Generation of a conditional mutant knock-in under the control of the natural promoter using CRISPR-Cas9 and Cre-Lox systems.

Modulation of gene activity by creating mutations has contributed significantly to the understanding of protein functions. Oftentimes, however, mutational analyses use overexpression studies, in which proteins are taken out of their normal contexts and stoichiometries. In the present work, we sought to develop an approach to simultaneously use the CRISPR/Cas9 and Cre-Lox techniques to modify the endogenous SAT1 gene to introduce mutant forms of the protein while still under the control of its natural gene promoter. We cloned the C-terminal portion of wild type (WT) SAT1, through the transcriptional stop elements, and flanked by LoxP sites in front of an identical version of SAT1 containing point mutations in critical binding sites. The construct was inserted into the endogenous SAT1 locus by Non-Homologous End Joining (NHEJ) after a CRISPR/Cas9 induced DNA double strand break. After validating that normal function of SAT1 was not altered by the insertional event, we were then able to assess the impact of point mutations by introduction of Cre recombinase. The system thus enables generation of cells in which endogenous WT SAT1 can be conditionally modified, and allow investigation of the functional consequences of site specific mutations in the context of the normal promoter and chromatin regulation.