Hiroshi Nishimasu

The RNA-guided endonuclease Cas9 cleaves its target DNA and is a powerful genome-editing tool. However, the widely used <i>Streptococcus pyogenes</i> 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

The CRISPR-associated endonuclease Cas9 can be targeted to specific genomic loci by single guide RNAs (sgRNAs). Here, we report the crystal structure of Streptococcus pyogenes Cas9 in complex with sgRNA and its target DNA at 2.5&amp;nbsp;Å resolution. The structure revealed a bilobed architecture composed of target recognition and nuclease lobes, accommodating the sgRNA:DNA heteroduplex in a positively charged groove at their interface. Whereas the recognition lobe is essential for binding sgRNA

The CRISPR-associated endonuclease Cas9 binds to a guide RNA and cleaves double-stranded DNA with a sequence complementary to the RNA guide. The Cas9-RNA system has been harnessed for numerous applications, such as genome editing. Here we use high-speed atomic force microscopy (HS-AFM) to visualize the real-space and real-time dynamics of CRISPR-Cas9 in action. HS-AFM movies indicate that, whereas apo-Cas9 adopts unexpected flexible conformations, Cas9-RNA forms a stable bilobed structure and in

In the prokaryotic CRISPR-Cas adaptive immune systems, a CRISPR RNA (crRNA) assembles with multiple or single Cas proteins to form crRNA ribonucleoprotein (crRNP) effector complexes, responsible for the destruction of invading genetic elements. Although the mechanisms of target recognition and cleavage by the crRNP effectors are quite diverse among the different types of CRISPR-Cas systems, the basic action principles of these crRNA-guided effector nucleases are highly conserved. In all of the c

tRNAs from all 3 phylogenetic domains have a 5-methyluridine at position 54 (T54) in the T-loop. The methyl group is transferred from S-adenosylmethionine by TrmA methyltransferase in most Gram-negative bacteria and some archaea and eukaryotes, whereas it is transferred from 5,10-methylenetetrahydrofolate (MTHF) by TrmFO, a folate/FAD-dependent methyltransferase, in most Gram-positive bacteria and some Gram-negative bacteria. However, the catalytic mechanism remains unclear, because the crystal

Hexokinase catalyzes the phosphorylation of glucose to glucose 6-phosphate by using ATP as a phosphoryl donor. Recently, we identified and characterized an ATP-dependent hexokinase (StHK) from the hyperthermophilic archaeon Sulfolobus tokodaii, which can phosphorylate a broad range of sugar substrates, including glucose, mannose, glucosamine, and N-acetylglucosamine. Here we present the crystal structures of StHK in four different forms: (i) apo-form, (ii) binary complex with glucose, (iii) bina