The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (CRISPR/Cas9) system,an RNA-guided DNA targeting technology,is triggering a revolution in the field of biology.CRISPR/ Cas9 has demonstrated great potential for genetic manipulation.In this review,we discuss the current development of CRISPR/Cas9 technologies for therapeutic applications,especially chimeric antigen receptor (CAR) T cell-based adoptive immunotherapy.Different methods used to facilitate efficient CRISPR delivery and gene editing in T cells are compared.The potential of genetic manipulation using CRISPR/Cas9 system to generate universal CAR T cells and potent T cells that are resistant to exhaustion and inhibition is explored.We also address the safety concems associated with the use of CRISPR/Cas9 gene editing and provide potential solutions and future directions of CRISPR application in the field of CAR T cell immunotherapy.As an integration-free gene insertion method,CRISPR/Cas9 holds great promise as an efficient gene knock-in platform.Given the tremendous progress that has been made in the past few years,we believe that the CRISPR/Cas9 technology holds immense promise for advancing immunotherapy.