enChIP systems using different CRISPR orthologues and epitope tags

Area of Science:

• Molecular Biology
• Genomics
• Biotechnology

Background:

• Engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) isolates specific genomic regions while preserving molecular interactions.
• Previous enChIP studies utilized a 3xFLAG-tagged CRISPR system from Streptococcus pyogenes (S. pyogenes).

Purpose of the Study:

• To increase the flexibility of enChIP technology.
• To explore the use of the CRISPR system from Staphylococcus aureus (S. aureus) with different epitope tags for enChIP.

Main Methods:

• Generation of a plasmid expressing S. aureus dCas9 (Sa-dCas9) fused to a nuclear localization signal (NLS) and a 3xFLAG-tag.
• Development of an enChIP system using S. pyogenes dCas9 fused with an NLS and a 2xAM-tag.

Main Results:

• enChIP yields using Sa-dCas9-3xFLAG were comparable to those obtained with 3xFLAG-S. pyogenes dCas9.
• High enChIP yields were achieved using the S. pyogenes dCas9 with a 2xAM-tag system.
• The developed tools demonstrated increased flexibility for enChIP analysis.

Conclusions:

• The new S. aureus-based CRISPR system and alternative epitope tags enhance the flexibility of enChIP.
• These advancements provide researchers with more options for targeted genomic region isolation and molecular interaction studies.