CRISPR Synthetic Guide RNA Services

Sep 15, 2021

Summary

Cas proteins are a group of proteins that function in the microbial immunological defense against viruses and plasmids. An encodable cysteine-containing sequence of amino acids Phe-Cys-Pro-Phe (FCPF), also known as π-clamp, offers selective modification of specific positions within a protein. In a recent publication, Rodrigo et al. utilized such technology to modify Cas proteins, including Cas9, Cas12, and Cas13, and reported that these engineered Cas proteins with the FCPF amino acid sequence insertion could be recognized by perfluoroaromatics and degraded through proteolysis-targeting chimera (PROTAC), indicating a potential strategy to reduce off-target edits related to hyperactivity of Cas proteins in genome editing.

Background

The CRISPR-Cas9 system has served as a tool to manipulate the genomes of multiple living organisms, accelerating the pace of basic research and enabling clinical breakthroughs. However, previous studies have revealed that overactivity of Cas proteins may lead to more off-target effects hindering its adoption in human clinical trials. Therefore, Rodrigo et al. sought to find a post-expression strategy to regulate Cas protein activity and improve the safety of the CRISPR-Cas9 technology. PROTAC, a chemical molecule capable of removing specific proteins, was what they investigated.

Experiment

Labeling Engineered Cas Proteins

As mentioned before, the investigators first modified Cas proteins with π-clamp. Insertion of the FCPF amino acid sequence into Cas proteins was performed and analyzed by GenScript. The expression of these modified proteins, such as Cas9^FCPF, was detected through a synthetic fluorescein (FITC)-conjugated perfluoroaromatic moiety. An electrophoresis analysis was applied to test whether FITC-FCPF molecules could mark Cas9^FCPF.

Degrading Engineered Cas Proteins

To degrade π-clamp containing Cas proteins , Rodrigo et al. created a molecule called PROTAC-FCPF, a modified PROTAC containing a perfluoroaromatic moiety. They tested different concentrations of the PROTAC-FCPF in various cell lines, such as HeLa, HEK293T, and Jopaca-1 cells after 48 hours of transfection with Cas9^FCPF. The efficiencies of delivery and genome editing of Cas9^FCPF and Cas9WT were compared to examine whether inserted π-clamp inhibited the function of the Cas9 protein. The degradation power of PROTAC-FCPF was also tested in cells transfected with other Cas proteins, includingCas12 and Cas13.

Results

The researchers observed a strong green fluorescence signal in cells expressing Cas9^FCPF protein and verified that FITC-FCPF molecules could exclusively label the protein. Six hours after treating with 10 μM PROTAC-FCPF, a reduction of Cas9^FCPF protein was observed in HeLacells. However, concentrations more than 500 μM led to poorer Cas9^FCPF protein degradation. The T7E1 assays of Cas9^FCPF and Cas9WT together showed analogous delivery and editing efficiency, indicating unaffected function of Cas9 protein even with FCPF insertion. Besides Cas9^FCPF, PROTAC-FCPF could also degrade catalytically inactive Cas9^FCPF and other Cas proteins, such as Cas12FCPF and Cas13FCPF.

In summary, these results suggest a potentially effective and flexible way to regulate any Cas proteins' stability, activity, and functionality with the π-clamp system. This new approach may enable the reduction of off-target mutations associated with the use of CRISPR-Cas9 for the development of in vivo and cell therapies. (e.g., CAR-T/TCR immunotherapies).

Reference

Gama-Brambila, Rodrigo A., et al. "A Chemical Toolbox for Labeling and Degrading Engineered Cas Proteins." JACS Au (2021).

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