Establishment of an in vitro investigation model to discover molecular mechanisms of B cell lymphoma development using CRISPR-Cas9 system

Abstract

Diffuse Large B-Cell Lymphoma (DLBCL) is the most common histologic subtype of non-Hodgkin lymphoma and a heterogeneous disease with a variety of molecular aberration and diverse clinical outcomes. Currently, therapeutic advances in DLBCL have been achieved, however; 40% of the patients fail to achieve durable remission and developed relapsed/refractory disease. To further improve and develop the therapeutic effects for patients of DLBCL, investigation on the gene functions of DLBCL is essential. However, up to now, many of the functions of genes in DLBCL was not completely identified due to the difficulty of gene manipulation in human B cells. Here, we established an in vitro investigation model to discover molecular mechanisms of B cell lymphoma development using measles virus glycoprotein displaying lentiviral transduction based CRISPR-Cas9 system. We transduced Cas9 into various DLBCLs, which are hard to transduce. The Cas9 expression was confirmed by western blot. Functionality of the transduced Cas9 was assessed by GFP knockout efficacy using GFP gRNA-GFP-BFP expression vector system. Transduction CD45-gRNA expressing vector knocked out by 70% of the cells, as well as transfection of CD45-sgRNA knock out by 20%. Thus, this gene functional analysis system is compatible with both cell lines for long-term culture and primary cells for short-term culture. Collectively, this system allowed us to manipulate genes in B cells with high efficiency and provide novel insight into therapeutic modalities for the DLBCL.