A Method for In Situ Reverse Genetic Analysis of Proteins Involved mtDNA Replication.

ABSTRACT: The unavailability of tractable reverse genetic analysis approaches represents an obstacle to a better understanding of mitochondrial DNA replication. Here, we used CRISPR-Cas9 mediated gene editing to establish the conditional viability of knockouts in the key proteins involved in mtDNA replication. This observation prompted us to develop a set of tools for reverse genetic analysis in situ, which we called the GeneSwap approach. The technique was validated by identifying 730 amino acid (aa) substitutions in the mature human TFAM that are conditionally permissive for mtDNA replication. We established that HMG domains of TFAM are functionally independent, which opens opportunities for engineering chimeric TFAMs with customized properties for studies on mtDNA replication, mitochondrial trans

SUBMITTER: Kozhukhar N 

PROVIDER: S-EPMC9316749 | biostudies-literature | 2022 Jul

REPOSITORIES: biostudies-literature