Genome editing holds promise for correcting pathogenic mutations. However, it is difficult to determine off-target effects of editing due to single nucleotide polymorphism in individuals. Here, we developed a method named GOTI (Genome-wide Off-target analysis by Two-cell embryo Injection) to detect off-target mutations by editing one blastomere of two-cell mouse embryos using either CRISPR-Cas9 or base editors. Comparison of the whole genome sequences of progeny cells of edited vs. non-edited blastomeres at E14.5 showed that off-target single nucleotide variants (SNVs) were rare in embryos edited by CRISPR-Cas9 or adenine base editor, with a frequency close to the spontaneous mutation rate. In contrast, cytosine base editing induced SNVs with over 20-fold higher frequencies, requiring a solution to address its fidelity.

Electron microscopy and crystallography studies of α4β7 integrin reveal the mechanism by which this atypical integrin enables rolling adhesion prior to integrin activation.

SUMMARY Leukocyte adhesion requires β 2 -integrin activation. Resting integrins exist in a bent-closed conformation—i.e., not extended (E − ) and not high affinity (H − )—unable to bind ligand. Fully activated E + H + integrin binds intercellular adhesion molecules (ICAMs) expressed on the opposing cell in trans . E − H − transitions to E + H + through E + H − or through EH + , which binds to ICAMs on the same cell in cis . Spatial patterning of activated integrins is thought to be required for effective arrest, but no high-resolution cell surface localization maps of activated integrins exist. Here, we developed Super-STORM by combining super-resolution microscopy with molecular modeling to precisely localize activated integrin molecules and identify the molecular patterns of activated integrins on primary human neutrophils. At the time of neutrophil arrest, E − H + integrins face each other to form oriented (non-random) nanoclusters. To address the mechanism causing this pattern, we blocked integrin binding to ICAMs in cis , which significantly relieved the face-to-face orientation.

Immune surveillance and host defense depend on the precisely regulated trafficking of lymphocytes. Integrin α4β7 mediates lymphocyte homing to the gut through its interaction with mucosal vascular address in cell adhesion molecule-1 (MAdCAM-1). α4β7 also binds vascular cell adhesion molecule-1 (VCAM-1), which is expressed in other tissues. To maintain the tissue specificity of lymphocyte homing, α4β7 must distinguish one ligand from the other. Here, we demonstrate that α4β7 is activated by different chemokines in a ligand-specific manner. CCL25 stimulation promotes α4β7-mediated lymphocyte adhesion to MAdCAM-1 but suppresses adhesion to VCAM-1, whereas CXCL10 stimulation has the opposite effect. Using separate pathways, CCL25 and CXCL10 stimulate differential phosphorylation states of the β7 tail and distinct talin and kindlin-3 binding patterns, resulting in different binding affinities of MAdCAM-1 and VCAM-1 to α4β7. Thus, our findings provide a mechanism for lymphocyte traffic control through the unique ligand-specific regulation of integrin adhesion by different chemokines.

No abstract

Rap1 is a major convergence point of the platelet signaling pathways that result in talin1 binding to the integrin b cytoplasmic domain and consequent integrin activation, platelet aggregation, and effective hemostasis. The nature of the connection between Rap1 and talin1 in integrin activation is an important remaining gap in our understanding of this process. Previous work identified a low affinity Rap1 binding site in talin1 F0 domain that makes a small contribution to integrin activation in platelets. We recently identified an additional Rap1 binding site in talin1 F1 domain that makes a greater contribution than F0 in model systems. Here we generated mice bearing point mutations, which block Rap1 binding without affecting talin1 expression, in either talin1 F1 domain (R118E) alone, which were viable, or both F0 and F1 domains (R35E,R118E), which were embryonic lethal. Loss of the Rap1-talin1 F1 interaction in platelets markedly decreases talin1-mediated activation of platelet b1- and b3-integrins. Integrin activation and platelet aggregation in mice whose platelets express only talin1(R35E,R118E) are even more impaired, resembling the defect seen in platelets lacking both Rap1a and Rap1b. Although Rap1 is important in thrombopoiesis, platelet secretion, and surface exposure of phosphatidylserine, loss of Rap1-talin interaction in talin1(R35E,R118E) platelets had little effect on these processes. These findings show that talin1 is the principal direct effector of Rap1 GTPases that regulates platelet integrin activation in hemostasis.

Platelets expressing a talin 1 mutant (R35E) that significantly reduces Rap1 affinity exhibit little change in GPIIb-IIIa activation.• Tln1 R35E/R35E mice are viable, apparently healthy, and fertile, with similar bleeding times as wild-type littermates.Activation of platelet glycoprotein IIb-IIIa (GPIIb-IIIa; integrin aIIbb3) leads to high-affinityfibrinogen binding and platelet aggregation during hemostasis. Whereas GTP-bound Rap1GTPase promotes talin 1 binding to the b3 cytoplasmic domain to activate platelet GPIIb-IIIa, the Rap1 effector that regulates talin association with b3 in platelets is unknown. Rap1binding to the talin 1 F0 subdomain was proposed to forge the talin 1-Rap1 link in platelets.Here, we report a talin 1 point mutant (R35E) that significantly reduces Rap1 affinity without a significant effect on its structure or expression. Talin 1 head domain (THD) (R35E) was of similar potency to wild-type THD in activating aIIbb3 in Chinese hamster ovary cells.Coexpression with activated Rap1b increased activation, and coexpression with Rap1GAP1 reduced activation caused by transfection of wild-type THD or THD(R35E). Furthermore, platelets from Tln1 R35E/R35E mice showed similar GPIIb-IIIa activation to those from wildtype littermates in response to multiple agonists. Tln1 R35E/R35E platelets exhibited slightly reduced platelet aggregation in response to low doses of agonists; however, there was not a significant hemostatic defect, as judged by tail bleeding times. Thus, the Rap1-talin 1 F0interaction has little effect on platelet GPIIb-IIIa activation and hemostasis and cannot account for the dramatic effects of loss of Rap1 activity on these platelet functions.

Proper chromosome segregation during meiosis requires cyclins associated with cyclin-dependent kinases. Li et al. generate Ccnb3 mutant mice via CRISPR/Cas9 and identify a requirement for cyclin B3 in female meiosis I.