Hiroki Komatsu

Bio: Hiroki Komatsu is an academic researcher from University of Tsukuba. The author has contributed to research in topics: Cas9 & Genome editing. The author has an hindex of 1, co-authored 2 publications receiving 400 citations.

Targeted base editing in rice and tomato using a CRISPR-Cas9 cytidine deaminase fusion

Abstract: Targeted editing of single base pairs is achieved in monocot rice and dicot tomato using Target-AID (Cas9 activation-induced cytidine deaminase fusion). We applied a fusion of CRISPR-Cas9 and activation-induced cytidine deaminase (Target-AID) for point mutagenesis at genomic regions specified by single guide RNAs (sgRNAs) in two crop plants. In rice, we induced multiple herbicide-resistance point mutations by multiplexed editing using herbicide selection, while in tomato we generated marker-free plants with homozygous heritable DNA substitutions, demonstrating the feasibility of base editing for crop improvement.

Modification of tomato breeding traits and plant hormone signaling by Target-AID, the genome-editing system inducing efficient nucleotide substitution

Abstract: Abstract Target activation-induced cytidine deaminase (Target-AID), a novel CRISPR/Cas9-based genome-editing tool, confers the base-editing capability on the Cas9 genome-editing system. It involves the fusion of cytidine deaminase (CDA), which catalyzes cytidine (C) to uridine (U) substitutions, to the mutated nickase-type nCas9 or deactivated-type dCas9. To confirm and extend the applicability of the Target-AID genome-editing system in tomatoes (Solanum lycopersicum L.), we transformed the model tomato cultivar “Micro-Tom” and commercial tomato cultivars using this system by targeting SlDELLA, which encodes a negative regulator of the plant phytohormone gibberellic acid (GA) signaling pathway. We confirmed that the nucleotide substitutions were induced by the Target-AID system, and we isolated mutants showing high GA sensitivity in both “Micro-Tom” and the commercial cultivars. Moreover, by successfully applying this system to ETHYLENE RECEPTOR 1 (SlETR1) with single sgRNA targeting, double sgRNA targeting, as well as dual-targeting of both SlETR1 and SlETR2 with a single sgRNA, we demonstrated that the Target-AID genome-editing system is a promising tool for molecular breeding in tomato crops. This study highlights an important aspect of the scientific and agricultural potential of the combinatorial use of the Target-AID and other base-editing systems.

Genome editing in PDS genes of tomatoes by non-selection method and of Nicotiana benthamiana by one single guide RNA to edit two orthologs.

Abstract: The CRISPR/Cas9 system is widely used for targeted mutagenesis in many organisms including plants. For application of this system, tissue culture methods need to be established. In this study, detailed methods for introduction of mutations in tomato and Nicotiana benthamiana plants using the CRISPR/Cas9 system are described. The methods include tissue culture protocols for tomato and N. benthamiana. We also demonstrate the methodology to generate Cas9-free genome edited tomato plants and use of one single guide RNA (sgRNA) to edit two orthologs in N. benthamiana. The examples of editing the PHYTOENE DESATURASE (PDS) genes in these plants are also provided. The Cas9-free tomato line was obtained when tomato plants were cultured on a non-selective medium after transformation with the CRISPR/Cas9 system. Two orthologs of PDS in N. benthamiana were mutated using a sgRNA, because these orthologs contain the same nucleotide sequences with PAM motif. These mutations were inherited to the next generation. The mutations in the PDS genes resulted in an albino phenotype in tomato and N. benthamiana plants. These results demonstrate that the non-selective method is one of the ways to obtain Cas9-free genome editing in tomato plants and that the two orthologs can be edited by one sgRNA in N. benthamiana.

Genome editing with CRISPR–Cas nucleases, base editors, transposases and prime editors

Abstract: The development of new CRISPR-Cas genome editing tools continues to drive major advances in the life sciences. Four classes of CRISPR-Cas-derived genome editing agents-nucleases, base editors, transposases/recombinases and prime editors-are currently available for modifying genomes in experimental systems. Some of these agents have also moved rapidly into the clinic. Each tool comes with its own capabilities and limitations, and major efforts have broadened their editing capabilities, expanded their targeting scope and improved editing specificity. We analyze key considerations when choosing genome editing agents and identify opportunities for future improvements and applications in basic research and therapeutics.

Base editing: precision chemistry on the genome and transcriptome of living cells

Abstract: RNA-guided programmable nucleases from CRISPR systems generate precise breaks in DNA or RNA at specified positions. In cells, this activity can lead to changes in DNA sequence or RNA transcript abundance. Base editing is a newer genome-editing approach that uses components from CRISPR systems together with other enzymes to directly install point mutations into cellular DNA or RNA without making double-stranded DNA breaks. DNA base editors comprise a catalytically disabled nuclease fused to a nucleobase deaminase enzyme and, in some cases, a DNA glycosylase inhibitor. RNA base editors achieve analogous changes using components that target RNA. Base editors directly convert one base or base pair into another, enabling the efficient installation of point mutations in non-dividing cells without generating excess undesired editing by-products. In this Review, we summarize base-editing strategies to generate specific and precise point mutations in genomic DNA and RNA, highlight recent developments that expand the scope, specificity, precision and in vivo delivery of base editors and discuss limitations and future directions of base editing for research and therapeutic applications.

Impact of Climate Change on Crops Adaptation and Strategies to Tackle Its Outcome: A Review.

Abstract: Agriculture and climate change are internally correlated with each other in various aspects, as climate change is the main cause of biotic and abiotic stresses, which have adverse effects on the agriculture of a region. The land and its agriculture are being affected by climate changes in different ways, e.g., variations in annual rainfall, average temperature, heat waves, modifications in weeds, pests or microbes, global change of atmospheric CO2 or ozone level, and fluctuations in sea level. The threat of varying global climate has greatly driven the attention of scientists, as these variations are imparting negative impact on global crop production and compromising food security worldwide. According to some predicted reports, agriculture is considered the most endangered activity adversely affected by climate changes. To date, food security and ecosystem resilience are the most concerning subjects worldwide. Climate-smart agriculture is the only way to lower the negative impact of climate variations on crop adaptation, before it might affect global crop production drastically. In this review paper, we summarize the causes of climate change, stresses produced due to climate change, impacts on crops, modern breeding technologies, and biotechnological strategies to cope with climate change, in order to develop climate resilient crops. Revolutions in genetic engineering techniques can also aid in overcoming food security issues against extreme environmental conditions, by producing transgenic plants.

Improved base excision repair inhibition and bacteriophage Mu Gam protein yields C:G-to-T:A base editors with higher efficiency and product purity.

Abstract: We recently developed base editing, the programmable conversion of target C:G base pairs to T:A without inducing double-stranded DNA breaks (DSBs) or requiring homology-directed repair using engineered fusions of Cas9 variants and cytidine deaminases. Over the past year, the third-generation base editor (BE3) and related technologies have been successfully used by many researchers in a wide range of organisms. The product distribution of base editing-the frequency with which the target C:G is converted to mixtures of undesired by-products, along with the desired T:A product-varies in a target site-dependent manner. We characterize determinants of base editing outcomes in human cells and establish that the formation of undesired products is dependent on uracil N-glycosylase (UNG) and is more likely to occur at target sites containing only a single C within the base editing activity window. We engineered CDA1-BE3 and AID-BE3, which use cytidine deaminase homologs that increase base editing efficiency for some sequences. On the basis of these observations, we engineered fourth-generation base editors (BE4 and SaBE4) that increase the efficiency of C:G to T:A base editing by approximately 50%, while halving the frequency of undesired by-products compared to BE3. Fusing BE3, BE4, SaBE3, or SaBE4 to Gam, a bacteriophage Mu protein that binds DSBs greatly reduces indel formation during base editing, in most cases to below 1.5%, and further improves product purity. BE4, SaBE4, BE4-Gam, and SaBE4-Gam represent the state of the art in C:G-to-T:A base editing, and we recommend their use in future efforts.

A Multipurpose Toolkit to Enable Advanced Genome Engineering in Plants

Abstract: We report a comprehensive toolkit that enables targeted, specific modification of monocot and dicot genomes using a variety of genome engineering approaches Our reagents, based on transcription activator-like effector nucleases (TALENs) and the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system, are systematized for fast, modular cloning and accommodate diverse regulatory sequences to drive reagent expression Vectors are optimized to create either single or multiple gene knockouts and large chromosomal deletions Moreover, integration of geminivirus-based vectors enables precise gene editing through homologous recombination Regulation of transcription is also possible A Web-based tool streamlines vector selection and construction One advantage of our platform is the use of the Csy-type (CRISPR system yersinia) ribonuclease 4 (Csy4) and tRNA processing enzymes to simultaneously express multiple guide RNAs (gRNAs) For example, we demonstrate targeted deletions in up to six genes by expressing 12 gRNAs from a single transcript Csy4 and tRNA expression systems are almost twice as effective in inducing mutations as gRNAs expressed from individual RNA polymerase III promoters Mutagenesis can be further enhanced 25-fold by incorporating the Trex2 exonuclease Finally, we demonstrate that Cas9 nickases induce gene targeting at frequencies comparable to native Cas9 when they are delivered on geminivirus replicons The reagents have been successfully validated in tomato (Solanum lycopersicum), tobacco (Nicotiana tabacum), Medicago truncatula, wheat (Triticum aestivum), and barley (Hordeum vulgare)