The most popular CRISPR-SpCas9 system recognizes canonical NGG protospacer adjacent motifs (PAMs). Previously engineered SpCas9 variants, such as Cas9-NG, favor G-rich PAMs in genome editing. In this ...manuscript, we describe a new plant genome-editing system based on a hybrid iSpyMacCas9 platform that allows for targeted mutagenesis, C to T base editing, and A to G base editing at A-rich PAMs. This study fills a major technology gap in the CRISPR-Cas9 system for editing NAAR PAMs in plants, which greatly expands the targeting scope of CRISPR-Cas9. Finally, our vector systems are fully compatible with Gateway cloning and will work with all existing single-guide RNA expression systems, facilitating easy adoption of the systems by others. We anticipate that more tools, such as prime editing, homology-directed repair, CRISPR interference, and CRISPR activation, will be further developed based on our promising iSpyMacCas9 platform.
This study reports the development of an iSpyMacCas9 genome-editing toolbox for targeted mutagenesis, C to T base editing, and A to G base editing at A-rich PAM sites. While the tools are demonstrated in rice, they are expected to aid genome-editing applications in other plant species as well with a broadened targeting scope.
MAD7 is an engineered nuclease of the Class 2 type V-A CRISPR-Cas (Cas12a/Cpf1) family with a low level of homology to canonical Cas12a nucleases. It has been publicly released as a royalty-free ...nuclease for both academic and commercial use. Here, we demonstrate that the CRISPR-MAD7 system can be used for genome editing and recognizes T-rich PAM sequences (YTTN) in plants. Its editing efficiency in rice and wheat is comparable to that of the widely used CRISPR-LbCas12a system. We develop two variants, MAD7-RR and MAD7-RVR that increase the target range of MAD7, as well as an M-AFID (a MAD7-APOBEC fusion-induced deletion) system that creates predictable deletions from 5′-deaminated Cs to the MAD7-cleavage site. Moreover, we show that MAD7 can be used for multiplex gene editing and that it is effective in generating indels when combined with other CRISPR RNA orthologs. Using the CRISPR-MAD7 system, we have obtained regenerated mutant rice and wheat plants with up to 65.6% efficiency.
Prime editing is an adaptation of the CRISPR-Cas system that uses a Cas9(H840A)-reverse transcriptase fusion and a guide RNA amended with template and primer binding site sequences to achieve ...RNA-templated conversion of the target DNA, allowing specified substitutions, insertions, and deletions. In the first report of prime editing in plants, a variety of edits in rice and wheat were described, including insertions up to 15 bp. Several studies in rice quickly followed, but none reported a larger insertion. Here, we report easy-to-use vectors for prime editing in dicots as well as monocots, their validation in
, rice, and Arabidopsis, and an insertion of 66 bp that enabled split-GFP fluorescent tagging.
The virus-induced genome editing (VIGE) system aims to induce targeted mutations in seeds without requiring any tissue culture. Here, we show that tobacco rattle virus (TRV) harboring guide RNA ...(gRNA) edits germ cells in a wild tobacco,
, that expresses
Cas9 (SpCas9). We first generated
transgenic plants expressing SpCas9 under the control of 35S promoter and infected rosette leaves with TRV carrying gRNA. Gene-edited seeds were not found in the progeny of the infected
. Next, the
ribosomal protein S5 A (RPS5A) promoter fused to SpCas9 was employed to induce the heritable gene editing with TRV. The RPS5A promoter-driven SpCas9 successfully produced monoallelic mutations at three target genes in
seeds with TRV-delivered guide RNA. These monoallelic mutations were found in 2%-6% seeds among M
progenies. This editing method provides an alternative way to increase the heritable editing efficacy of VIGE.
Facing the challenges of the world's food sources posed by a growing global population and a warming climate will require improvements in plant breeding and technology. Enhancing crop resiliency and ...yield via genome engineering will undoubtedly be a key part of the solution. The advent of new tools, such as CRIPSR/Cas, has ushered in significant advances in plant genome engineering. However, several serious challenges remain in achieving this goal. Among them are efficient transformation and plant regeneration for most crop species, low frequency of some editing applications, and high attrition rates. On March 8 and 9, 2021, experts in plant genome engineering and breeding from academia and industry met virtually for the Keystone eSymposium “Plant Genome Engineering: From Lab to Field” to discuss advances in genome editing tools, plant transformation, plant breeding, and crop trait development, all vital for transferring the benefits of novel technologies to the field.
Facing the challenges of the world's food sources posed by a growing global population and a warming climate will require improvements in plant breeding and technology. The advent of new tools, such as CRIPSR/Cas, has ushered in significant advances in plant genome engineering. However, several serious challenges remain in achieving this goal. Among them are efficient transformation and plant regeneration for most crop species, low frequency of some editing applications, and high attrition rates. On Demand content: https://keysym.us/EK25NYAS
Plant genome editing has the potential to become another chapter in the intractable debate that has dogged agricultural biotechnology. In 2016, 107 Nobel Laureates accused Greenpeace of emotional and ...dogmatic campaigning against agricultural biotechnology and called for governments to defy such campaigning. The Laureates invoke the authority of science to argue that Greenpeace is putting lives at risk by opposing agricultural biotechnology and Golden Rice and is notable in framing Greenpeace as unethical and its views as marginal. This paper examines environmental, food and farming NGOs’ social and ethical concerns about genome editing, situating these concerns in comparison to alternative ethical assessments provided by the Nuffield Council on Bioethics, a key actor in this policy debate. In doing so, we show that participant NGOs and the Nuffield Council on Bioethics share considerable concerns about the social and ethical implications of genome editing. These concerns include choices over problem/solution framing and broader terminology, implications of regulatory and research choices on consumer choice and relations of power. However, GM-engaged NGOs and the Nuffield Council on Bioethics diverge on one important area: the NGOs seek to challenge the existing order and broaden the scope of debate to include deeply political questions regarding agricultural and technological choices. This distinction between the ethical positions means that NGOs provide valuable ethical insight and a useful lens to open up debate and discussion on the role of emerging technologies, such as genome editing, and the future of agriculture and food sovereignty.
CRISPR has brought a revolutionary impetus to the field of life sciences. The abundance of CRISPR arrays found in nature and the programmability of Cas nucleases have catalyzed the development of ...gene editing tools based on CRISPR. CRISPR technology has seen applications in agriculture and has greatly accelerated the process of crop breeding. We review a number of CRISPR-based genome editing approaches having been used in plants. Then we present the contributions these approaches have made to the breeding of crops resistant to abiotic stresses that prominently decline crop production. The principles, characteristics and scope of application of different gene modification methods are summarized and compared. Regarding abiotic stress-related genes, we respectively introduced what strategies can be used for the two types of genes, abiotic stress-sensitive and -tolerance gene, to obtain resistant crops. Besides, we summarized the application of precision genome editing in abiotic stress, including CRISPR-based NHEJ, HDR and base-editing methods. We also point out the technical bottlenecks faced by CRISPR-based breeding of abiotic stress-resistant crops and discuss the potential applications of CRISPR in crop improvement.
The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR‐associated protein 9 (Cas9) system is a fast‐growing, genome editing technology that has wide applications in identifying ...gene functions as well as improving agricultural production and crop breeding. Here, we summarized recent advances in the development and applications of genome editing technologies in plants. We briefly described CRISPR/Cas9 technology and examined the base and prime editing techniques that have been developed from CRISPR technology. Some new prime editing‐derived techniques were assessed.
The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR‐associated protein 9 (Cas9) system is a fast‐growing, genome editing technology that has wide applications in identifying gene functions as well as improving agricultural production and crop breeding. Here, we summarized recent advances in the development and applications of genome editing technologies in plants. We briefly described CRISPR/Cas9 technology and examined the base and prime editing techniques that have been developed from CRISPR technology. Some new prime editing‐derived techniques were assessed.
In virus‐induced gene‐editing system, subgenomic promoters have been used to express guide RNAs (gRNAs). However, the transcription initiation site of the subgenomic promoters remains elusive. Here, ...we examined the sequence of gRNAs expressed by subgenomic promoters and found the variable length of overhangs at 5′‐end of gRNAs. The overhangs at 5′‐end of gRNA decrease the cleavage activity of SpCas9. To overcome this problem, we inserted hammerhead ribozyme between the subgenomic promoter and gRNA and confirmed that gRNAs with a precise 5′‐end increase the editing efficacy in wild tobacco. This system will be widely used for editing target genes in plants with high efficiency.
Graphical and Lay Summary
In virus‐induced genome editing system, subgenomic promoters of virus have been used to express guide RNAs (gRNAs). In this study, we inserted hammerhead (HH) ribozyme sequence between the subgenomic promoter and gRNA to express gRNA with a precise 5′‐end. Furthermore, we showed that ribozyme‐processed gRNAs increase the mutation frequency in wild tobacco. RdRP, RNA‐dependent RNA polymerase.
Plant genome editing, particularly CRISPR-Cas biotechnologies, has rapidly evolved and drawn enormous attention all around the world in the last decade. The cutting-edge technologies have had ...substantial impact on precise genome editing for manipulating gene expression, stacking gene mutations, and improving crop agronomic traits. Following the global trends, investigations on CRISPR-Cas have been thriving in Australia, especially in agriculture sciences. Importantly, CRISPR-edited plants, classified as SDN-1 organisms (SDN: site-directed nuclease), have been given a green light in Australia, with regulatory bodies indicating they will not be classified as a genetically modified organism (GMO) if no foreign DNA is present in an edited plant. As a result, genome-edited products would not attract the onerous regulation required for the introduction of a GMO, which could mean more rapid deployment of new varieties and products that could be traded freely in Australia, and potentially to export markets. In the present review, we discuss the current status and prospects of plant genome editing in Australia by highlighting several species of interest. Using these species as case studies, we discuss the priorities and potential of plant genome editing, as well as the remaining challenges.
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