Sondergaard, J. N. | Search bioRxiv

bioRxiv2022

Control of NK cell tolerance in MHC class I-deficiency by regulated SHP-1 localization to the activating immune synapse

Signaling via inhibitory KIR/Ly49 receptors preserves natural killer (NK) cell self-tolerance but also conveys NK cell reactivity towards MHC class-I low target cells in an education process. Here, we demonstrate that mouse NK cell education by H-2Dd regulates transcription of several genes in Ly49A+ NK cells including Ptpn6, encoding the phosphatase SHP-1. SHP-1 was highly expressed in uneducated NK cells, in which knock-out of Ptpn6 increased responsiveness. Following NKp46 triggering of uneducated NK cells, a higher synaptic abundance of phosphorylated SHP-1 was found relative to educated NK cells, concomitant with reduced phosphorylation of several signaling molecules, including PLC-g2, SLP-76, ZAP70/Syk and ERK1/2. SHP-1 overlapped extensively with F-actin and SLP-76 in the uneducated activating synapse of Ly49A+ NK cells, whereas a greater association between Ly49A and SHP-1 was observed in educated NK cells. Thus, our results indicate that in addition to transcriptional regulation, a distinct SHP-1 patterning in NK cell activating synapses can determine their tolerance.

immunology↗

bioRxiv2021

CRISPR/Cas9 deletions induce adverse on-target genomic effects leading to functional DNA in human cells

The CRISPR/Cas9 system is widely used to permanently delete genomic regions by inducing double-strand breaks via dual guide RNAs. However, on-target consequences of Cas9 deletion events have yet to be fully investigated. To characterize Cas9-induced genotypic abnormalities in human cells, we utilized an innovative droplet-based target enrichment approach followed by long-read sequencing and coupled it to a customized de novo sequence assembly. This approach enabled us to dissect the sequence content at kilobase scale within an on-target genomic locus. We here describe extensive genomic disruptions by Cas9, involving a genomic duplication and inversion of the target region as well as integrations of exogenous DNA and interchromosomal DNA fragment rearrangements at the double-strand break sites often at the same time. Although these events altered the genomic composition of the on-target region, we found that the aberrant DNA fragments are still functional, marked by active histones and bound by RNA polymerase III. In HAP1 cells, the integration of the target-derived fragments accelerated cell proliferation in deletion clones. Our findings broaden the consequential spectrum of the Cas9 deletion system, reinforce the necessity of meticulous genomic validations and rationalize extra caution when interpreting results from a deletion event.

molecular biology↗