Project Summary Lynch syndrome (LS) is a hereditary disease that predisposes patients to colorectal, endometrial, ovarian and other cancers. Definitive diagnosis of LS depends on detection of a deleterious, germline mutation in one of the DNA mismatch repair (MMR) genes. A problem arises for clinicians and genetic counselors, however, when the sequencing reveals a variation, such as a missense mutation, whose effect on gene function is not immediately obvious. These variants of uncertain significance (VUS) cannot be used to confirm nor rule out LS resulting in uncertainty about how to manage the patient and their family members. Laboratory studies to determine whether a VUS disrupts protein function has become one important part of a strategy for determining their relevance to disease. Studying the function of a VUS in human cells is the most thorough approach to determining its effects on MMR function. We propose that advances in the culture of human pluripotent stem cells (PSC) along with the development of CRISPR-Cas9 mediated gene editing provide an ideal model system for testing the functional effects of MMR gene variants. PSCs are immortal, which makes them a practical model system in the laboratory, yet non-transformed, thus they may more closely resemble the environment in which the variant protein first influences early transformation events in LS patient cells. In addition, they are pluripotent which means they can be differentiated into multiple cell types to examine cell- type specific effects. As LS patients are mostly affected by colorectal cancer, we will further examine subsets of VUS in human colonic organoids (HCOs) derived from the PSCs. Our goal is to perform a large-scale screen of VUS in the MMR genes MSH2, MSH6 and MLH1 under the following aims: 1) Examining whether cancer-associated variants in the MMR genes disrupt cellular repair and response functions in PSCs. We will perform CRISPR-Cas9-mediated gene targeting at the endogenous gene loci to create non-transformed cell culture models expressing different VUS. 2) For those VUS-expressing PSCs with intermediate MMR effects, we will differentiate the cells into HCOs and examine their ability to induce an apoptotic or senescent response to damage in intestinal cells. 3) For VUS HCOs that still display inconclusive, intermediate function, we will examine longer-term effects on genome stability and cell survival including through the use of mixed wild- type/variant HCOs to assess whether a survival advantage exists for the variant-expressing cells. This proposal will assist in the disease significance classification for a large number of MMR gene VUS which can be used by clinicians and genetic counselors world-wide to assist in managing and preventing cancer in patients and their families.