Gene disruption by cell-penetrating peptide-mediated delivery of Cas9 protein and guide RNA

By S. Ramakrishna, Abu-Bonsrah Kwaku Dad, J. Beloor, Ramu Gopalappa, Sang-Kyung Lee, H. Kim. Genome Research, 2014

651 citations recorded by the source corpus.

RNA-guided endonucleases (RGENs) derived from the CRISPR/Cas system represent an efficient tool for genome editing. RGENs consist of two components: Cas9 protein and guide RNA. Plasmid-mediated delivery of these components into cells can result in uncontrolled integration of the plasmid sequence into the host genome, and unwanted immune responses and potential safety problems that can be caused by the bacterial sequences. Furthermore, this delivery method requires transfection tools. Here we show that simple treatment with cell-penetrating peptide (CPP)–conjugated recombinant Cas9 protein and CPP-complexed guide RNAs leads to endogenous gene disruptions in human cell lines. The Cas9 protein was conjugated to CPP via a thioether bond, whereas the guide RNA was complexed with CPP, forming condensed, positively charged nanoparticles. Simultaneous and sequential treatment of human cells, including embryonic stem cells, dermal fibroblasts, HEK293T cells, HeLa cells, and embryonic carcinoma cells, with the modified Cas9 and guide RNA, leads to efficient gene disruptions with reduced off-target mutations relative to plasmid transfections, resulting in the generation of clones containing RGEN-induced mutations. Our CPP-mediated RGEN delivery process provides a plasmid-free and additional transfection reagent–free method to use this tool with reduced off-target effects. We envision that our method will facilitate RGEN-directed genome editing.

Subfields: Molecular Biology

View Paper: https://www.semanticscholar.org/paper/854b1cdcbf20cfe65852987cc4600bf18ddcb65a

How researchers see this paper

The researchers whose trusted networks rank it highest, strongest advocate first. Every researcher gets a different order.

In Molecular Biology

Citations in the corpus

The new frontier of genome engineering with CRISPR-Cas9 (2014, positive): “cell-penetrating peptides to enable rapid and timed editing (89, 152), and transgenic organisms that express Cas9 from inducible promoters are being tested.”
Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins (2014, background): “In an accompanying paper published in this issue, Ramakrishna et al. (2014) report that a cell-penetrating peptide can be used to deliver both the Cas9 protein and sgRNA into hu-”
Delivering CRISPR: a review of the challenges and approaches (2018, background): “As an example, the PAM from Neisseria meningitidis Cas9 is reported to be 50-NNNNGATT-30 (Jiang et al., 2013; Ma et al., 2014).”
Engineering the Delivery System for CRISPR-based Genome Editing (2018, background): “Conjugating Cas9 protein and gRNA with CPPs was demonstrated as a means to enhance protein delivery [55, 56].”
Non-viral delivery systems for CRISPR/Cas9-based genome editing: challenges and opportunities (2018, background): “This system led to efficient disruptions of CCR5 gene with minimal off-target mutations [96].”
Target specificity of the CRISPR-Cas9 system (2014, positive): “Compared to plasmid transfection based delivery, direct delivery of recombinant Cas9 protein and in vitro transcribed sgRNA, either individually or as purified complex, reduces off-targets in cells [66,67].”
CRISPR/Cas9 in Genome Editing and Beyond. (2016, methodology): “(258) used cell-penetrating peptides (CPPs) to induce gene editing through Cas9 conjugated with CPPs and through the sgRNA in complex with CPPs.”