The RNA-guided endonuclease Cas9 cleaves its target DNA and is a powerful genome-editing tool. However, the widely used Cas9 enzyme (SpCas9) requires an NGG protospacer adjacent motif (PAM) for target recognition, thereby restricting the targetable genomic loci. Here, we report a rationally engineered SpCas9 variant (SpCas9-NG) that can recognize relaxed NG PAMs. The crystal structure revealed that the loss of the base-specific interaction with the third nucleobase is compensated by newly introduced non-base-specific interactions, thereby enabling the NG PAM recognition. We showed that SpCas9-NG induces indels at endogenous target sites bearing NG PAMs in human cells. Furthermore, we found that the fusion of SpCas9-NG and the activation-induced cytidine deaminase (AID) mediates the C-to-T conversion at target sites with NG PAMs in human cells.

This is a PDF file of a peer-reviewed paper that has been accepted for publication. Although unedited, the content has been subjected to preliminary formatting. Nature is providing this early version of the typeset paper as a service to our authors and readers. The text and figures will undergo copyediting and a proof review before the paper is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers apply.Attenuated replication and pathogenicity of SARS-CoV-2 B.1.1.529 Omicron

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Soon after the emergence and global spread of a new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron lineage, BA.1 (ref1, 2), another Omicron lineage, BA.2, has initiated outcompeting BA.1. Statistical analysis shows that the effective reproduction number of BA.2 is 1.4-fold higher than that of BA.1. Neutralisation experiments show that the vaccine-induced humoral immunity fails to function against BA.2 like BA.1, and notably, the antigenicity of BA.2 is different from BA.1. Cell culture experiments show that BA.2 is more replicative in human nasal epithelial cells and more fusogenic than BA.1. Furthermore, infection experiments using hamsters show that BA.2 is more pathogenic than BA.1. Our multiscale investigations suggest that the risk of BA.2 for global health is potentially higher than that of BA.1.

Water deficit and the resulting osmotic stress affect plant growth. To understand how plant cells monitor and respond to osmotic change from water stress, we isolated a cDNA from dehydrated Arabidopsis plants. This cDNA encodes a novel hybrid-type histidine kinase, ATHK1. Restriction fragment length polymorphism mapping showed that the ATHK1 gene is on chromosome 2. The predicted ATHK1 protein has two putative transmembrane regions in the N-terminal half and has structural similarity to the yeast osmosensor synthetic lethal of N-end rule 1 (SLN1). The ATHK1 transcript was more abundant in roots than other tissues under normal growth conditions and accumulated under conditions of high or low osmolarity. Histochemical analysis of ␤ -glucuronidase activities driven by the ATHK1 promoter further indicates that the ATHK1 gene is transcriptionally upregulated in response to changes in external osmolarity. Overexpression of the ATHK1 cDNA suppressed the lethality of the temperature-sensitive osmosensing-defective yeast mutant sln1-ts . By contrast, ATHK1 cDNAs in which conserved His or Asp residues had been substituted failed to complement the sln1-ts mutant, indicating that ATHK1 functions as a histidine kinase. Introduction of the ATHK1 cDNA into the yeast double mutant sln1 ⌬ sho1 ⌬ , which lacks two osmosensors, suppressed lethality in high-salinity media and activated the highosmolarity glycerol response 1 (HOG1) mitogen-activated protein kinase (MAPK). These results imply that ATHK1 functions as an osmosensor and transmits the stress signal to a downstream MAPK cascade. INTRODUCTIONPlants have multiple physiological and biochemical systems that enable them to tolerate environmental stresses. Water deficit is the most serious factor limiting plant growth and productivity, and it occurs not only during drought but also with high salinity and low temperature. A change in osmotic potential in cells caused by water loss triggers various molecular responses in plants (Bray, 1997). To date, many genes induced by drought, salinity, or cold stress have been identified and studied (Ingram and Bartels, 1996; Yamaguchi-Shinozaki, 1996, 1997). However, little is known about how plant cells detect water deficits.In bacteria, histidine kinases function as sensor molecules that transduce extracellular signals (including chemotactic factors, changes in osmolarity, and nutrient deficiency) to the cytoplasm. This transduction is mediated by phosphotransfer to the cognate response regulator (Parkinson and Kofoid, 1992;Parkinson, 1993;Alex and Simon, 1994;Swanson et al., 1994;Chang and Meyerowitz, 1995;Mizuno et al., 1996;Mizuno, 1997Mizuno, , 1998Wurgler-Murphy et al., 1997;Chang and Stewart, 1998). This simple signaling unit is called a twocomponent system. Typically, the two-component system is composed of two types of proteins, a sensory histidine kinase and a response regulator. A typical histidine kinase contains an N-terminal input domain and a C-terminal transmitter domain with an invariant histidine residue. A typical respon...

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We present here the annotation of the complete genome of rice Oryza sativa L. ssp. japonica cultivar Nipponbare. All functional annotations for proteins and non-protein-coding RNA (npRNA) candidates were manually curated. Functions were identified or inferred in 19,969 (70%) of the proteins, and 131 possible npRNAs (including 58 antisense transcripts) were found. Almost 5000 annotated protein-coding genes were found to be disrupted in insertional mutant lines, which will accelerate future experimental validation of the annotations. The rice loci were determined by using cDNA sequences obtained from rice and other representative cereals. Our conservative estimate based on these loci and an extrapolation suggested that the gene number of rice is ∼32,000, which is smaller than previous estimates. We conducted comparative analyses between rice and Arabidopsis thaliana and found that both genomes possessed several lineage-specific genes, which might account for the observed differences between these species, while they had similar sets of predicted functional domains among the protein sequences. A system to control translational efficiency seems to be conserved across large evolutionary distances. Moreover, the evolutionary process of protein-coding genes was examined. Our results suggest that natural selection may have played a role for duplicated genes in both species, so that duplication was suppressed or favored in a manner that depended on the function of a gene.

Streptococcus pyogenes strains can be divided into two classes, one capable and the other incapable of producing H 2 O 2 (M. Saito, S. Ohga, M. Endoh, H. Nakayama, Y. Mizunoe, T. Hara, and S. Yoshida, Microbiology 147: [2469][2470][2471][2472][2473][2474][2475][2476][2477] 2001). In the present study, this dichotomy was shown to parallel the presence or absence of H 2 O 2 -producing lactate oxidase activity in permeabilized cells. Both lactate oxidase activity and H 2 O 2 production under aerobic conditions were detectable only after glucose in the medium was exhausted. Thus, the glucose-repressible lactate oxidase is likely responsible for H 2 O 2 production in S. pyogenes. Of the other two potential H 2 O 2 -producing enzymes of this bacterium, NADH and ␣-glycerophosphate oxidase, only the former exhibited low but significant activity in either class of strains. This activity was independent of the growth phase, suggesting that the protein may serve in vivo as a subunit of the H 2 O 2 -scavenging enzyme NAD(P)H-linked alkylhydroperoxide reductase. The activity of lactate oxidase was associated with the membrane while that of NADH oxidase was in the soluble fraction, findings consistent with their respective physiological roles, i.e., the production and scavenging of H 2 O 2 . Analyses of fermentation end products revealed that the concentration of lactate initially increased with time and decreased on glucose exhaustion, while that of acetate increased during the culture. These results suggest that the lactate oxidase activity of H 2 O 2 -producing cells oxidizes lactate to pyruvate, which is in turn converted to acetate. This latter process proceeds presumably via acetyl coenzyme A and acetyl phosphate with formation of extra ATP.The gram-positive microorganism Streptococcus pyogenes is the causative agent of a variety of important human diseases. These include not only the direct consequences of infections such as pharyngitis, impetigo, cellulitis, necrotizing fasciitis, and toxic-shock-like syndrome but also secondary pathologies called poststreptococcal sequelae. The physiological features of S. pyogenes place it as a member of lactic acid bacteria, which are generally believed to be almost totally dependent for growth on the lactic mode of fermentation under both aerobic and anaerobic conditions.In a number of species of lactic acid bacteria, it has been known that cells growing aerobically produce and excrete copious amounts of H 2 O 2. In a previous study, it was found that S. pyogenes strains could be divided into two classes with respect to the production of H 2 O 2 , i.e., producers and nonproducers (18). However, the metabolic basis and biological significance of bacterial H 2 O 2 production are largely unexplored. The presence of H 2 O 2 -producing oxidases in lactic acid bacteria has been demonstrated, and the possibility of their involvement in this phenomenon has been suggested (6,14,15,21). Notably, a homology search against the S. pyogenes genome database revealed that putative H 2 O 2 -produc...