Toward the Development of a Tissue Engineered Gradient Utilizing CRISPR-Guided Gene Modulation

Cellular, compositional, and mechanical gradients are found throughout biological tissues, especially in transition zones between tissue types. Yet, strategies to engineer such gradients have proven difficult due to the complex nature of these tissues. Current strategies for tissue engineering complex gradients often utilize stem cells; however, these multipotent cells require direction from environmental cues, which can be difficult to control both in vitro and in vivo. In this study, we utilize clustered regularly-interspaced short palindromic repeats (CRISPR)-guided gene modulation to direct the differentiation of multipotent adipose-derived stem cells (ASCs) to demonstrate the effectiveness of CRISPR-engineered cells in tissue engineering applications. Specifically, we screen CRISPR-interference (CRISPRi) constructs targeting the promotors of selected osteogenic inhibitors and demonstrate that ASC osteogenic differentiation and mineral deposition can be regulated with CRISPRi targeting of Noggin without the use of exogenous growth factors in tissue engineered constructs. As a proof of concept, we combine three technologies developed out of our laboratories to demonstrate the controlled deposition of these engineered cells in a gradient with CRISPR-activation multiplex-engineered aggrecan/collagen type-II-chondrogenic ASCs on a high density anisotropic type I collagen construct to create a cell and tissue gradient similar to the fibrocartilage-to-mineralized-fibrocartilage gradient in the enthesis. Our results display the promise of CRISPR-engineered ASCs to produce tissue gradients, similar to what is observed in native tissue. Impact statement Tissue gradients are found throughout biological tissues, especially in transition zones between tissue types. Yet, strategies to engineer such gradients have fallen short due to the complex nature of these tissues. The tendon enthesis gradient is composed of a number of cell types resulting in a fibrocartilage-to-mineralized-fibrocartilage gradient, connecting muscle to bone. Clustered regularly-interspaced short palindromic repeats (CRISPR)-guided cell engineering presents a tool to engineer and control stem cell differentiation for use in gradient tissue engineering applications. By controlling the differentiation potential of stem cells, utilizing CRISPR-guided gene modulation, we display a high degree of spatial resolution for tissue engineering an enthesis-like gradient.