Cell-penetrating peptide-mediated delivery of Cas9 protein and guide RNA for genome editing
- Authors
- Suresh, B.; Ramakrishna, S.; Kim, H.
- Issue Date
- 2017
- Publisher
- Humana Press Inc.
- Keywords
- Cas9 conjugation; Cas9 protein purification; Dialysis; In vitro sgRNA synthesis; Protein delivery; T7E1 assay
- Citation
- Methods in Molecular Biology, v.1507, pp.81 - 94
- Indexed
- SCOPUS
- Journal Title
- Methods in Molecular Biology
- Volume
- 1507
- Start Page
- 81
- End Page
- 94
- URI
- https://scholarworks.bwise.kr/hanyang/handle/2021.sw.hanyang/153275
- DOI
- 10.1007/978-1-4939-6518-2_7
- ISSN
- 1064-3745
- Abstract
- The clustered, regularly interspaced, short palindromic repeat (CRISPR)-associated (Cas) system represents an efficient tool for genome editing. It consists of two components: the Cas9 protein and a guide RNA. To date, delivery of these two components has been achieved using either plasmid or viral vectors or direct delivery of protein and RNA. Plasmid- and virus-free direct delivery of Cas9 protein and guide RNA has several advantages over the conventional plasmid-mediated approach. Direct delivery results in shorter exposure time at the cellular level, which in turn leads to lower toxicity and fewer off-target mutations with reduced host immune responses, whereas plasmid- or viral vector-mediated delivery can result in uncontrolled integration of the vector sequence into the host genome and unwanted immune responses. Cellpenetrating peptide (CPP), a peptide that has an intrinsic ability to translocate across cell membranes, has been adopted as a means of achieving efficient Cas9 protein and guide RNA delivery. We developed a method for treating human cell lines with CPP-conjugated recombinant Cas9 protein and CPP-complexed guide RNAs that leads to endogenous gene disruption. Here we describe a protocol for preparing an efficient CPP-conjugated recombinant Cas9 protein and CPP-complexed guide RNAs, as well as treatment methods to achieve safe genome editing in human cell lines. ? Springer Science+Business Media New York 2017.
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