Genome Engineering Core

Core B will provide expertise in genome engineering to support Projects 1,3, and 4 and Core C. Gene editing has advanced tremendously in recent years, allowing targeted gene modification using CRISPR-Cas technology and engineered base editors (BEs). Cas proteins, reprogrammable molecular scissors, create DNA breaks in an RNA-guided manner, while BEs use RNA-guided DNA binding by a catalytic-deficient Cas protein to direct DNA deaminases for site-specific genome editing. Coupled with lentiviral delivery, this technology enables loss-of- function, gain-of-function, and transgene introduction in primary cells. The available toolset for genome modification is impressive, but no technology is perfect. Delivery of the editing machinery can be challenging, efficiency can be limiting, and optimizing on-target editing in the proper cellular and genomic location can be difficult. Genome modification methods can disrupt needed host functions, resulting in genotoxicity. Thus, Core B will focus on optimizing genome modification methods for use across projects, implement model-independent methods for quantifying off-target genome modification, and advance the project through the following Specific Aims: 1) Optimize and implement methods for genome editing of T cells and hematopoietic stem cells (HSCs), including a) traditional indel-mediated gene inactivation with Cas9 and Cas12a or multiplex editing, and b) peptide-assisted genome engineering (PAGE) to direct protein editing with high efficiency and minimal toxicity. 2) Optimize and implement methods for base editing of T cells and HSCs, including a) base editing with optimized constructs spanning diverse target site requirements and editing windows, and b) inclusion of controllable split- engineered base editors to allow for inducible editing or PAGE technology for non-toxic protein-based editing. 3) Optimize and implement methods for tracking consequences of cell engineering, including a) analysis of vector integration site distributions and clonal behavior, b) analysis of specificity of CRISPR-Cas9 cleavage, and c) novel methods for quantifying off-target base editing by CRISPR-BE.