B cells exert multiple effector functions, and dysfunctions of B cells often lead to initiation and progression of diseases, including autoimmune and inflammatory diseases. Therefore, B cell intervention may be an effective strategy to treat diseases involving B cells. The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 gene editing system has been widely used for DNA deletion, insertion, and replacement. Nanocarriers have been developed as relatively mature systems and may be applied to deliver the CRISPR-Cas9 system to B cells in vivo. In this study, we created a library of nanoparticles (NPs) with different polyethylene glycol densities and zeta potentials and screened an optimal NP for in vivo B cell targeting. The selected NP could deliver the CRISPR-Cas9 system to B cells and induce Cas9 expression inside the cell environment. Injection of the NP encapsulated with Cas9/gB220 (NP_Cas9/gB220) into mice could disrupt B220 expression in B cells, suggestive of its applications to intervene the expression of the target molecule in B cells. Moreover, the treatment with NP_Cas9/gBAFFR could decrease the number of B cells and exert therapeutic effect in rheumatoid arthritis, as B-cell activating factor receptor (BAFFR) is vital for the survival and functions of B cells. In conclusion, we developed a carrier for the delivery of the CRISPR-Cas9 gene editing system for B cell intervention that could be used for the treatment of diseases related to B cell dysfunctions.