Breast cancer is the leading cause of cancer-related deaths in women. The aggressive breast cancer subtype is commonly linked to the genetic alterations in the TP53 tumor suppressor gene, predominantly the missense mutations. Robust experimental models are needed to gain better insights into these mutations’ molecular properties and implications in tumorigenesis. The generation of such models harboring the alterations is feasible with the CRISPR-based gene editing technology. Moreover, the development of new CRISPR applications, particularly DNA base and prime editing, has considerably improved the precision and versatility of gene editing. Here, we employed the prime editing tool to revert a TP53 missense C > T mutation (L194F) in a T47D luminal A breast cancer cell line. In parallel, this prime editing tool was also utilized to introduce the L194F mutation in HEK293T cells. To assess the prime editing efficiency in both cell lines, we first performed Sanger sequencing in the prime-edited cells pool and single cell-derived clones. However, the Sanger sequencing approach did not detect any base substitution in these cell lines. Next, by employing the more sensitive amplicon target sequencing, we managed to identify the expected substitution in these T47D and HEK293T cells, albeit the editing efficiency was low. In light of these findings, we discussed the technical aspects and provided suggestions for improve the prime editing workflow and efficiency for future experiments.

Cells encounter a myriad of endogenous and exogenous stresses that could perturb cellular physiological processes. Therefore, cells are equipped with several adaptive and stress-response machinery to overcome and survive these insults. One such machinery is the heat shock response (HSR) program that is governed by the heat shock factors (HSFs) family in response towards elevated temperature, free radicals, oxidants, and heavy metals. HSF4 is a member of this HSFs family that could exist in two predominant isoforms, either the transcriptional repressor HSFa or transcriptional activator HSF4b. HSF4 is constitutively active due to the lack of oligomerization negative regulator domain. HSF4 has been demonstrated to play roles in several physiological processes and not only limited to regulating the classical heat shock- or stress-responsive transcriptional programs. In this review, we will revisit and delineate the recent updates on HSF4 molecular properties. We also comprehensively discuss the roles of HSF4 in health and diseases, particularly in lens cell development, cataract formation, and cancer pathogenesis. Finally, we will posit the potential direction of HSF4 future research that could enhance our knowledge on HSF4 molecular networks as well as physiological and pathophysiological functions.