBs. This allowed rescuing IDLV-mediated site-directed mutagenesis to levels approaching those achieved by using their isogenic chromosomally integrating counterparts. Hence, HDAC inhibition constitutes an efficacious expedient to incorporate in genome-editing strategies based on transient IDLV-mediated ZFN expression. Finally, we compared two of the most commonly used readout systems to measure targeted gene knockout activities based on restriction and mismatch-sensitive endonucleases. These experiments indicate that these enzymatic assays display a similar performance.
1 Department of Molecular Cell Biology, Leiden University Medical Center , 2333 ZC Leiden, The Netherlands .
Laetitia P L
LP
Pelascini
Ignazio
I
Maggio
Jin
J
Liu
Maarten
M
Holkers
Toni
T
Cathomen
Manuel A F V
MA
Gonçalves
D053903:DNA Breaks, Double-Stranded; D004706:Endodeoxyribonucleases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D055786:Gene Knockout Techniques / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics*; D057809:HEK293 Cells; D006367:HeLa Cells; D056572:Histone Deacetylase Inhibitors / Q000494:pharmacology; D006655:Histone Deacetylases / Q000378:metabolism*; D006801:Humans; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics; D019426:Integrases / Q000235:genetics*; D016086:Lentivirus / Q000235:genetics*; D059630:Mesenchymal Stem Cells / Q000378:metabolism; D032446:Myoblasts / Q000378:metabolism; D016335:Zinc Fingers
D056572:Histone Deacetylase Inhibitors; D007041:Hypoxanthine Phosphoribosyltransferase; D019426:Integrases; D004706:Endodeoxyribonucleases; D006655:Histone Deacetylases