*Review* **Application of CRISPR-Cas9 System to Study Biological Barriers to Drug Delivery**

**Ji He <sup>1</sup> , Riya Biswas <sup>1</sup> , Piyush Bugde <sup>1</sup> , Jiawei Li <sup>1</sup> , Dong-Xu Liu 1,2 and Yan Li 1,2,3,\***


**Abstract:** In recent years, sequence-specific clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas) systems have been widely used in genome editing of various cell types and organisms. The most developed and broadly used CRISPR-Cas system, CRISPR-Cas9, has benefited from the proof-of-principle studies for a better understanding of the function of genes associated with drug absorption and disposition. Genome-scale CRISPR-Cas9 knockout (KO) screen study also facilitates the identification of novel genes in which loss alters drug permeability across biological membranes and thus modulates the efficacy and safety of drugs. Compared with conventional heterogeneous expression models or other genome editing technologies, CRISPR-Cas9 gene manipulation techniques possess significant advantages, including ease of design, costeffectiveness, greater on-target DNA cleavage activity and multiplexing capabilities, which makes it possible to study the interactions between membrane proteins and drugs more accurately and efficiently. However, many mechanistic questions and challenges regarding CRISPR-Cas9 gene editing are yet to be addressed, ranging from off-target effects to large-scale genetic alterations. In this review, an overview of the mechanisms of CRISPR-Cas9 in mammalian genome editing will be introduced, as well as the application of CRISPR-Cas9 in studying the barriers to drug delivery.

**Keywords:** CRISPR-Cas9; blood-brain barrier; intestinal epithelial barrier; drug permeability
