Chapter title |
Cell-Penetrating Peptide-Mediated Delivery of Cas9 Protein and Guide RNA for Genome Editing.
|
---|---|
Chapter number | 7 |
Book title |
Eukaryotic Transcriptional and Post-Transcriptional Gene Expression Regulation
|
Published in |
Methods in molecular biology, January 2017
|
DOI | 10.1007/978-1-4939-6518-2_7 |
Pubmed ID | |
Book ISBNs |
978-1-4939-6516-8, 978-1-4939-6518-2
|
Authors |
Bharathi Suresh, Suresh Ramakrishna, Hyongbum Kim, Suresh, Bharathi, Ramakrishna, Suresh, Kim, Hyongbum |
Editors |
Narendra Wajapeyee, Romi Gupta |
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. 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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Russia | 1 | 20% |
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Members of the public | 4 | 80% |
Scientists | 1 | 20% |
Mendeley readers
Geographical breakdown
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Unknown | 88 | 100% |
Demographic breakdown
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Student > Ph. D. Student | 12 | 14% |
Student > Bachelor | 12 | 14% |
Researcher | 11 | 13% |
Student > Master | 10 | 11% |
Other | 6 | 7% |
Other | 8 | 9% |
Unknown | 29 | 33% |
Readers by discipline | Count | As % |
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Agricultural and Biological Sciences | 13 | 15% |
Pharmacology, Toxicology and Pharmaceutical Science | 4 | 5% |
Chemistry | 4 | 5% |
Engineering | 3 | 3% |
Other | 6 | 7% |
Unknown | 35 | 40% |