Engineering High Specificity DNA Base Editors for Programmable Genome Editing

• Applicant: Professor Dr. Julian Grünewald
• Subject Area: General Genetics and Functional Genome Biology
• Term: from 2018 to 2021
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 416375182

CRISPR-Cas nucleases have transformed the field of genome editing and synthetic biology. This technology is based on an adaptive prokaryotic DNA defense mechanism that provides resistance to bacteriophages and other foreign DNA. Recently, it has been repurposed to edit single bases in DNA of mammalian cells, a technology referred to as "base editing". Using this platform, so-called guide RNAs can be designed to target specific DNA sequences of interest which are then efficiently edited by engineered base editor proteins. Base editors consist of a catalytically inactive or nicking version of a CRISPR-Cas nuclease, which can target and unwind DNA, fused to a cytidine deaminase that can then access the single stranded DNA induced by the CRISPR-Cas protein. A targeted cytidine gets deaminated to uracil followed by DNA repair that then yields stable and efficient alteration of cytidine to thymine. While the ease of use and capability to edit DNA efficiently without inducing DNA breaks have led to swift adoption of base editing in biomedical research, an important consideration for safe therapeutic application of this technology is to define and minimize potential off-target effects. DNA base editors that accomplish C G to T A editing contain a cytidine deaminase of the APOBEC family. Many of these deaminases have been shown to edit both single strand RNA and DNA. While DNA off-target effects have been studied extensively, the RNA off-target profile of DNA base editors has not been investigated yet. This represents an important issue to be addressed prior to the translation of base editing into the clinic. Unwanted RNA editing and hypermutation could have detrimental effects and indeed this process has been linked to both cancer and autoimmunity. Our preliminary data suggests that RNA is mutated by the cytidine deaminase part of base editors, independent of the sequence-specific function of Cas9, thereby resulting in APOBEC-associated off-target RNA editing by base editors intended to edit DNA. Here, I propose to more comprehensively characterize this unwanted RNA editing by performing whole transcriptome and genome sequencing. Further, I intend to use rational protein engineering to design base editors with diminished or abolished RNA base editing activity but that still retain the ability to edit DNA with high precision. These experiments will comprehensively define the on-target/off-target profiles of base editors. Most importantly, the pipeline established here will improve the safety profile of base editors and pave the way for their application as true precision therapeutics.

• DFG Programme: Research Fellowships
• International Connection: USA
• Host: Professor J. Keith Joung, Ph.D.