ERK contributes to B cell receptor-induced cell spreading in the A20 mouse B cell line.

B cells provide protective immunity by secreting antibodies. When a B cell encounters its specific antigen, B-cell receptor (BCR) signaling initiates actin remodeling. This allows B cells to spread on antigen-bearing surfaces and find more antigen, which increases BCR signaling and facilitates B cell activation. The BCR activates multiple signaling pathways that target actin-regulatory proteins. Although the extracellular signal-regulated kinases ERK1 and ERK2 regulate actin-dependent processes in adherent cells, their role in BCR-induced actin remodeling had not been investigated. Here, we show that targeting ERK with chemical inhibitors or siRNA inhibits BCR-induced spreading in a murine B cell line.

Off-target effects of CRISPRa on interleukin-6 expression.

Inactive fusion variants of the CRISPR-Cas9 system are increasingly being used as standard methodology to study transcription regulation. Their ability to readily manipulate the native genomic loci is particularly advantageous. In this work, we serendipitously uncover the key cytokine IL6 as an off-target of the activating derivative of CRISPR (CRISPRa) while studying RP11-326A19.4, a novel long-non coding RNA (lncRNA). Increasing RP11-326A19.4 expression in HEK293T cells via CRISPRa-mediated activation of its promoter region induced genome-wide transcriptional changes, including upregulation of IL6, an important cytokine. IL6 was increased in response to distinct sgRNA targeting the RP11-326A19.4 promoter region, suggesting specificity. Loss of the cognate sgRNA recognition sites failed to abolish CRISPRa mediated activation of IL6 however, pointing to off-target effects. Bioinformatic approaches did not reveal predicted off-target binding sites. Off-target activation of IL6 was sustained and involved low level activation of known IL6 regulators. Increased IL6 remained sensitive to further activation by TNFα, consistent with the existence of independent mechanisms. This study provides experimental evidence that CRISPRa has discrete, unpredictable off-targeting limitations that must be considered when using this emerging technology.