Targeted base editing in rice and tomato using a CRISPR-Cas9 cytidine deaminase fusion
TL;DR: 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 demonstrates the feasibility of base editing for crop improvement.
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.
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TL;DR: This work analyzes key considerations when choosing genome editing agents and identifies opportunities for future improvements and applications in basic research and therapeutics.
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.
1,068 citations
TL;DR: A comprehensive account of the state of the art of base editing of DNA and RNA is provided, including the progressive improvements to methodologies, understanding and avoiding unintended edits, cellular and organismal delivery of editing reagents and diverse applications in research and therapeutic settings.
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.
989 citations
30 Jan 2019
TL;DR: The causes ofClimate change, stresses produced due to climate change, impacts on crops, modern breeding technologies, and biotechnological strategies to cope with climate change are summarized in order to develop climate resilient crops.
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.
742 citations
Cites background from "Targeted base editing in rice and t..."
...Without any transformation of a gene, wheat has been developed with a low gluten level by using CRISPR/Cas9 technique [246]....
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...CRISPR/Cas9 was applied in rice for producing triplet mutants....
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...The SlAGL6 gene was knocked down by Klap et al. (2017) by using the CRISPR/Cas9 system to develop parthenocarpic fruits in response to heat stress....
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...Herbicide tolerance was developed by knocking down PmCDA1 gene in mutant rice lines with the help of CRISPR/Cas9 [250]....
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...CRISPR/Cas9 technology was applied for mutant rice lines, resulting in the enhancement of tolerance under cold stress....
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TL;DR: Fourth-generation base editors (BE4 and SaBE4) are engineered 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, and recommend their use in future efforts.
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.
560 citations
TL;DR: An integrated reagent toolkit and streamlined protocols work across diverse plant species to enable sophisticated genome edits and it is demonstrated that Cas9 nickases induce gene targeting at frequencies comparable to native Cas9 when they are delivered on geminivirus replicons.
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)
418 citations
References
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TL;DR: E engineered fusions of CRISPR/Cas9 and a cytidine deaminase enzyme that retain the ability to be programmed with a guide RNA, do not induce dsDNA breaks, and mediate the direct conversion of cytidine to uridine, thereby effecting a C→T (or G→A) substitution.
Abstract: Current genome-editing technologies introduce double-stranded (ds) DNA breaks at a target locus as the first step to gene correction. Although most genetic diseases arise from point mutations, current approaches to point mutation correction are inefficient and typically induce an abundance of random insertions and deletions (indels) at the target locus resulting from the cellular response to dsDNA breaks. Here we report the development of 'base editing', a new approach to genome editing that enables the direct, irreversible conversion of one target DNA base into another in a programmable manner, without requiring dsDNA backbone cleavage or a donor template. We engineered fusions of CRISPR/Cas9 and a cytidine deaminase enzyme that retain the ability to be programmed with a guide RNA, do not induce dsDNA breaks, and mediate the direct conversion of cytidine to uridine, thereby effecting a C→T (or G→A) substitution. The resulting 'base editors' convert cytidines within a window of approximately five nucleotides, and can efficiently correct a variety of point mutations relevant to human disease. In four transformed human and murine cell lines, second- and third-generation base editors that fuse uracil glycosylase inhibitor, and that use a Cas9 nickase targeting the non-edited strand, manipulate the cellular DNA repair response to favour desired base-editing outcomes, resulting in permanent correction of ~15-75% of total cellular DNA with minimal (typically ≤1%) indel formation. Base editing expands the scope and efficiency of genome editing of point mutations.
3,384 citations
TL;DR: CCTop provides the bench biologist with a tool for the rapid and efficient identification of high quality target sites and was experimentally validated for gene inactivation, non-homologous end-joining as well as homology directed repair.
Abstract: Engineering of the CRISPR/Cas9 system has opened a plethora of new opportunities for site-directed mutagenesis and targeted genome modification. Fundamental to this is a stretch of twenty nucleotides at the 5’ end of a guide RNA that provides specificity to the bound Cas9 endonuclease. Since a sequence of twenty nucleotides can occur multiple times in a given genome and some mismatches seem to be accepted by the CRISPR/Cas9 complex, an efficient and reliable in silico selection and evaluation of the targeting site is key prerequisite for the experimental success. Here we present the CRISPR/Cas9 target online predictor (CCTop, http://crispr.cos.uni-heidelberg.de) to overcome limitations of already available tools. CCTop provides an intuitive user interface with reasonable default parameters that can easily be tuned by the user. From a given query sequence, CCTop identifies and ranks all candidate sgRNA target sites according to their off-target quality and displays full documentation. CCTop was experimentally validated for gene inactivation, non-homologous end-joining as well as homology directed repair. Thus, CCTop provides the bench biologist with a tool for the rapid and efficient identification of high quality target sites.
771 citations
TL;DR: It is shown that only the nuclease but not the nickase is an efficient tool for NHEJ-mediated mutagenesis in plants and the Cas9 nickase has the potential to become an important tool for genome engineering in plants.
Abstract: Engineered nucleases can be used to induce site-specific double-strand breaks (DSBs) in plant genomes Thus, homologous recombination (HR) can be enhanced and targeted mutagenesis can be achieved by error-prone non-homologous end-joining (NHEJ) Recently, the bacterial CRISPR/Cas9 system was used for DSB induction in plants to promote HR and NHEJ Cas9 can also be engineered to work as a nickase inducing single-strand breaks (SSBs) Here we show that only the nuclease but not the nickase is an efficient tool for NHEJ-mediated mutagenesis in plants We demonstrate the stable inheritance of nuclease-induced targeted mutagenesis events in the ADH1 and TT4 genes of Arabidopsis thaliana at frequencies from 25 up to 700% Deep sequencing analysis revealed NHEJ-mediated DSB repair in about a third of all reads in T1 plants In contrast, applying the nickase resulted in the reduction of mutation frequency by at least 740-fold Nevertheless, the nickase is able to induce HR at similar efficiencies as the nuclease or the homing endonuclease I-SceI Two different types of somatic HR mechanisms, recombination between tandemly arranged direct repeats as well as gene conversion using the information on an inverted repeat could be enhanced by the nickase to a similar extent as by DSB-inducing enzymes Thus, the Cas9 nickase has the potential to become an important tool for genome engineering in plants It should not only be applicable for HR-mediated gene targeting systems but also by the combined action of two nickases as DSB-inducing agents excluding off-target effects in homologous genomic regions
622 citations
TL;DR: This work was supported by the National Basic Research Program of China (863 program) ( 2012AA10A304), the Program for New Century Excellent Talents in University ( NCET-13-0807 ), and the Fundamental Research Funds for the Central Universities ( 2013PY004, 2014PY018 ).
524 citations
TL;DR: Analysis of the processing of recombinant FMDV polyproteins shows that a 19 amino acid sequence spanning 2A is sufficient to mediate polyprotein cleavage at a site immediately C-terminal to 2A, whereas deletions extending into the 2A region prevent cleavage.
Abstract: The 2A region of the foot-and-mouth disease virus (FMDV) polyprotein is only 16 amino acids in length. During synthesis of the FMDV polyprotein a primary proteolytic processing event occurs between the 2A and 2B regions of the polyprotein. The activity responsible for this cleavage is not known but it is thought that either an unidentified virus-encoded proteinase may be responsible, or that 2A acts as a substrate for a host cell proteinase. A series of recombinant FMDV polyproteins has been constructed in which sequences to the N- or C-terminal side of the 2A region have been deleted. Analysis of the processing of these polyproteins shows that a 19 amino acid sequence spanning 2A is sufficient to mediate polyprotein cleavage at a site immediately C-terminal to 2A, whereas deletions extending into the 2A region prevent cleavage.
495 citations