Lentivirus pre-packed with Cas9 protein for safer gene editing

J. G. Choi, Y. Dang, S. Abraham, H. Ma, J. Zhang, H. Guo, Y. Cai, J. G. Mikkelsen, H. Wu, P. Shankar, N. Manjunath

Research output: Contribution to journalArticlepeer-review

135 Scopus citations

Abstract

The CRISPR/Cas9 system provides an easy way to edit specific site/s in the genome and thus offers tremendous opportunity for human gene therapy for a wide range of diseases. However, one major concern is off-target effects, particularly with long-term expression of Cas9 nuclease when traditional expression methods such as via plasmid/viral vectors are used. To overcome this limitation, we pre-packaged Cas9 protein (Cas9P LV) in lentiviral particles for transient exposure and showed its effectiveness for gene disruption in cells, including primary T cells expressing specific single guide RNAs (sgRNAs). We then constructed an 'all in one virus' to express sgRNAs in association with pre-packaged Cas9 protein (sgRNA/Cas9P LV). We successfully edited CCR5 in TZM-bl cells by this approach. Using an sgRNA-targeting HIV long terminal repeat, we also were able to disrupt HIV provirus in the J-LAT model of viral latency. Moreover, we also found that pre-packaging Cas9 protein in LV particle reduced off-target editing of chromosome 4:-29134166 locus by CCR5 sgRNA, compared with continued expression from the vector. These results show that sgRNA/Cas9P LV can be used as a safer approach for human gene therapy applications.

Original languageEnglish
Pages (from-to)627-633
Number of pages7
JournalGene therapy
Volume23
Issue number7
DOIs
StatePublished - Jul 1 2016

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