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Updated: Jun 5, 2025
09:51
Enhanced Genome Editing with Cas9 Ribonucleoprotein in Diverse Cells and Organisms
Published on: May 25, 2018
33.7K
Dual pH-responsive CRISPR/Cas9 ribonucleoprotein xenopeptide complexes for genome editing
Xianjin Luo1, Janin Germer1, Tobias Burghardt1
1Pharmaceutical Biotechnology, Department of Pharmacy, Ludwig-Maximilians-Universität Munich, Butenandtstrasse 5-13, 81377 Munich, Germany.
Summary
New xenopeptides (XPs) efficiently deliver CRISPR/Cas9 gene editing tools, enhancing cellular uptake and genome editing. These pH-responsive carriers show promise for treating genetic diseases like Duchenne muscular dystrophy.
Area of Science:
- Biotechnology
- Molecular Biology
- Genetic Engineering
Background:
- Clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR associated (Cas) protein systems are powerful tools for genetic disease treatment.
- Effective delivery carriers for CRISPR/Cas9 ribonucleoprotein (RNP) complexes are limited, hindering broader applications.
- Dual pH-responsive amphiphilic xenopeptides (XPs) offer a potential solution for CRISPR/Cas9 RNP delivery.
Purpose of the Study:
- To evaluate dual pH-responsive amphiphilic xenopeptides (XPs) as carriers for CRISPR/Cas9 RNP delivery.
- To assess the efficiency of these XPs in cellular uptake, endosomal disruption, and genome editing across various cell lines.
- To optimize XP structures for enhanced gene editing efficiency and explore their utility in homology-directed repair (HDR).
Main Methods:
- Designed and synthesized artificial lipo-xenopeptides (lipo-XPs) with varying ratios of lipoamino fatty acid (LAF) and oligoaminoethylene acid (Stp) units.
- Screened lipo-XPs for functional Cas9/sgRNA RNP delivery in four reporter cell lines, including a Duchenne muscular dystrophy (DMD) model.
- Compared Cas9/sgRNA RNP complexes with Cas9 mRNA/sgRNA polyplexes and tested modified XP analogues for improved gene editing.
Main Results:
- Xenopeptides demonstrated significantly enhanced cellular uptake and effective endosomal disruption in HeLa cells.
- Several Cas9/sgRNA RNP complexes delivered by XPs showed potent genome editing in reporter cell lines at low sgRNA concentrations (5 nM).
- Optimized XP analogues achieved high gene editing efficiency (EC50 down to 0.51 nM) and facilitated significant homology-directed repair (HDR) up to 43%.
Conclusions:
- Dual pH-responsive amphiphilic xenopeptides represent a tunable and effective delivery system for CRISPR/Cas9 RNP complexes and RNP/donor DNA polyplexes.
- These carriers offer a promising strategy for gene editing applications, including the potential treatment of genetic disorders.
- The developed xenopeptide system shows high efficiency and applicability for various gene editing modalities.
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