The Komor Lab

Prior to the development of base editing in 2016, genome editing technologies functioned by introducing double stranded DNA breaks (DSBs) at a target genomic locus as the first step of genome editing. This is typically accomplished using Cas9 (a programmable endonuclease) and a piece of RNA called a guide RNA (gRNA) that encodes for the genomic location at which Cas9 will bind and cleave using simple Watson-Crick-Franklin base pairing rules. The cell primarily processes DSBs through the non-homologous end joining (NHEJ) pathway, resulting in the accumulation of insertions and deletions (indels) or translocations at the site of the DSB. Actively dividing cells can also process the DSB through the homology-directed repair (HDR) pathway, which incorporates into the genome a user-supplied donor DNA template containing homology to the regions surrounding the DSB. HDR allows researchers to manipulate genome sequences in a precise and predicable manner, in contrast to processing by NHEJ, but DSBs are typically repaired more efficiently by NHEJ, resulting in a mixture of desired editing products with undesired indel sequences. The high frequency of indels versus HDR products has been a long-standing challenge in the genome editing field since its inception in the 1990’s. A major research focus of the Komor Lab is aimed at developing new genome editing methodologies with improved efficiency and precision. These include the development of new base editor tools, as well as new methods to improve the precision of DSB-reliant methods.

Base editors (BEs) are comprised of a catalytically inactivated Cas9 (dCas9 or Cas9n) tethered to a single-stranded DNA (ssDNA) modifying enzyme, which directly chemically modifies target nucleobases within a “bae editing window”. Two classes of base editors exist, which use cytosine and adenine deamination chemistries to catalyze the conversion of C•G base pairs to T•A (CBEs), and A•T base pairs to G•C (ABEs), respectively. These transition mutations (purine-purine or pyrimidine-pyrimidine) are mediated by uracil (cytosine deamination) or inosine (adenine deamination) intermediates, and occur with high efficiencies (up to 90% conversion) with little to no competing indel formation. Expansion of the BE toolbox to include transversion editors will require engineering of new nucleic acid editing enzymes. As ABEs were developed by engineering and evolving a tRNA adenosine deaminase enzyme, TadA, into a ssDNA adenosine deaminase enzyme, TadA7.10, the development of future BEs may be accomplished by converting additional tRNA modifying enzymes into ssDNA editing enzymes. A second major research focus of the Komor Lab aims to mechanistically understand how current BE enzymes function. The enhanced understanding of how known DNA editing enzymes function, and in particular how wtTadA was converted into TadA7.10, can then guide the development of future DNA editing enzymes.

The development of “nontraditional” genome editing tools has expanded the types of intermediates used for genome editing beyond DSBs. Cellular processing of DSB genome editing intermediates has been well-studied since the field’s inception in the 1990s, and has uncovered a variety of methods for improving the efficiency and precision of DSB-reliant tools. In contrast, the cellular processing mechanisms of “nontraditional” genome editing intermediates are largely unknown, and a third research focus of the Komor Lab is to mechanistically study how base editor and prime editor intermediates are processed by the cell.

Technological advances are making the routine sequencing of human genomes increasingly ubiquitous, including in clinical settings. However, our inability to interpret the disease-relevance of genetic variants discovered by sequencing remains a critical obstacle to the progress of precision medicine: there are currently over 685 million human single nucleotide variants (SNVs) identified from sequencing data, and less than 1% have a defined clinical characterization. New laboratory-based methods capable of interpreting SNVs and predicting the clinical relevance of previously unobserved mutations would not only enhance the efficacy of current therapies by better informing patient selection strategies, but also accelerate the development of new approaches to combat diseases with a genetic component. The final research focus of the Komor Lab aims to develop new methods and strategies to characterize the impact of SNVs in cellular contexts.