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Updated: Mar 3, 2026
09:51
Enhanced Genome Editing with Cas9 Ribonucleoprotein in Diverse Cells and Organisms
Published on: May 25, 2018
36.0K
Synthetically modified guide RNA and donor DNA are a versatile platform for CRISPR-Cas9 engineering
Kunwoo Lee1, Vanessa A Mackley1, Anirudh Rao2
1GenEdit Inc, Berkeley, United States.
Elife
|May 3, 2017
Summary
Chemical modifications to guide RNA (gRNA) and donor DNA enhance gene editing with Cas9 and Cpf1. Conjugating gRNA and donor DNA improves cell transfection and homology-directed repair (HDR) efficiency.
Area of Science:
- Molecular Biology
- Biochemistry
- Genetic Engineering
Background:
- Gene editing technologies like Cas9 and Cpf1 offer powerful tools for genetic manipulation.
- Optimizing the efficiency of gene editing, particularly through chemical modifications of key components, remains an active area of research.
Purpose of the Study:
- To investigate the impact of chemical modifications on guide RNA (gRNA) and donor DNA activity in Cas9 and Cpf1 systems.
- To explore novel strategies for enhancing gene editing efficiency and cell targeting using modified nucleic acids.
Main Methods:
- Chemical modification of gRNA and donor DNA at terminal positions.
- Utilizing 5' fluorescently labeled donor DNA for cell enrichment via cell sorting.
- Direct conjugation of gRNA and donor DNA into a single molecule (gRNA-donor DNA conjugate).
- Evaluating transfection and homology-directed repair (HDR) efficiency using cationic polymers.
Main Results:
- Terminal chemical modifications of gRNA and donor DNA did not diminish their gene editing activity.
- 5' fluorescently labeled donor DNA enabled a twofold enrichment of HDR-edited cells through cell sorting.
- The gRNA-donor DNA conjugate demonstrated a threefold improvement in cell transfection and HDR induction compared to unconjugated components.
Conclusions:
- gRNA and donor DNA exhibit tolerance to chemical modifications, opening avenues for improved genome engineering.
- Chemical modifications and direct conjugation represent promising strategies to enhance the efficiency and applicability of CRISPR-based gene editing systems.
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