Enhanced Genome Editing with Cas9 Ribonucleoprotein in Diverse Cells and Organisms

作者： Behnom Farboud*1,2, Erin Jarvis*1, Theodore L. Roth*3,4,5,6, Jiyung Shin*1,3, Jacob E. Corn1,3, Alexander Marson3,5,6,7,8,9, Barbara J. Meyer1,2, Nipam H. Patel1,10, Megan L. Hochstrasser3 1Department of Molecular Cell Biology,University of California, Berkeley, 2Howard Hughes Medical Institute,University of California, Berkeley, 3Innovative Genomics Institute,University of California, Berkeley, 4Biomedical Sciences Graduate Program,University of California, San Francisco, 5Department of Microbiology and Immunology,University of California, San Francisco, 6Diabetes Center,University of California, San Francisco, 7Chan Zuckerberg Biohub, 8Department of Medicine,University of California, San Francisco, 9UCSF Helen Diller Family Comprehensive Cancer Center,University of California, San Francisco, 10Department of Integrative Biology,University of California, Berkeley

简介：

Overview

This protocol describes the use of CRISPR/Cas9 ribonucleoprotein complexes for targeted genomic changes in various cells and organisms. It emphasizes the advantages of using RNPs over plasmid DNA, including higher efficiency and reduced off-target effects.

Key Study Components

Area of Science

• Genome editing
• CRISPR technology
• Cell biology

Background

• CRISPR/Cas9 is a revolutionary tool for genome editing.
• Ribonucleoprotein complexes offer improved efficiency.
• This method can be applied to a wide range of organisms.
• It has potential applications in ex vivo therapies for diseases.

Purpose of Study

• To enable precise genomic modifications in various cell types.
• To facilitate research on fundamental biological questions.
• To enhance the ability of cells to combat diseases.

Methods Used

• Preparation of guide RNA and Cas9 protein to form RNP complexes.
• Electroporation of human hematopoietic stem and progenitor cells (HSPCs).
• Microinjection techniques for C.elegans and Parhyale embryos.
• Use of specific protocols for embryo handling and injection.

Main Results

• Successful genomic modifications in primary human cells.
• Effective application in model organisms like C.elegans.
• Demonstrated potential for addressing genetic diseases.
• Enhanced understanding of genome editing techniques.

Conclusions

• RNP complexes are a superior method for genome editing.
• This approach broadens the scope of genetic research.
• It holds promise for therapeutic applications in medicine.

Frequently Asked Questions