Cysteine-independent CRISPR-Associated Protein Labeling for Presentation and Co-delivery of Molecules Toward Genetic and Epigenetic Regulations.
Chembiochem
; 25(10): e202400149, 2024 May 17.
Article
em En
| MEDLINE
| ID: mdl-38530114
ABSTRACT
Labeling of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) associated proteins (Cas) remains an immense challenge for their genome engineering applications. To date, cysteine-mediated bioconjugation is the most efficient strategy for labeling Cas proteins. However, introducing a cysteine residue in the protein at the right place might be challenging without perturbing the enzymatic activity. We report a method that does not require cysteine residues for small molecule presentation on the CRISPR-associated protein SpCas9 for inâ
vitro protein detection, probing cellular protein expression, and nuclear co-delivery of molecules in mammalian cells. We repurposed a simple protein purification tag His6 peptide for non-covalent labeling of molecules on the CRISPR enzyme SpCas9. The small molecule labeling enabled us to rapidly detect SpCas9 in a biochemical assay. We demonstrate that small molecule labeling can be utilized for probing bacterial protein expression in realtime. Furthermore, we coupled SpCas9's nuclear-targeting ability in co-delivering the presenting small molecules to the mammalian cell nucleus for prospective genome engineering applications. Furthermore, we demonstrate that the method can be generalized to label oligonucleotides for multiplexing CRISPR-based genome editing and template-mediated DNA repair applications. This work paves the way for genomic loci-specific bioactive small molecule and oligonucleotide co-delivery toward genetic and epigenetic regulations.
Palavras-chave
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Cisteína
/
Epigênese Genética
/
Sistemas CRISPR-Cas
Limite:
Humans
Idioma:
En
Revista:
Chembiochem
Ano de publicação:
2024
Tipo de documento:
Article