Purification of Cas9 - RNA Complexes by Ultrafiltration

The discovery of CRISPR-Cas9 has revolutionized molecular biology, greatly accelerating the introduction of genetic modifications into organisms and facilitating the development of novel therapeutics and diagnostics. For many applications, guide RNA and Cas9 protein are expressed, combined, and purified to produce a ribonucleic enzyme complex that is then added into a diagnostic device or delivered into cells. The objective of this work was to develop an ultrafiltration process for the selective purification of Cas9 ribonucleoprotein by removal of excess guide RNA. A His-tagged S. pyogenes Cas9 protein was produced in E. coli, purified by metal affinity chromatography, and complexed with a 40 kDa (124 nucleotide) single guide RNA. Ultrafiltration experiments were first performed on solutions containing either guide RNA or Cas9 protein to identify the effect of filtration conditions and membrane pore size on the selectivity. Shear-induced aggregation of the Cas9 led to significant fouling under some conditions. A diafiltration process was then developed using a Biomax® 300 kDa polyethersulfone membrane to selectively remove excess guide RNA from a solution containing Cas9-bound guide RNA and free guide RNA. These results demonstrate the potential of using ultrafiltration for the removal of excess RNA during the production of functional ribonucleoprotein complexes.


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Work Title Purification of Cas9 - RNA Complexes by Ultrafiltration
Open Access
  1. Jesus Ivan Manzano Garcia
  2. Neil Taylor
  3. Matthew Csordas
  5. Howard Salis
  6. Andrew Zydney
  1. CRISPR, Cas9, single guide RNA, gene editing, ultrafiltration, membrane fouling
License CC BY-NC-ND 4.0 (Attribution-NonCommercial-NoDerivatives)
Work Type Article
  1. Wiley
Publication Date November 25, 2020
Publisher Identifier (DOI)
  1. 10.1002/btpr.3104
Deposited March 07, 2021




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Work History

Version 1

  • Created
  • Added Creator Jesus Ivan Manzano Garcia
  • Added Creator Neil Taylor
  • Added Creator Matthew Csordas
  • Added Creator GRACE E VEZEAU
  • Added Creator Howard Salis
  • Added Creator Andrew Zydney
  • Added RNP purification-final.docx
  • Updated License Show Changes
    • https://creativecommons.org/licenses/by-nc-nd/4.0/
  • Published
  • Updated