Transient and DNA‐free in vivo CRISPR/Cas9 genome editing for flexible modeling of endometrial carcinogenesis

The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) (CRISPR/Cas9)-mediated generation of somatically genetically engineered mouse models have emerged as a new approach for in vivo modeling of cancer [1].Here, we describe a novel DNA-free, easy, rapid, flexible, multiplexable, and robust method to model endometrial neoplasia by CRISPR/Cas9 ribonucleoprotein (RNP) electroporation into the uterus of mice.First, we tested our CRISPR/Cas9 editing approach with an in vitro CRISPR/Cas9 assay using a plasmid encoding a Locus of X-over P1 (LoxP)-flanked membrane-targeted tandem dimer Tomato (mT) followed by membrane-targeted Green Fluorescent protein (GFP) (pCA-mT/mG).The material and methods used in this study are described in the Supplementary Material file.Upon Cre recombination, LoxP-flanked mT was deleted, and we observed that the cells shifted from red to green fluorescence.Then, we designed a CRISPR RNA (crRNA) targeting the Protospacer Adjacent Motif (PAM) sequence adjacent to LoxP sites flanking mT (Supplementary Figure S1A).The pCA-mT/mG plasmid was incubated with the LoxP-targeting RNP, and the Cas9-mediated cleavage of the target DNA was assessed by agarose gel electrophoresis (Supplementary Figure S1B, arrowhead).Next, we tested the ability of the LoxP-targeting Cas9 RNP to induce gene editing into living cells.Skin fibroblasts isolated from mT/mG reporter mouse [2] were transfected with the LoxP-targeting Cas9 RNP.