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Updated: Aug 31, 2025
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Using Sniper-Cas9 to Minimize Off-target Effects of CRISPR-Cas9 Without the Loss of On-target Activity Via Directed Evolution
Published on: February 26, 2019
9.8K
Comprehensive UHPLC- and CE-based methods for engineered Cas9 characterization
Julien Camperi1, Gary Console2, Laura Zheng2
1Cell Therapy Engineering and Development, Genentech, 1 DNA Way, South San Francisco, CA, 94080, USA.
Talanta
|August 21, 2022
Summary
Engineered CRISPR-Cas9 variants, like high-fidelity SpyFi Cas9, offer improved gene editing. New UHPLC and CE methods fully characterize these variants, ensuring purity, modifications, and function for therapeutic applications.
Area of Science:
- Biochemistry and Molecular Biology
- Analytical Chemistry
- Gene Editing Technologies
Background:
- CRISPR-Cas9 systems are crucial gene-editing tools with therapeutic potential.
- Minimizing off-target effects of CRISPR-Cas9 is essential for safe therapeutic applications.
- Engineered Cas9 variants, such as high-fidelity SpyFi Cas9, aim to enhance specificity and reduce unwanted mutations.
Purpose of the Study:
- To develop and validate ultra-high-performance liquid chromatography (UHPLC) and capillary electrophoresis (CE) methods for comprehensive characterization of engineered Cas9 variants.
- To assess purity, size variants, isoelectric points (pI), post-translational modifications (PTMs), and functional activities of different Cas9 variants.
- To establish methods for differentiating engineered variants like SpyFi Cas9 from wild-type Streptococcus pyogenes Cas9 (SpCas9).
Main Methods:
- Purity and size variants determined by CE-sodium dodecyl sulfate (SDS).
- Functional activity assessed via in vitro DNA cleavage assays using ribonucleoprotein (RNP) complexes.
- Isoelectric points (pI) measured by imaged capillary isoelectric focusing (icIEF); intact mass analysis by reversed-phase (RP)-UHPLC coupled with high-resolution mass spectrometry (HRMS).
- Peptide mapping using LC-UV-MS/MS with endoproteinase Lys-C for sequence confirmation.
Main Results:
- Developed robust UHPLC and CE methods for full characterization of engineered Cas9 variants.
- Successfully differentiated SpyFi Cas9 from WT SpCas9 using peptide mapping and mass spectrometry.
- Demonstrated the utility of these analytical techniques for assessing critical quality attributes of gene-editing proteins.
Conclusions:
- The developed UHPLC and CE methods provide a comprehensive analytical toolkit for characterizing engineered Cas9 variants.
- These methods are crucial for ensuring the quality, consistency, and safety of Cas9-based therapeutics.
- Low-resolution mass spectrometry may serve as a cost-effective method for identifying specific variants like SpyFi Cas9 in GMP environments.
