Efficient DNA- and virus-free engineering of cellular transcriptomic states using dCas9 ribonucleoprotein (dRNP)...

Area of Science:

• * Molecular Biology
• * Gene Regulation
• * Synthetic Biology

Background:

• * CRISPR ribonucleoprotein (RNP) complexes are superior for genome editing compared to plasmid or viral methods.
• * dCas9 fusion protein RNPs for transcriptome and epigenome manipulation are less accessible.
• * Second-generation CRISPR activation RNPs (dRNPs) offer improved accessibility and functionality.

Purpose of the Study:

• * To describe the production, delivery, and optimization of second-generation CRISPRa RNPs (dRNPs).
• * To characterize the transcriptional and cellular effects of dRNP treatments in human cells.
• * To evaluate the efficiency of dRNPs for gene activation, multiplexing, and cell fate conversion.

Main Methods:

• * Production and optimization of dCas9 fusion protein RNPs (dRNPs).
• * Delivery and characterization of dRNP treatments in various human cell types.
• * Assessment of gene activation potency, temporal precision, and multiplexing capabilities.
• * Demonstration of cell fate conversion using dRNPs in stem and differentiated cells.

Main Results:

• * dRNPs demonstrated highly efficient cellular uptake and potent gene activation, even for silenced genes.
• * Gene activation was immediate and temporally precise, unlike DNA-based CRISPRa strategies.
• * dRNPs enabled high-target multiplexing with undiminished gene activation efficiency.
• * Intensive multiplexing at single promoters synergistically increased gene transcription.
• * dRNPs efficiently instructed and converted cell fates in human stem and differentiated cells.

Conclusions:

• * Second-generation dRNPs are a powerful and accessible tool for precise gene activation.
• * dRNPs offer advantages over DNA-based methods, including immediate temporal control and high multiplexing capacity.
• * dRNPs facilitate efficient cell fate manipulation without requiring DNA delivery or viral vectors.