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The prokaryote-derived CRISPR–Cas genome editing technology has altered plant molecular biology beyond all expectations. Characterized by robustness and high target specificity and programmability, CRISPR–Cas allows precise genetic manipulation of crop species, which provides the opportunity to create germplasms with beneficial traits and to develop novel, more sustainable agricultural systems. Furthermore, the numerous emerging biotechnologies based on CRISPR–Cas platforms have expanded the toolbox of fundamental research and plant synthetic biology. In this Review, we first briefly describe gene editing by CRISPR–Cas, focusing on the newest, precise gene editing technologies such as base editing and prime editing. We then discuss the most important applications of CRISPR–Cas in increasing plant yield, quality, disease resistance and herbicide resistance, breeding and accelerated domestication. We also highlight the most recent breakthroughs in CRISPR–Cas-related plant biotechnologies, including CRISPR–Cas reagent delivery, gene regulation, multiplexed gene editing and mutagenesis and directed evolution technologies. Finally, we discuss prospective applications of this game-changing technology.The newest CRISPR–Cas genome editing technologies enable precise and simplified formation of crops with increased yield, quality, disease resistance and herbicide resistance, as well as accelerated domestication. Recent breakthroughs in CRISPR–Cas plant biotechnologies improve reagent delivery, gene regulation, multiplexed gene editing and directed evolution.

Gene expression is regulated by multiple processes, and the translation of mRNAs into proteins is an especially critical step. Upstream open reading frames (uORFs) are widespread cis -elements in eukaryotic genes that usually suppress the translation of downstream primary ORFs (pORFs). Here, we describe a protocol for fine-tuning gene translation in plants by editing endogenous uORFs with the CRISPR–Cas9 system. The method we present readily yields transgene-free uorf mutant offspring. We provide detailed protocols for predicting uORFs and testing their effects on downstream pORFs using a dual-luciferase reporter system, designing and constructing single guide RNA (sgRNA)–Cas9 vectors, identifying transgene-free uorf mutants, and finally comparing the mRNA, protein and phenotypic levels of target genes in uorf mutants and controls. Predicting uORFs and confirming their effects in protoplasts takes only 2–3 weeks, and transgene-free mutants with edited target uORFs controlling different levels of pORF translation can be obtained within 4 months. Unlike previous methods, our strategy achieves fine-tuning of gene translation in transgene-free derivatives, which accelerates the analysis of gene function and the improvement of crop traits. In this protocol, the authors describe a method to fine-tune gene expression in plants by editing endogenous upstream ORFs with the CRISPR–Cas9 system to prevent their inhibition of the translation of primary ORFs. This protocol yields transgene-free uorf mutant offspring.

Genome-editing tools provide advanced biotechnological techniques that enable the precise and efficient targeted modification of an organism’s genome. Genome-editing systems have been utilized in a wide variety of plant species to characterize gene functions and improve agricultural traits. We describe the current applications of genome editing in plants, focusing on its potential for crop improvement in terms of adaptation, resilience, and end-use. In addition, we review novel breakthroughs that are extending the potential of genome-edited crops and the possibilities of their commercialization. Future prospects for integrating this revolutionary technology with conventional and new-age crop breeding strategies are also discussed.

Plant agriculture is poised at a technological inflection point. Recent advances in genome engineering make it possible to precisely alter DNA sequences in living cells, providing unprecedented control over a plant's genetic material. Potential future crops derived through genome engineering include those that better withstand pests, that have enhanced nutritional value, and that are able to grow on marginal lands. In many instances, crops with such traits will be created by altering only a few nucleotides among the billions that comprise plant genomes. As such, and with the appropriate regulatory structures in place, crops created through genome engineering might prove to be more acceptable to the public than plants that carry foreign DNA in their genomes. Public perception and the performance of the engineered crop varieties will determine the extent to which this powerful technology contributes towards securing the world's food supply.

Precise genome engineering of a handful of genes enables rapid domestication of wild tomato plants. Crop improvement by inbreeding often results in fitness penalties and loss of genetic diversity. We introduced desirable traits into four stress-tolerant wild-tomato accessions by using multiplex CRISPR–Cas9 editing of coding sequences, cis -regulatory regions or upstream open reading frames of genes associated with morphology, flower and fruit production, and ascorbic acid synthesis. Cas9-free progeny of edited plants had domesticated phenotypes yet retained parental disease resistance and salt tolerance.

Cytosine and adenine base editors (CBEs and ABEs) are promising new tools for achieving the precise genetic changes required for disease treatment and trait improvement. However, genome-wide and unbiased analyses of their off-target effects in vivo are still lacking. Our whole-genome sequencing analysis of rice plants treated with the third-generation base editor (BE3), high-fidelity BE3 (HF1-BE3), or ABE revealed that BE3 and HF1-BE3, but not ABE, induce substantial genome-wide off-target mutations, which are mostly the C→T type of single-nucleotide variants (SNVs) and appear to be enriched in genic regions. Notably, treatment of rice with BE3 or HF1-BE3 in the absence of single-guide RNA also results in the rise of genome-wide SNVs. Thus, the base-editing unit of BE3 or HF1-BE3 needs to be optimized in order to attain high fidelity.

Nucleotide base editors in plants have been limited to conversion of cytosine to thymine. Here, we describe a new plant adenine base editor based on an evolved tRNA adenosine deaminase fused to the nickase CRISPR/Cas9, enabling A•T to G•C conversion at frequencies up to 7.5% in protoplasts and 59.1% in regenerated rice and wheat plants. An endogenous gene is also successfully modified through introducing a gain-of-function point mutation to directly produce an herbicide-tolerant rice plant. With this new adenine base editing system, it is now possible to precisely edit all base pairs, thus expanding the toolset for precise editing in plants.

Editing plant genomes is technically challenging in hard-to-transform plants and usually involves transgenic intermediates, which causes regulatory concerns. Here we report two simple and efficient genome-editing methods in which plants are regenerated from callus cells transiently expressing CRISPR/Cas9 introduced as DNA or RNA. This transient expression-based genome-editing system is highly efficient and specific for producing transgene-free and homozygous wheat mutants in the T0 generation. We demonstrate our protocol to edit genes in hexaploid bread wheat and tetraploid durum wheat, and show that we are able to generate mutants with no detectable transgenes. Our methods may be applicable to other plant species, thus offering the potential to accelerate basic and applied plant genome-engineering research. Plant genome editing typically relies upon transgenic intermediates, which is a concern given the current regulatory requirements concerning GMOs. Here, Zhang et al . describe a method to edit wheat genomes by transiently expressing CRISPR/Cas9 DNA or RNA, and are able to generate mutant plants with no detectable transgenes.

Prime editors, which are CRISPR–Cas9 nickase (H840A)–reverse transcriptase fusions programmed with prime editing guide RNAs (pegRNAs), can edit bases in mammalian cells without donor DNA or double-strand breaks. We adapted prime editors for use in plants through codon, promoter, and editing-condition optimization. The resulting suite of plant prime editors enable point mutations, insertions and deletions in rice and wheat protoplasts. Regenerated prime-edited rice plants were obtained at frequencies of up to 21.8%. Prime editors are optimized for use in plants.

TALEN-induced mutation of all homologous copies of a gene that represses resistance to an important wheat pathogen confers a trait that has eluded plant breeders for decades. Sequence-specific nucleases have been applied to engineer targeted modifications in polyploid genomes 1 , but simultaneous modification of multiple homoeoalleles has not been reported. Here we use transcription activator–like effector nuclease (TALEN) 2 , 3 and clustered, regularly interspaced, short palindromic repeats (CRISPR)-Cas9 (refs. 4 , 5 ) technologies in hexaploid bread wheat to introduce targeted mutations in the three homoeoalleles that encode MILDEW-RESISTANCE LOCUS (MLO) proteins 6 . Genetic redundancy has prevented evaluation of whether mutation of all three MLO alleles in bread wheat might confer resistance to powdery mildew, a trait not found in natural populations 7 . We show that TALEN-induced mutation of all three TaMLO homoeologs in the same plant confers heritable broad-spectrum resistance to powdery mildew. We further use CRISPR-Cas9 technology to generate transgenic wheat plants that carry mutations in the TaMLO-A1 allele. We also demonstrate the feasibility of engineering targeted DNA insertion in bread wheat through nonhomologous end joining of the double-strand breaks caused by TALENs. Our findings provide a methodological framework to improve polyploid crops.