Cell-Penetrating Peptide-Mediated Delivery of Cas9 Protein and Guide RNA for Genome Editing.
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.
Department of Pharmacology and Brain Korea 21 PLUS Project for Medical Science, Yonsei University College of Medicine, Seoul, South Korea.|Graduate Program of Nano Science and Technology, Yonsei University, Seoul, South Korea.
Bharathi
B
Suresh
Graduate School of Biomedical Science and Engineering, Hanyang University, Seoul, South Korea. suri28@hanyang.ac.kr.|College of Medicine, Hanyang University, Seoul, South Korea. suri28@hanyang.ac.kr.
Suresh
S
Ramakrishna
Department of Pharmacology and Brain Korea 21 PLUS Project for Medical Science, Yonsei University College of Medicine, Seoul, South Korea. hkim1@yuhs.ac.|Graduate Program of Nano Science and Technology, Yonsei University, Seoul, South Korea. hkim1@yuhs.ac.|Department of Pharmacology, Yonsei University College of Medicine, ABMRC Room 504, 50-1 Yonsei-ro, Seodaemoon-gu, Seoul, 120-752, South Korea. hkim1@yuhs.ac.
Hyongbum
H
Kim
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D000076987:CRISPR-Associated Protein 9; D064113:CRISPR-Cas Systems; D057846:Cell-Penetrating Peptides / Q000737:chemistry / Q000378:metabolism*; D003001:Cloning, Molecular; D004720:Endonucleases / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli; D000072669:Gene Editing / Q000379:methods*; D057809:HEK293 Cells; D006801:Humans; D010957:Plasmids; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014169:Transformation, Bacterial
D001426:Bacterial Proteins; D057846:Cell-Penetrating Peptides; D000094704:RNA, Guide, CRISPR-Cas Systems; D000076987:CRISPR-Associated Protein 9; C000606107:Cas9 endonuclease Streptococcus pyogenes; D004720:Endonucleases
Cas9 conjugation; Cas9 protein purification; Dialysis; In vitro sgRNA synthesis; Protein delivery; T7E1 assay