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CRISPR-Cas12a is a promising genome editing system for targeting AT-rich genomic regions. Comprehensive genome engineering requires simultaneous targeting of multiple genes at defined locations. Here, to expand the targeting scope of Cas12a, we screen nine Cas12a orthologs that have not been demonstrated in plants, and identify six, ErCas12a, Lb5Cas12a, BsCas12a, Mb2Cas12a, TsCas12a and MbCas12a, that possess high editing activity in rice. Among them, Mb2Cas12a stands out with high editing efficiency and tolerance to low temperature. An engineered Mb2Cas12a-RVRR variant enables editing with more relaxed PAM requirements in rice, yielding two times higher genome coverage than the wild type SpCas9. To enable large-scale genome engineering, we compare 12 multiplexed Cas12a systems and identify a potent system that exhibits nearly 100% biallelic editing efficiency with the ability to target as many as 16 sites in rice. This is the highest level of multiplex edits in plants to date using Cas12a. Two compact single transcript unit CRISPR-Cas12a interference systems are also developed for multi-gene repression in rice and Arabidopsis . This study greatly expands the targeting scope of Cas12a for crop genome engineering. CRISPR-Cas12a is a promising system for targeting AT-rich regions of the genome. Here the authors identify Cas12a orthologs with expanded targeting scope and develop a highly multiplexable editing system in rice.

Members of the piggyBac superfamily of DNA transposons are widely distributed in host genomes ranging from insects to mammals. The human genome has retained five piggyBac -derived genes as domesticated elements although they are no longer mobile. Here, we have investigated the transposition properties of piggyBat from Myotis lucifugus , the only known active mammalian DNA transposon, and show that its low activity in human cells is due to subterminal inhibitory DNA sequences. Activity can be dramatically improved by their removal, suggesting the existence of a mechanism for the suppression of transposon activity. The cryo-electron microscopy structure of the piggyBat transposase pre-synaptic complex showed an unexpected mode of DNA binding and recognition using C-terminal domains that are topologically different from those of the piggyBac transposase. Here we show that structure-based rational re-engineering of the transposase through the removal of putative phosphorylation sites and a changed domain organization - in combination with truncated transposon ends - results in a transposition system that is at least 100-fold more active than wild-type piggyBat . piggyBat is the only known DNA transposon active in mammals. Here the authors used cellular assays and 3D structure to show that sequences within the transposon ends restrict activity, and also how transposition activity can be substantially increased.

Full understanding of the functions of the polycystin proteins, PC1 and PC2, in renal epithelial cells is obscured by signaling complexity and renal injury that occurs in Autosomal Dominant Polycystic Kidney Disease (ADPKD). The polycystins likely function as a complex in the primary cilium, yet previous work hinted at a critical role for PC1 function outside of the primary cilium (extra-ciliary) during tubule development. Here, we investigate an extra-ciliary role for the polycystins in regulating renal cell and tubular morphology. First, we found acute loss of polycystins significantly increased the circularity of renal epithelial cells and tubuloids grown in 3D culture. Next, we demonstrated that both PC1 and PC2 can immunoprecipitate Ezrin, an ERM protein important for apical compartment shape. In human ADPKD renal cystic tissue, and after acute inducible knockout of or , we found that Ezrin protein abundance is significantly reduced, with the remaining Ezrin protein mis-localized. Immunofluorescence in 2D cells and 3D tubuloids suggested acute polycystin loss specifically reduced the active form of Ezrin at the apical surface, leaving inactive Ezrin colocalized with ZO1 in the cell junctions. A specific ERM phosphorylation inhibitor, NSC668394, phenocopied the increased circularity observed in the knockout spheroids, as did inhibition of PKC activity, implicating the polycystin complex in regulating Ezrin phosphorylation. Our data strongly support a role for the polycystin complex in regulating renal cell and tubular shape via interactions with the ERM protein Ezrin, interactions that do not require trafficking to the primary cilium.