RESUMO
The widespread use of Cas12a (formerly Cpf1) nucleases for genome engineering is limited by their requirement for a rather long TTTV protospacer adjacent motif (PAM) sequence. Here we have aimed to loosen these PAM constraints and have generated new PAM mutant variants of the four Cas12a orthologs that are active in mammalian and plant cells, by combining the mutations of their corresponding RR and RVR variants with altered PAM specificities. LbCas12a-RVRR showing the highest activity was selected for an in-depth characterization of its PAM preferences in mammalian cells, using a plasmid-based assay. The consensus PAM sequence of LbCas12a-RVRR resembles a TNTN motif, but also includes TACV, TTCV CTCV and CCCV. The D156R mutation in improved LbCas12a (impLbCas12a) was found to further increase the activity of that variant in a PAM-dependent manner. Due to the overlapping but still different PAM preferences of impLbCas12a and the recently reported enAsCas12a variant, they complement each other to provide increased efficiency for genome editing and transcriptome modulating applications.
Assuntos
Proteínas Associadas a CRISPR/genética , Proteínas Associadas a CRISPR/metabolismo , Endodesoxirribonucleases/genética , Endodesoxirribonucleases/metabolismo , Edição de Genes , Animais , Linhagem Celular Tumoral , Células HEK293 , Humanos , Camundongos , Mutação , Especificidade por SubstratoRESUMO
Cortical pathology contributes to chronic cognitive impairment of patients suffering from the neuroinflammatory disease multiple sclerosis (MS). How such gray matter inflammation affects neuronal structure and function is not well understood. In the present study, we use functional and structural in vivo imaging in a mouse model of cortical MS to demonstrate that bouts of cortical inflammation disrupt cortical circuit activity coincident with a widespread, but transient, loss of dendritic spines. Spines destined for removal show local calcium accumulations and are subsequently removed by invading macrophages or activated microglia. Targeting phagocyte activation with a new antagonist of the colony-stimulating factor 1 receptor prevents cortical synapse loss. Overall, our study identifies synapse loss as a key pathological feature of inflammatory gray matter lesions that is amenable to immunomodulatory therapy.