Genome Engineering Shared Resource Summary The goals of the Genome Engineering Shared Resource (GESR) are to utilize state-of-the-art CRISPR/Cas9- mediated precise genome engineering to efficiently generate desired modifications in mammalian (most frequently human) cells in an accurate and timely manner. This will be done through the following specific aims: 1) To provide CRISPR/Cas9 gene editing design services coordinated with the individual researcher with the goal of generating the desired genetic modification in their model cell line; 2) To provide CRISPR/Cas9 targeting reagents to be used by clients if they have the personnel and expertise already in place in their own laboratory for gene-targeting experiments; 3) To use the CRISPR/Cas9 system to generate cell lines of an investigator's choice containing a) knockouts of non-essential genes, b) conditional knockouts of essential genes, c) knock-in mutations, or d) gene tagging; 4) To work with the Mouse Genetics Laboratory (MGL) Shared Resource to create mutant mice using CRISPR/Cas9-mediated gene targeting; and 5) To work with the Stem Cell Institute with the goal of offering CRISPR/Cas9 genome editing of human induced pluripotent stem cells for researchers requiring this service. The GESR provides Masonic Cancer Center (MCC) members with convenient, cost-effective access to these reagents and services for genetic modification that can only rarely (or very expensively) be obtained from commercial sources. Access to these human cell lines and associated CRISPR/Cas9 technologies is critical to the ongoing work of the MCC, especially to members of the Genetic Mechanisms, Immunology, and Cellular Mechanisms Programs. Over the past 7 years, the GESR has successfully generated 61 independent knockouts of non-essential genes, 5 conditional knockouts, 20 single-nucleotide knock-ins, and 11 gene-tagged cell lines and has assisted MGL in the generation of 8 knockout, 12 conditional knockout, 9 knock-in, and 10 gene-tagged mouse strains. The GESR is co-led by Drs. Branden Moriarity (Genetic Mechanisms [GM] and Transplant and Cellular Therapy [TCT] Programs) and Dr. Beau Webber (GM and TCT). Dr. Moriarity has over 10 years of experience in gene editing using TALENs and the CRISPR/Cas9 system. His laboratory focuses on gene editing in primary human immune cells for research and therapy. Dr. Webber has over 6 years of experience in gene editing. His focus is to synergize genome engineering, stem cell biology, and adoptive cellular therapy to develop cures for genetic disease and cancer. The GESR is coordinated by Walker Lahr, who brings 7 years of experience to GESR. In fiscal year 2022, 32 individuals used GESR services, of whom 28 were MCC members.