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.
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The authors apologize to those colleagues whose work was not cited due to restrictions on the number of references. This work was supported by grants from the National Natural Science Foundation of China (31788103), the Strategic Priority Research Program of the Chinese Academy of Sciences (Precision Seed Design and Breeding, XDA24020102) and the Chinese Academy of Sciences (QYZDY-SSW-SMC030).
The authors declare no competing interests.
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- Green revolution
Refers to a great increase in crop production in the second half of the twentieth century through the use of fertilizers, the sue of agrochemicals, cultivation of high-yield crop varieties and mechanization.
A nucleic acid (single-stranded DNA, double-stranded DNA or RNA) that has some homology with the region flanking a CRISPR–Cas-generated DNA break that can serve as a template during homology-directed repair.
- Gene targeting
A genome editing technology that creates genome modifications, such as gene substitutions, insertions and deletions, through homology-directed repair.
- Cas9 nickase
(nCas9). A term for catalytically defective Cas9 variants that cut only one strand of the target DNA; they include Cas9 bearing the mutations D10A or H840A, which cut the target strand and non-target strand, respectively.
- Base transitions
Single-nucleotide changes that substitute one pyrimidine for another or one purine for another.
- Single guide RNA
(sgRNA). An artificial fusion of CRISPR RNA and trans-activating CRISPR RNA, which guides Cas9 to the target site through DNA–RNA recognition.
A nucleic acid structure formed when the Cas9–single guide RNA (sgRNA) complex invades the target DNA and the sgRNA forms a DNA–RNA hybrid with the target strand while displacing the non-target strand.
- Editing windows
Regions of the target DNA in which base substitutions are induced by base editors; the window is usually numbered in ascending order from the distal end of the protospacer adjacent motif.
A two- to six-nucleotide sequence within the guide RNA that determines the target site of CRISPR–Cas. It is located at the 5′ terminus of the single guide RNA in Cas9 and the 3′ terminus of the CRISPR RNA in Cas12a.
- Protospacer adjacent motif
(PAM). The DNA motif flanking the target sequence, which is indispensable for target recognition and cleavage by CRISPR–Cas. For Streptococcus pyogenes Cas9, the PAM is 5′-NGG-3′.
- Editing scope
The length of the genomic sequence that can be targeted for editing given the requirements of the particular protospacer adjacent motif.
- sgRNA scaffold
(scRNA). A single guide RNA harbouring RNA aptamer hairpins in its tetraloop, stem loop 2 or 3′ end.
- Directed evolution
A protein engineering method that generates user-defined proteins or DNA by mimicking the process of natural selection.
- Hybrid vigour
The phenomenon of heterozygotes formed from homozygous parents often exhibiting better agronomic performance than either parent.
Situations in which female and male gametes are both fertile but the pollen cannot germinate on stigmas with the same or a similar genotype.
- Orphan crops
Crops cultivated and consumed regionally, which are generally not fully domesticated and are especially essential in developing countries.
Particles with specific nanoscale structures that can load biomacromolecules and deliver them into intact plant cells.
- Polyethylene glycol
A high molecular weight polymer that enables uptake by plant protoplasts of biomacromolecules, including DNA, RNA and protein.
- RNA aptamers
RNA oligonucleotides that form a secondary structure to bind a specific protein with high specificity and affinity.
- CRISPR library
A high-throughput tool for functional genomics studies, comprising a collection of single guide RNAs or CRISPR RNAs, which target a set of predefined loci.
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Zhu, H., Li, C. & Gao, C. Applications of CRISPR–Cas in agriculture and plant biotechnology. Nat Rev Mol Cell Biol 21, 661–677 (2020). https://doi.org/10.1038/s41580-020-00288-9
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