Engineered transcription-associated Cas9 targeting in eukaryotic cells.

Area of Science:

• Molecular Biology
• Gene Editing Technologies
• Biochemistry

Background:

• Class 2 CRISPR-Cas systems, like Cas9, use DNA sequence recognition (PAM and guide RNA) for targeting.
• Current CRISPR-Cas systems offer spatial but not temporal control over DNA targeting.
• Constitutive nature of PAM and guide RNA interactions limits dynamic regulation of Cas9 activity.

Purpose of the Study:

• To engineer a CRISPR-Cas9 system with temporal control over DNA targeting.
• To couple Cas9 DNA targeting activity with active gene transcription.
• To develop a novel gene editing mechanism termed Transcription-associated Cas9 Targeting (TraCT).

Main Methods:

• Engineered Cas9 fusion proteins that bind to nascent RNAs.
• Utilized suboptimal PAM interactions to limit basal Cas9 activity.
• Demonstrated TraCT in eukaryotic yeast and human cells.
• Applied TraCT for selective gene editing in identical target loci and differentially transcribed allelic loci.

Main Results:

• Successfully engineered Cas9 fusion proteins for Transcription-associated Cas9 Targeting (TraCT).
• TraCT enables spatiotemporal coupling between transcription and DNA targeting.
• Demonstrated selective editing of specific DNA targets in yeast.
• Showcased conditional stimulation of CRISPR-Cas DNA targeting via co-transcriptional RNA binding.

Conclusions:

• Temporal control of Cas9 targeting can be achieved without altering core Cas9 or guide RNA components.
• Co-transcriptional RNA binding serves as a cis-acting mechanism to conditionally activate CRISPR-Cas DNA targeting.
• TraCT offers a new paradigm for precise and regulated gene editing in eukaryotic cells.