Investigating Fanconi anemia Gene Editing Using Two Different CRISPR/Cas9-Based Approaches in vitro
Fanconi anemia (FA) occurs in approximately 1 in 130,000 births in the United States and is the result of mutations in 23 known genes that make up the FA pathway, an important DNA damage repair pathway for maintaining genomic integrity. The most common mutation found in FA patients is the Spanish founder mutation (SFM) located in the FANCA gene. The SFM changes a cytosine (C) to a thymine (T) resulting in an early stop codon and a nonfunctional protein product. Our lab previously demonstrated that editing the mutated codon TAG to the TGG codon (encoding trytophan) leads to phenotypic rescue in humans however this does not appear to be true for humanized mouse models. Therefore, our research goal was to revert the mutated stop codon (i.e. the FANCA SFM) back to the original CAG sequence using adenosine base editors (ABEs). Notably, ABE constructs rely on SpCas9 targeting, which requires an NGG protospacer adjacent motif (PAM), which is not available at the target site. Instead, we developed novel ABE constructs (i.e. ABE9-SpRY and ABE9-iSpyMac) with less restrictive PAM requirements to revert the SFM back to the WT FANCA sequence. By combining TadA9 (the adenosine deaminase) with SpRY Cas9 (NNN) or iSpyMac Cas9 (NAA), we achieved an average of 20-30% correction of the SFM in primary mouse embryonic fibroblasts (MEFs) and patient-derived lymphoblastoid cells (LCLs), with no off-target editing at the target site. Moreover, we showed that correction of the FANCA SFM rescued FA-associated phenotypes, including sensitivity to DNA damage and activation of the FA pathway. Our next goals are to further assess off-target editing (e.g. off-target translocations) and develop a safe and effective way to deliver our ABE reagents to cells in vivo.