US20240200059
2024-06-20
Chemistry; metallurgy
C12N15/11
Photocleavable guide RNAs (gRNAs) are designed to enhance CRISPR/Cas gene editing techniques by incorporating a targeting domain and a CRISPR RNA (crRNA) sequence, along with at least one photocleavable moiety. These gRNAs allow for precise control over gene editing by utilizing light to activate the cleavage function, potentially reducing off-target effects and improving safety in therapeutic applications.
The gRNAs feature a targeting domain that aligns with specific target site sequences adjacent to a protospacer-adjacent motif (PAM). They can include multiple photocleavable moieties, such as 2-nitrobenzyl bonds, strategically placed within the targeting or crRNA sequences. This structural flexibility allows for customization in targeting specific genes while maintaining functionality.
To create an effective CRISPR system, the photocleavable gRNA is combined with a CRISPR/Cas nuclease, such as Cas12a or Cas9. This combination forms a ribonucleoprotein (RNP) complex that can bind to designated target sites in the genome of a cell. The methods for forming these complexes can vary, including the introduction of pre-formed RNPs or nucleic acids encoding the gRNA and nuclease into the cells.
The photocleavable moieties within the gRNAs can be activated using light at specific wavelengths, typically around 365 nm. This activation leads to cleavage of the moiety, which can significantly reduce the activity of the CRISPR/Cas system at the target site. Such control mechanisms enable researchers to fine-tune gene editing processes and minimize unwanted genetic modifications.
The innovative design of these photocleavable gRNAs opens up new possibilities for genetic engineering, particularly in hematopoietic cells and immune cells. By utilizing these methods, researchers can create genetically engineered cell populations with improved safety profiles and enhanced precision in gene editing, paving the way for potential therapeutic applications.