Engineered transcription-associated Cas9 targeting in eukaryotic cells

Area of Science:

• Molecular Biology
• Gene Editing
• Biotechnology

Background:

• Class 2 CRISPR-Cas effector nucleases, like Cas9, utilize protospacer-adjacent motif (PAM) and guide RNA interactions for DNA targeting.
• These interactions provide spatial but not temporal control, leading to constitutive DNA binding and cleavage.
• Existing CRISPR-Cas systems lack inherent mechanisms for temporal regulation of DNA targeting.

Purpose of the Study:

• To engineer a CRISPR-Cas9 system with spatiotemporal control over DNA targeting, coupling it with active transcription.
• To demonstrate the feasibility of Transcription-associated Cas9 Targeting (TraCT) in eukaryotic cells.
• To explore applications of TraCT for selective gene editing.

Main Methods:

• Engineered Cas9 fusion proteins designed to bind nascent RNAs near target DNA sequences.
• Utilized suboptimal PAM interactions to limit basal Cas9 activity.
• Tested the TraCT system in eukaryotic yeast and human cells, and applied it to selective gene editing scenarios.

Main Results:

• Demonstrated successful spatiotemporal coupling between transcription and DNA targeting using engineered Cas9 fusion proteins (TraCT).
• TraCT functions in yeast and human cells when suboptimal PAM interactions are present and nascent RNA is tethered to DNA.
• Achieved selective editing of identical targets in distinct gene loci and differentially transcribed allelic loci in yeast.

Conclusions:

• Temporal control over Cas9 targeting can be engineered without altering core Cas9 domains or guide RNA components.
• Co-transcriptional RNA binding serves as a cis-acting mechanism to conditionally activate CRISPR-Cas DNA targeting.
• The TraCT system offers a novel approach for precise and regulated gene editing in eukaryotic systems.