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Abstract BackgroundThe use of 3D imaging techniques, such as X-ray CT, in root phenotyping has become more widespread in recent years. However, due to the complexity of the root structure, analyzing the resulting 3D volumes to obtain detailed architectural root traits remains a challenging computational problem. When it comes to image-based phenotyping of excavated maize root crowns, two types of root features that are notably missing from existing methods are the whorls and soil line. Whorls refer to the distinct areas located at the base of each stem node from which roots sprout in a circular pattern (Liu S, Barrow CS, Hanlon M, Lynch JP, Bucksch A. Dirt/3D: 3D root phenotyping for field-grown maize (zea mays). Plant Physiol. 2021;187(2):739–57.https://doi.org/10.1093/plphys/kiab311.). The soil line is where the root stem meets the ground. Knowledge of these features would give biologists deeper insights into the root system architecture (RSA) and the below- and above-ground root properties. ResultsWe developed TopoRoot+, a computational pipeline that produces architectural traits from 3D X-ray CT volumes of excavated maize root crowns. Building upon the TopoRoot software (Zeng D, Li M, Jiang N, Ju Y, Schreiber H, Chambers E, et al. Toporoot: A method for computing hierarchy and fine-grained traits of maize roots from 3D imaging. Plant Methods. 2021;17(1).https://doi.org/10.1186/s13007-021-00829-z.) for computing fine-grained root traits, TopoRoot + adds the capability to detect whorls, identify nodal roots at each whorl, and compute the soil line location. The new algorithms in TopoRoot + offer an additional set of fine-grained traits beyond those provided by TopoRoot. The addition includes internode distances, root traits at every hierarchy level associated with a whorl, and root traits specific to above or below the ground. TopoRoot + is validated on a diverse collection of field-grown maize root crowns consisting of nine genotypes and spanning across three years. TopoRoot + runs in minutes for a typical volume size of$$\:40{0}^{3}$$on a desktop workstation. Our software and test dataset are freely distributed on Github. ConclusionsTopoRoot + advances the state-of-the-art in image-based phenotyping of excavated maize root crowns by offering more detailed architectural traits related to whorls and soil lines. The efficiency of TopoRoot + makes it well-suited for high-throughput image-based root phenotyping. 

Abstract Despite the availability of Cas9 variants with varied protospacer-adjacent motif (PAM) compatibilities, some genomic loci—especially those with pyrimidine-rich PAM sequences—remain inaccessible by high-activity Cas9 proteins. Moreover, broadening PAM sequence compatibility through engineering can increase off-target activity. With directed evolution, we generated four Cas9 variants that together enable targeting of most pyrimidine-rich PAM sequences in the human genome. Using phage-assisted noncontinuous evolution and eVOLVER-supported phage-assisted continuous evolution, we evolved Nme2Cas9, a compact Cas9 variant, into variants that recognize single-nucleotide pyrimidine-PAM sequences. We developed a general selection strategy that requires functional editing with fully specified target protospacers and PAMs. We applied this selection to evolve high-activity variants eNme2-T.1, eNme2-T.2, eNme2-C and eNme2-C.NR. Variants eNme2-T.1 and eNme2-T.2 offer access to N₄TN PAM sequences with comparable editing efficiencies as existing variants, while eNme2-C and eNme2-C.NR offer less restrictive PAM requirements, comparable or higher activity in a variety of human cell types and lower off-target activity at N₄CN PAM sequences.