Kiyotaka Y. Hara

Bio: Kiyotaka Y. Hara is an academic researcher from University of Shizuoka. The author has contributed to research in topics: Glutathione & Metabolic engineering. The author has an hindex of 24, co-authored 55 publications receiving 2725 citations. Previous affiliations of Kiyotaka Y. Hara include Tokyo Institute of Technology & Kobe University.

Targeted nucleotide editing using hybrid prokaryotic and vertebrate adaptive immune systems

Abstract: INTRODUCTION To combat invading pathogens, cells develop an adaptive immune response by changing their own genetic information. In vertebrates, the generation of genetic variation (somatic hypermutation) is an essential process for diversification and affinity maturation of antibodies that function to detect and sequester various foreign biomolecules. The activation-induced cytidine deaminase (AID) carries out hypermutation by modifying deoxycytidine bases in the variable region of the immunoglobulin locus that produces antibody. AID-generated deoxyuridine in DNA is mutagenic as it can be miss-recognized as deoxythymine, resulting in C to T mutations. CRISPR (clustered regularly interspaced short palindromic repeats)/Cas (CRISPR-associated) is a prokaryotic adaptive immune system that records and degrades invasive foreign DNA or RNA. The CRISPR/Cas system cleaves and incorporates foreign DNA/RNA segments into the genomic region called the CRISPR array. The CRISPR array is transcribed to produce crispr-RNA that serves as guide RNA (gRNA) for recognition of the complementary foreign DNA/RNA in a ribonucleoprotein complex with Cas proteins, which degrade the target. The CRISPR/Cas system has been repurposed as a powerful genome editing tool, because it can be programmed to cleave specific DNA sequence by providing custom gRNAs. RATIONALE Although the precise mechanism by which AID specifically mutates the immunoglobulin locus remains elusive, targeting of AID activity is facilitated by the formation of a single-stranded DNA region, such as a transcriptional RNA/DNA hybrid (R-loop). The CRISPR/Cas system can be engineered to be nuclease-inactive. The nuclease-inactive form is capable of unfolding the DNA double strand in a protospacer adjacent motif (PAM) sequence-dependent manner so that the gRNA binds to complementary target DNA strand and forms an R-loop. The nuclease-deficient CRISPR/Cas system may serve as a suitable DNA-targeting module for AID to catalyze site-specific mutagenesis. RESULTS To determine whether AID activity can be specifically targeted by the CRISPR/Cas system, we combined dCas9 (a nuclease-deficient mutant of Cas9) from Streptococcus pyogenes and an AID ortholog, PmCDA1 from sea lamprey, to form a synthetic complex (Target-AID) by either engineering a fusion between the two proteins or attaching a SH3 (Src 3 homology) domain to the C terminus of dCas9 and a SHL (SH3 interaction ligand) to the C terminus of PmCDA1. Both of these complexes performed highly efficient site-directed mutagenesis. The mutational spectrum was analyzed in yeast and demonstrated that point mutations were dominantly induced at cytosines within the range of three to five bases surrounding the –18 position upstream of the PAM sequence on the noncomplementary strand to gRNA. The toxicity associated with the nuclease-based CRISPR/Cas9 system was greatly reduced in the Target-AID complexes. Combination of PmCDA1 with the nickase Cas9(D10A) mutant, which retains cleavage activity for noncomplementary single-stranded DNA, was more efficient in yeast but also induced deletions as well as point mutations in mammalian cells. Addition of the uracil DNA glycosylase inhibitor protein, which blocks the initial step of the uracil base excision repair pathway, suppressed collateral deletions and further improved targeting efficiency. Potential off-target effects were assessed by whole-genome sequencing of yeast as well as deep sequencing of mammalian cells for regions that contain mismatched target sequences. These results showed that off-target effects were comparable to those of conventional CRISPR/Cas systems, with a reduced risk of indel formation. CONCLUSION By expanding the genome editing potential of the CRISPR/Cas9 system by deaminase-mediated hypermutation, Target-AID demonstrated a very narrow range of targeted nucleotide substitution without the use of template DNA. Nickase Cas9 and uracil DNA glycosylase inhibitor protein can be used to boost the targeting efficiency. The reduced cytotoxicity will be beneficial for use in cells that are sensitive to artificial nucleases. Use of other types of nucleotide-modifying enzymes and/or other CRISPR-related systems with different PAM requirements will expand our genome-editing repertoire and capacity.

Catalysis and rotation of F1 motor: Cleavage of ATP at the catalytic site occurs in 1 ms before 40° substep rotation

Abstract: F1, a water-soluble portion of FoF1-ATP synthase, is an ATP hydrolysis-driven rotary motor. The central γ-subunit rotates in the α3β3 cylinder by repeating the following four stages of rotation: ATP-binding dwell, rapid 80° substep rotation, interim dwell, and rapid 40° substep rotation. At least two 1-ms catalytic events occur in the interim dwell, but it is still unclear which steps in the ATPase cycle, except for ATP binding, correspond to these events. To discover which steps, we analyzed rotations of F1 subcomplex (α3β3γ) from thermophilic Bacillus PS3 under conditions where cleavage of ATP at the catalytic site is decelerated: hydrolysis of ATP by the catalytic-site mutant F1 and hydrolysis of a slowly hydrolyzable substrate ATPγS (adenosine 5′-[γ-thio]triphosphate) by wild-type F1. In both cases, interim dwells were extended as expected from bulk phase kinetics, confirming that cleavage of ATP takes place during the interim dwell. Furthermore, the results of ATPγS hydrolysis by the mutant F1 ensure that cleavage of ATP most likely corresponds to one of the two 1-ms events and not some other faster undetected event. Thus, cleavage of ATP on F1 occurs in 1 ms during the interim dwell, and we call this interim dwell catalytic dwell.

Pause and rotation of F(1)-ATPase during catalysis.

Abstract: F1-ATPase is a rotary motor enzyme in which a single ATP molecule drives a 120° rotation of the central γ subunit relative to the surrounding α3β3 ring. Here, we show that the rotation of F1-ATPase spontaneously lapses into long (≈30 s) pauses during steady-state catalysis. The effects of ADP-Mg and mutation on the pauses, as well as kinetic comparison with bulk-phase catalysis, strongly indicate that the paused enzyme corresponds to the inactive state of F1-ATPase previously known as the ADP-Mg inhibited form in which F1-ATPase fails to release ADP-Mg from catalytic sites. The pausing position of the γ subunit deviates from the ATP-waiting position and is most likely the recently found intermediate 90° position.

Sequence and comparative analysis of the chicken genome provide unique perspectives on vertebrate evolution

Abstract: We present here a draft genome sequence of the red jungle fowl, Gallus gallus. Because the chicken is a modern descendant of the dinosaurs and the first non-mammalian amniote to have its genome sequenced, the draft sequence of its genome--composed of approximately one billion base pairs of sequence and an estimated 20,000-23,000 genes--provides a new perspective on vertebrate genome evolution, while also improving the annotation of mammalian genomes. For example, the evolutionary distance between chicken and human provides high specificity in detecting functional elements, both non-coding and coding. Notably, many conserved non-coding sequences are far from genes and cannot be assigned to defined functional classes. In coding regions the evolutionary dynamics of protein domains and orthologous groups illustrate processes that distinguish the lineages leading to birds and mammals. The distinctive properties of avian microchromosomes, together with the inferred patterns of conserved synteny, provide additional insights into vertebrate chromosome architecture.

Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage

Abstract: The spontaneous deamination of cytosine is a major source of transitions from C•G to T•A base pairs, which account for half of known pathogenic point mutations in humans. The ability to efficiently convert targeted A•T base pairs to G•C could therefore advance the study and treatment of genetic diseases. The deamination of adenine yields inosine, which is treated as guanine by polymerases, but no enzymes are known to deaminate adenine in DNA. Here we describe adenine base editors (ABEs) that mediate the conversion of A•T to G•C in genomic DNA. We evolved a transfer RNA adenosine deaminase to operate on DNA when fused to a catalytically impaired CRISPR-Cas9 mutant. Extensive directed evolution and protein engineering resulted in seventh-generation ABEs that convert targeted A•T base pairs efficiently to G•C (approximately 50% efficiency in human cells) with high product purity (typically at least 99.9%) and low rates of indels (typically no more than 0.1%). ABEs introduce point mutations more efficiently and cleanly, and with less off-target genome modification, than a current Cas9 nuclease-based method, and can install disease-correcting or disease-suppressing mutations in human cells. Together with previous base editors, ABEs enable the direct, programmable introduction of all four transition mutations without double-stranded DNA cleavage.

Multimodal fast optical interrogation of neural circuitry

Abstract: Our understanding of the cellular implementation of systems-level neural processes like action, thought and emotion has been limited by the availability of tools to interrogate specific classes of neural cells within intact, living brain tissue. Here we identify and develop an archaeal light-driven chloride pump (NpHR) from Natronomonas pharaonis for temporally precise optical inhibition of neural activity. NpHR allows either knockout of single action potentials, or sustained blockade of spiking. NpHR is compatible with ChR2, the previous optical excitation technology we have described, in that the two opposing probes operate at similar light powers but with well-separated action spectra. NpHR, like ChR2, functions in mammals without exogenous cofactors, and the two probes can be integrated with calcium imaging in mammalian brain tissue for bidirectional optical modulation and readout of neural activity. Likewise, NpHR and ChR2 can be targeted together to Caenorhabditis elegans muscle and cholinergic motor neurons to control locomotion bidirectionally. NpHR and ChR2 form a complete system for multimodal, high-speed, genetically targeted, all-optical interrogation of living neural circuits.

Artificial Molecular Machines

Abstract: The widespread use of molecular machines in biology has long suggested that great rewards could come from bridging the gap between synthetic molecular systems and the machines of the macroscopic world. In the last two decades, it has proved possible to design synthetic molecular systems with architectures where triggered large amplitude positional changes of submolecular components occur. Perhaps the best way to appreciate the technological potential of controlled molecular-level motion is to recognize that nanomotors and molecular-level machines lie at the heart of every significant biological process. Over billions of years of evolution, nature has not repeatedly chosen this solution for performing complex tasks without good reason. When mankind learns how to build artificial structures that can control and exploit molecular level motion and interface their effects directly with other molecular-level substructures and the outside world, it will potentially impact on every aspect of functional molecule and materials design. An improved understanding of physics and biology will surely follow. The first steps on the long path to the invention of artificial molecular machines were arguably taken in 1827 when the Scottish botanist Robert Brown observed the haphazard motion of tiny particles under his microscope.1,2 The explanation for Brownian motion, that it is caused by bombardment of the particles by molecules as a consequence of the kinetic theory of matter, was later provided by Einstein, followed by experimental verification by Perrin.3,4 The random thermal motion of molecules and its implications for the laws of thermodynamics in turn inspired Gedankenexperiments (“thought experiments”) that explored the interplay (and apparent paradoxes) of Brownian motion and the Second Law of Thermodynamics. Richard Feynman’s famous 1959 lecture “There’s plenty of room at the bottom” outlined some of the promise that manmade molecular machines might hold.5,6 However, Feynman’s talk came at a time before chemists had the necessary synthetic and analytical tools to make molecular machines. While interest among synthetic chemists began to grow in the 1970s and 1980s, progress accelerated in the 1990s, particularly with the invention of methods to make mechanically interlocked molecular systems (catenanes and rotaxanes) and control and switch the relative positions of their components.7−24 Here, we review triggered large-amplitude motions in molecular structures and the changes in properties these can produce. We concentrate on conformational and configurational changes in wholly covalently bonded molecules and on catenanes and rotaxanes in which switching is brought about by various stimuli (light, electrochemistry, pH, heat, solvent polarity, cation or anion binding, allosteric effects, temperature, reversible covalent bond formation, etc.). Finally, we discuss the latest generations of sophisticated synthetic molecular machine systems in which the controlled motion of subcomponents is used to perform complex tasks, paving the way to applications and the realization of a new era of “molecular nanotechnology”. 1.1. The Language Used To Describe Molecular Machines Terminology needs to be properly and appropriately defined and these meanings used consistently to effectively convey scientific concepts. Nowhere is the need for accurate scientific language more apparent than in the field of molecular machines. Much of the terminology used to describe molecular-level machines has its origins in observations made by biologists and physicists, and their findings and descriptions have often been misinterpreted and misunderstood by chemists. In 2007 we formalized definitions of some common terms used in the field (e.g., “machine”, “switch”, “motor”, “ratchet”, etc.) so that chemists could use them in a manner consistent with the meanings understood by biologists and physicists who study molecular-level machines.14 The word “machine” implies a mechanical movement that accomplishes a useful task. This Review concentrates on systems where a stimulus triggers the controlled, relatively large amplitude (or directional) motion of one molecular or submolecular component relative to another that can potentially result in a net task being performed. Molecular machines can be further categorized into various classes such as “motors” and “switches” whose behavior differs significantly.14 For example, in a rotaxane-based “switch”, the change in position of a macrocycle on the thread of the rotaxane influences the system only as a function of state. Returning the components of a molecular switch to their original position undoes any work done, and so a switch cannot be used repetitively and progressively to do work. A “motor”, on the other hand, influences a system as a function of trajectory, meaning that when the components of a molecular motor return to their original positions, for example, after a 360° directional rotation, any work that has been done is not undone unless the motor is subsequently rotated by 360° in the reverse direction. This difference in behavior is significant; no “switch-based” molecular machine can be used to progressively perform work in the way that biological motors can, such as those from the kinesin, myosin, and dynein superfamilies, unless the switch is part of a larger ratchet mechanism.14