Functional investigation of human genetic variation and development of precision genome editing methodologies

The long-term goal of the Komor Lab research is to combat the variant interpretation problem that looms over the field of precision medicine: there are currently over 685 million human single nucleotide variants (SNVs) identified, and less than 1% have a defined clinical interpretation. This issue is particularly endemic to rare genetic variants and those discovered in minoritized populations and indigenous people, highlighting the need for a significant increase in studies that functionally assess human genetic variants in a more equitable manner. We have therefore initiated a research program aimed at developing new laboratory-based strategies and systems to study how mutations in DNA repair genes (our area of expertise) affect enzymatic activity, cellular function, and ultimately human health.

How do mutations in DNA-repair proteins contribute to disease phenotypes?

Advances in DNA sequencing technologies have ushered in a new era in human genetics research and have provided researchers with entire databases of mutations that cause human disease. A variety of DNA repair proteins from the nucleotide excision repair and homologous recombination repair pathways have been implicated in human disease. In many cases, a single repair protein can give rise to a diverse set of disease phenotypes depending on the mutation. We plan to systematically biochemically characterize these mutant repair proteins in order to elucidate, at the molecular level, the contributions of different types of DNA lesions to the progression of different disease phenotypes such as cancer and aging.

How and why do different types of DNA damage lead to different outcomes?

The existence of multiple, orthogonal DNA repair pathways combined with the vast structural diversity of DNA lesions indicates that different types of damage can lead to different outcomes at both the genomic and cellular levels (including mutations, cellular senescence, or cell death). In addition, the location within the genome of a particular type of damage and the DNA sequence surrounding it can govern its recognition and resolution. Programmable DNA endonucleases such as CRISPR-Cas9 allow researchers to introduce specific types of damage into the genome at a genomic locus of their choosing and can be utilized to facilitate the study of DNA damage with a degree of precision and control not previously available. We will use these genome editing tools as “programmable precision DNA damaging tools” to characterize, in a high-throughput manner, the effects of lesion identity, as well as chromosomal location and surrounding sequence on repair outcomes of DNA damage.

Can we engineer DNA-repair proteins to better "protect" the genome?

The precise molecular details of DNA damage detection and repair have yet to be elucidated. In addition, a given set of DNA repair proteins can be responsible for repair of a very chemically diverse set of lesions. We plan to use high-throughput screening methods to identify key residues within DNA repair proteins that are responsible for lesion detection and repair. In particular, we will use these screens to illuminate, at the amino acid residue level, how certain proteins are able to recognize and repair such structurally diverse substrates. Such detailed mechanistic information can guide rational design-based engineering efforts in the development of therapeutically-relevant DNA repair proteins with enhanced activities. Many species exhibit a remarkably low cancer incidence, or extreme longevity, such as the bowhead whale and hydra vulgaris. These extraordinary characteristics could be due to enhanced DNA lesion repair capabilities, and the DNA repair proteins of these species display unusually high sequence identity to those of humans. We will characterize the ability of these species’ DNA repair proteins to repair specific types of DNA damage, and compare them to the human homologs.

How can we manipulate DNA-repair pathways to better control genome editing outcomes?

All genome editing techniques employ a two-step process to introduce modifications of interest into the genomic DNA of a cell: 1) Introduce DNA damage at a genomic locus of interest, followed by 2) Manipulate the DNA repair pathways of the cell to process the damage in a specific way that results in the desired modification. Current strategies to precisely modify eukaryotic genomes under therapeutically relevant conditions suffer from low efficiencies and frequently result in stochastic mixtures of genome modifications. We aim to develop methods for the comprehensive identification of genes involved in repair of DNA lesions. We will use these systems to better understand current genome editing mechanisms and to identify potential new strategies for the manipulation of mammalian genomes.