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Purification of Cas9-RNA complexes by ultrafiltration
Ivan Manzano1, Neil Taylor1, Matthew Csordas1
1Department of Chemical Engineering, The Pennsylvania State University, University Park, Pennsylvania, USA.
Biotechnology Progress
|December 4, 2020
Summary
This study developed an ultrafiltration method to purify Cas9 ribonucleoprotein complexes by removing excess guide RNA. This technique enhances the production of functional CRISPR-Cas9 complexes for research and diagnostics.
Area of Science:
- Molecular Biology
- Biotechnology
- Biochemistry
Background:
- CRISPR-Cas9 technology has transformed genetic engineering and therapeutic development.
- Cas9 ribonucleoprotein complexes, essential for CRISPR applications, require precise purification.
- Current purification methods can be complex and time-consuming.
Purpose of the Study:
- To develop an efficient ultrafiltration process for selective purification of Cas9 ribonucleoprotein (RNP).
- To remove excess guide RNA (gRNA) from Cas9-RNP complexes.
- To optimize filtration conditions for Cas9-RNP production.
Main Methods:
- Produced and purified His-tagged Streptococcus pyogenes Cas9 protein using metal affinity chromatography.
- Complexed purified Cas9 protein with a 40 kDa single guide RNA.
- Utilized ultrafiltration and diafiltration with a 300 kDa polyethersulfone membrane to separate Cas9-RNP from excess gRNA.
Main Results:
- Identified optimal filtration conditions and membrane pore sizes for selective purification.
- Demonstrated effective removal of excess guide RNA from Cas9-RNP solutions.
- Observed shear-induced aggregation of Cas9, necessitating careful process control.
Conclusions:
- Ultrafiltration is a viable method for selectively removing excess guide RNA during Cas9-RNP production.
- This process can improve the efficiency and scalability of CRISPR reagent manufacturing.
- The developed method holds promise for enhancing the production of functional ribonucleoprotein complexes for diverse applications.
