ABSTRACT
The genome-editing efficiency of the CRISPR-Cas9 system hinges on the recognition of the protospacer adjacent motif (PAM) sequence, which is essential for Cas9 binding to DNA. The commonly used Streptococcus pyogenes (SpyCas9) targets the 5'-NGG-3' PAM sequence, which does not cover all the potential genomic-editing sites. To expand the toolbox for genome editing, SpyCas9 has been engineered to recognize flexible PAM sequences and Cas9 orthologs have been used to recognize novel PAM sequences. In this study, Abyssicoccus albus Cas9 (AalCas9, 1059 aa), which is smaller than SpyCas9, was found to recognize a unique 5'-NNACR-3' PAM sequence. Modification of the guide RNA sequence improved the efficiency of AalCas9-mediated genome editing in both plant and human cells. Predicted structure-assisted introduction of a point mutation in the putative PAM recognition site shifted the sequence preference of AalCas9. These results provide insights into Cas9 diversity and novel tools for genome editing.
Subject(s)
CRISPR-Associated Protein 9 , CRISPR-Cas Systems , Gene Editing , RNA, Guide, CRISPR-Cas Systems , Streptococcus pyogenes , Gene Editing/methods , CRISPR-Associated Protein 9/metabolism , CRISPR-Associated Protein 9/genetics , Humans , RNA, Guide, CRISPR-Cas Systems/genetics , Streptococcus pyogenes/genetics , Streptococcus pyogenes/enzymology , Nucleotide Motifs , Clustered Regularly Interspaced Short Palindromic RepeatsABSTRACT
Surface states of topological materials provide extreme electronic states for unconventional superconducting states. CaAg1-xPdxP is an ideal candidate for a nodal-line Dirac semimetal with drumhead surface states and no additional bulk bands. Here, we report that CaAg1-xPdxP has surface states that exhibit unconventional superconductivity (SC) around 1.5 K. Extremely sharp magnetoresistance, tuned by surface-sensitive gating, determines the surface origin of the ultrahigh-mobility "electrons." The Pd-doping elevates the Fermi level towards the surface states, and as a result, the critical temperature (Tc) is increased up to 1.7 K from 1.2 K for undoped CaAgP. Furthermore, a soft point-contact study at the surface of Pd-doped CaAgP proved the emergence of unconventional SC on the surface. We observed the bell-shaped conductance spectra, a hallmark of the unconventional SC. Ultrahigh mobility carriers derived from the surface flat bands generate a new class of unconventional SC.
ABSTRACT
CRISPR/Cas9-based genome editing represents an unprecedented potential for plant breeding. Unlike animal cells, plant cells contain a rigid cell wall, genome editing tool delivery into plant cells is thus challenging. In particular, the delivery of the Cas9-gRNA ribonucleoprotein (RNP) into plant cells is desired since the transgene insertion into the genome should be avoided for industrial applications in plants. In this study, we present a novel RNP delivery approach in rice. We applied the sonication-assisted whisker method, conventionally developed for DNA delivery in plants, for RNP delivery in rice. Combined with marker gene delivery, we successfully isolated OsLCYß genome-edited lines generated by RNPs. The calli and regenerated shoot of the OsLCYß mutant showed abnormal carotenoid accumulation. In addition, we also detected, although at a low frequency, genome editing events in rice calli cells by RNP delivery using the sonication-assisted whisker method without any additional. Therefore, the sonication-assisted whisker method could be an attractive way to create RNP-based genome-edited lines in plants.
Subject(s)
Callosities , Oryza , Animals , Oryza/genetics , CRISPR-Cas Systems , Gene Editing , Sonication , Vibrissae , Plant Breeding , Ribonucleoproteins/geneticsABSTRACT
A multiplexed glyco-bead array method for determining the sugar-binding specificities of plant lectins was described by a bead-based flow cytometric assay. Glycopeptides with N- and O-glycans were immobilized on multiplexed beads, and the specificities of several kinds of sugar chains were measured in a single reaction. This strategy is easy, rapid, reproducible, and suitable for small samples, and allows the reliable elucidation of sugar-binding properties of lectins under identical conditions.
Subject(s)
Carbohydrate Metabolism , Lectins/metabolism , Microarray Analysis/methods , Microspheres , Animals , Cattle , Flow Cytometry , Glycopeptides/metabolism , Substrate Specificity , SwineABSTRACT
Carbohydrate binding underlies many cell recognition events. Here, we describe a multiplexed glyco-bead array method for determining the carbohydrate-binding specificities of plant lectins using a bead-based flow cytometric analysis. N-glycans including high mannose, hybrid, and complex types and O-glycans from glycoproteins were immobilized on multiplexed beads, and the specificities of 13 kinds of sugar chains were monitored within 2 h in a single reaction. This strategy is easy, rapid, reproducible, and suitable for small samples and allows the reliable and simultaneous elucidation of sugar-binding properties under identical conditions.