ABSTRACT
The manipulation of gene activity through the creation of hypomorphic mutants has been a long-standing tool in examining gene function. Our previous studies have indicated that hypomorphic mutants could be created through the insertion of cis-regulatory sequences composed of consecutive adenosine nucleotides called polyA tracks. Here we confirm that this method can be used for the creation of hypomorphic mutants and functional characterization of membrane, secretory and endogenous proteins. Insertion of polyA tracks into the sequences of interleukin-2 and membrane protein CD20 results in a programmable reduction of mRNA stability and attenuation of protein expression regardless of the presence of signaling sequence. Likewise, CRISPR/Cas9 targeted insertion of polyA tracks in the coding sequence of endogenous human genes AUF1 and TP53 results in a programmable reduction of targeted protein and mRNA levels. Functional analyses of AUF1 engineered hypomorphs indicate a direct correlation between AUF1 gene levels and the stability of AUF1-regulated mRNAs. Hypomorphs of TP53 affect the expression of the downstream target genes differentially depending upon the severity of the hypomorphic mutation. Finally, decreases in TP53 protein affect the same cellular pathways in polyA track engineered cells as in cancer cells, indicating these variants’ biological relevance. These results highlight this technology’s power to create predictable, stable hypomorphs in recombinant or endogenous genes in combination with CRISPR/Cas9 engineering tools.
Competing Interest Statement
The authors have declared no competing interest.
