Targeted genome editing by lentiviral protein transduction of zinc-finger and TAL-effector nucleases.
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.
Department of Biomedicine, Aarhus University, Aarhus, Denmark.
Yujia
Y
Cai
Department of Biomedicine, Aarhus University, Aarhus, Denmark.
Rasmus O
RO
Bak
Department of Biomedicine, Aarhus University, Aarhus, Denmark giehm@hum-gen.au.dk.
Jacob Giehm
JG
Mikkelsen
D001483:Base Sequence; D004720:Endonucleases / Q000096:biosynthesis* / Q000235:genetics; D005786:Gene Expression Regulation; D005818:Genetic Engineering / Q000379:methods*; D005822:Genetic Vectors; D015894:Genome, Human; D057809:HEK293 Cells; D006801:Humans; D016086:Lentivirus / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D019713:Receptors, CCR5 / Q000235:genetics / Q000378:metabolism; D013997:Time Factors; D014157:Transcription Factors / Q000096:biosynthesis* / Q000235:genetics; D014161:Transduction, Genetic; D014162:Transfection / Q000379:methods*; D014771:Virion; D016335:Zinc Fingers
C584933:CCR5 protein, human; D019713:Receptors, CCR5; D014157:Transcription Factors; D004720:Endonucleases
Gag; lentiviral vector; protein transduction; transcription activator-like effector nucleases; zinc-finger nucleases