NEW YORK (GenomeWeb) – Jennifer Doudna and Feng Zhang have received separate grants from the National Human Genome Research Institute for CRISPR technology-based projects they both say have the potential to advance the tool's usefulness for human health purposes.
The University of California, Berkeley's Doudna will receive $2.1 million to set up and run the Center for Genome Editing and Recording (CGER). According to Doudna's grant proposal, the center will address the challenges of accurately interrogating and manipulating DNA sequences in situ "at a scale and level of accuracy not currently available" by developing technologies based on CRISPR-Cas9 that can "detect, alter and record the sequence and output of the genome in individual cells and tissues."
The CGER researchers will combine Cas9's DNA cleavage activity with strategies that enhance the placement of DNA replacement sequences using homology-directed double-strand break repair. The researchers will also conjugate Cas9 to DNA base editing domains to enable accurate introduction or correction of point mutations without double-stranded DNA cleavage, and will further use cell-based assays to interrogate specific disease-associated loci in human cells, the grant proposal noted.
Finally, the researchers will develop a high-throughput pipeline to test the functional gene expression impacts of sequence variants responsible for human disease and identify the relationships between variants, and will create new methods for permanently recording cell state changes in DNA so that they can be read out in a single-cell RNA-seq format.
"Together, these approaches will enable the creation of any desired sequence alteration at any locus with high specificity and efficiency, with profound implications for both genome science and practical therapeutic intervention," the proposal added. "[The] development of molecular cell recorders will focus primarily on an evolving lineage tracer that, by enabling the generation of fate maps at unprecedented resolutions, holds the promise to revolutionize studies of normal development and disease progression."
The Broad Institute's Zhang will receive $1.1 million for a project that aims to develop a suite of tools for the interrogation of RNA based on CRISPR-Cas enzymes that target RNA in a programmable manner.
"Tools for transcript knockdown, translation upregulation, and transcript sensing will be developed, which, together, will enable dissection of genetic circuits in a dynamic, high-throughput manner, accelerating nearly all areas of biomedical science," Zhang's grant proposal noted.
The researchers intend to functionally and biochemically characterize RNA-targeting Cas enzymes, use these enzymes to edit transcripts in eukaryotic cells, and develop novel RNA-targeting CRISPR-Cas enzymes as platforms for transcript knockdown and translational upregulation. They further plan to improve RNA knockdown screens, create programmable reporter systems for transcript sensing by engineering catalytically inactive RNA-targeting Cas enzymes to serve as programmable RNA-binding scaffolds, and develop genome-wide screens to dissect genetic regulatory circuits.
"These tools, which will be openly shared, will be broadly applicable across species and systems and will serve as a general framework for the expansion of the RNA-targeting toolbox. They will radically transform existing approaches for studying gene expression dynamics and exploring the significance of isoforms and non-coding transcripts," the proposal said. "These tools will provide researchers with methods to fine-tune and detect gene expression events in a dynamic, flexible way, dramatically accelerating the study of the complex genetic and epigenetic circuits that underlie specific phenotypes, including disease."