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Cell-Penetrating Peptide-Mediated Delivery of Cas9 Protein and Guide RNA for Genome Editing

 Bharathi Suresh  ;  Suresh Ramakrishna  ;  Hyongbum Kim 
 Methods in Molecular Biology (Clifton, N.J.), Vol.1507 : 81-94, 2017 
Journal Title
Methods in Molecular Biology (Clifton, N.J.)
Issue Date
Bacterial Proteins/genetics ; Bacterial Proteins/metabolism* ; CRISPR-Cas Systems* ; Cell-Penetrating Peptides/chemistry ; Cell-Penetrating Peptides/metabolism* ; Cloning, Molecular ; Endonucleases/genetics ; Endonucleases/metabolism* ; Escherichia coli ; Gene Editing/methods* ; HEK293 Cells ; Humans ; Plasmids ; RNA, Guide/chemistry ; RNA, Guide/genetics ; RNA, Guide/metabolism* ; Transformation, Bacterial
Cas9 conjugation ; Cas9 protein purification ; Dialysis ; In vitro sgRNA synthesis ; Protein delivery ; T7E1 assay
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. Cell-penetrating 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.
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1. College of Medicine (의과대학) > Dept. of Pharmacology (약리학교실) > 1. Journal Papers
Yonsei Authors
Kim, Hyongbum(김형범) ORCID logo https://orcid.org/0000-0002-4693-738X
Suresh, Bharathi(호사발레랑가파바라티)
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