Background/Purpose:

Autoimmunity is a complex, poly-genic disorder that culminates in multi-organ damage. In systemic lupus erythematosus (SLE), the prototypic autoimmune disorder, a breakdown of tolerance leads to erroneous immune cell activation and the production of disease-causing autoantibodies. B cell functional abnormalities appear to be central to this process and many of the lupus susceptibility genes identified by genome wide association studies (GWAS) are predicted to impact B cell selection, differentiation, signaling, and proliferation. However, their precise mechanisms of action in human B cells remain to be defined. A major challenge preventing the application of GWAS data to therapeutic potential is that animal models often do not recapitulate human phenotypes. In fact, a number of identified autoimmune disease-related genes are known to have differential expression in mice and humans. For this reason, it is important to investigate gene function directly in primary human B cells. To date, the methods for investigating gene function in human B cells has been limited by the relative rarity of many susceptibility alleles and ineffective or transient RNA silencing approaches. CRISPR-Cas9 technology, which uses customizable guide RNAs (gRNA) to direct the cutting of DNA by Cas9 enzymes, presents an unexplored opportunity for studying gene function directly in primary human lymphocytes. The aim of this study was to develop a robust CRISPR-Cas9 method for studying gene function directly in peripheral human B cells isolated from PBMCs.

Methods:

Primary human B cells were isolated from healthy donors using Ficoll-Paque gradients followed by negative magnetic sorting of B cells. Isolated B cells were nucleofected with CRISPR-Cas9 ribonuclear protein complexes and cultured on BAFF and CD40L expressing OP9 stroma for 7 days. Function was assessed by calcium flux assays and phoshosignaling using a flow cytometer.

Results:

Taking advantage of recent advances in genome editing technologies, we were able to successfully and robustly knockout CD22 in primary human B cells from healthy donors, achieving an average efficiency of 35% as measured by loss of cell surface expression. Using this highly adaptable system, we show that gRNA selection can have a profound impact on knockout efficiency and that B cell lines are not an effective proxy for this process. Consistent with the literature, knockout of CD22 in primary B cells resulted in the generation of activated/memory B cells with significantly increased expression of CD86. Furthermore, knockout of CD22 resulted in increased basal activation and as a consequence, total B cells had a reduced capacity for pSyk and pPLCγ signaling. Supporting this observation, IgD⁺ B cells were hyperresponsive to IgM stimulation with an increased capacity for calcium fluxing.

Conclusion: These findings demonstrate, for the first time, the ability to robustly knockout genes directly in primary human B cells. Using this approach, the role of CD22 in maintaining naïve B cells was confirmed in healthy donors. This methodology allows for the targeted study of autoimmune-related genes and their impact on B cell function.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/generation-of-an-efficient-crispr-cas9-editing-technique-in-human-primary-b-cells-for-the-targeted-study-of-autoimmune-susceptibility-genes/