Preparation of B2M– Cell By CRISPR/Cas9 Technology

Abstract: The major histocompatibility complex I (MHC I) is an extremely important molecule that causes immune rejection. In this study， they used CRISPR/Cas9 technology to target its B2M (beta-2 microglobulin) subunit to achieve MHC I knockout， and verify the deletion efficiency on molecular and cellular level. The guide RNA (gRNA) of B2M gene was designed and constructed to the CRISPR/Cas9 knockout plasmid containing both Cas9 protein and gRNA. Then the plasmid was transfected into HEK293 cells by electroporation systems. After that， HEK293-B2M– cells were sorted by fluorescence activated cell sorting， followed by extracting genomic DNA from B2M knockout HEK293 cells. The polymerase chain reaction (PCR) was performed for amplifying either the gRNA targeted or potential off-target regions of genome. The PCR product was inserted into the T vector and analyzed of targeted and off-target efficiency using genetic sequencing. At the same time， the expression of B2M gene in RNA level was detected by reverse transcription-polymerase chain reaction (RT-PCR) and Real-time polymerase chain reaction (Real-time PCR). In conclusion， this study developed a rapid and convenient method for knocking out B2M gene using CRISPR/Cas9 technology. After detecting， it demonstrated to successfully introducing frameshift， insertion， deletion and single base mutations in B2M gene， and off-target phenomenon was not found. It could be a great application in solving the problem of immune rejection in the future.

CSTR: 32200.14.cjcb.2018.08.0009