Targeted genome editing by lentiviral protein transduction of zinc-finger and TAL-effector nucleases.

Yujia Cai,Jacob Giehm Mikkelsen,Rasmus O Bak

doi:10.7554/elife.01911

Abstract

Future therapeutic use of engineered site-directed nucleases, like zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), relies on safe and effective means of delivering nucleases to cells. In this study, we adapt lentiviral vectors as carriers of designer nuclease proteins, providing efficient targeted gene disruption in vector-treated cell lines and primary cells. By co-packaging pairs of ZFN proteins with donor RNA in 'all-in-one' lentiviral particles, we co-deliver ZFN proteins and the donor template for homology-directed repair leading to targeted DNA insertion and gene correction. Comparative studies of ZFN activity in a predetermined target locus and a known nearby off-target locus demonstrate reduced off-target activity after ZFN protein transduction relative to conventional delivery approaches. Additionally, TALEN proteins are added to the repertoire of custom-designed nucleases that can be delivered by protein transduction. Altogether, our findings generate a new platform for genome engineering based on efficient and potentially safer delivery of programmable nucleases.DOI: http://dx.doi.org/10.7554/eLife.01911.001.

Highlights

The capacity of designed nucleases, like ZFNs and transcription activator-like effector nucleases (TALENs), to generate DNA double-stranded breaks (DSBs) at desired positions in the genome has created optimism for therapeutic translation of locusdirected genome engineering
We describe the use of lentivirus-derived particles as carriers of designer nucleases for safe administration of ZFN and TALEN proteins fused to lentiviral Gag precursors
To incorporate ZFN proteins in Lentiviral particles (LPs), we fused ZFNs to the N-terminus of Gag containing an intervening heterologous phospholipase C-δ1 pleckstrin homology (PH) domain thought to promote the recruitment of Gag and GagPol to the membrane (Urano et al, 2008)

Summary

Introduction

The capacity of designed nucleases, like ZFNs and TALENs, to generate DNA double-stranded breaks (DSBs) at desired positions in the genome has created optimism for therapeutic translation of locusdirected genome engineering. ZFNs and TALENs are chimeric nucleases composed of a custom-designed DNA binding domain fused to the DNA-cleavage domain from the FokI endonuclease that upon dimer formation cleaves the DNA. ZFNs and TALENs have been administered to cells by transfection or electroporation of nucleic acids, DNA or RNA, encoding a pair of nuclease proteins (Urnov et al, 2005; Miller et al, 2011; Carlson et al, 2012) or by exploiting viral gene vehicles such as integrase-deficient lentiviral vectors (IDLVs) (Lombardo et al, 2007), adeno-associated virus-derived vectors (AAV vectors) (Ellis et al, 2013), or adenoviral vectors (Holkers et al, 2013). ZFNs have been fused to destabilizing domains regulated by small

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