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.

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

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.

Applications of CRISPR–Cas in agriculture and plant biotechnology

Genome engineering for crop improvement and future agriculture

Enhanced prime editing systems by manipulating cellular determinants of editing outcomes

The rapid expansion of the CRISPR toolbox through tagging effector domains to either enzymatically inactive Cas9 (dCas9) or Cas9 nickase (nCas9) has led to several promising new gene editing strategies. Recent additions include CRISPR cytosine or adenine base editors (CBEs and ABEs) and the CRISPR prime editors (PEs), in which a deaminase or reverse transcriptase are fused to nCas9, respectively. These tools hold great promise to model and correct disease-causing mutations in animal and plant models. But so far, no widely-available tools exist to automate the design of both BE and PE reagents. We developed PnB Designer, a web-based application for the design of pegRNAs for PEs and guide RNAs for BEs. PnB Designer makes it easy to design targeting guide RNAs for single or multiple targets on a variant or reference genome from organisms spanning multiple kingdoms. With PnB Designer, we designed pegRNAs to model all known disease causing mutations available in ClinVar. Additionally, PnB Designer can be used to design guide RNAs to install or revert a SNV, scanning the genome with one CBE and seven different ABE PAM variants and returning the best BE to use. PnB Designer is publicly accessible at http://fgcz-shiny.uzh.ch/PnBDesigner/
 With PnB Designer we created a user-friendly design tool for CRISPR PE and BE reagents, which should simplify choosing editing strategy and avoiding design complications.

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PnB Designer: a web application to design prime and base editor guide RNAs for animals and plants.

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 genome editing in rice and wheat

Short insertions and deletions can be produced in plant genomes using CRISPR-Cas editors, but reliable production of larger deletions in specific target sites has proven difficult to achieve We report the development of a series of APOBEC-Cas9 fusion-induced deletion systems (AFIDs) that combine Cas9 with human APOBEC3A (A3A), uracil DNA-glucosidase and apurinic or apyrimidinic site lyase In rice and wheat, AFID-3 generated deletions from 5'-deaminated C bases to the Cas9-cleavage site Approximately one-third of deletions produced using AFID-3 in rice and wheat protoplasts (302%) and regenerated plants (348%) were predictable We show that eAFID-3, in which the A3A in AFID-3 is replaced with truncated APOBEC3B (A3Bctd), produced more uniform deletions from the preferred TC motif to the double-strand break AFIDs could be applied to study regulatory regions and protein domains to improve crop plants

Precise, predictable multi-nucleotide deletions in rice and wheat using APOBEC-Cas9.

Antagonistic control of seed dormancy in rice by two bHLH transcription factors

A precise and efficient adenine base editor.

Provided is a gene editing system for editing a target sequence in the genome of a cell, comprising a CRISPR nuclease, a cytosine deaminase, an AP lyase, a guide RNA and optionally an uracil-DNA glycosylase. Also provided are a method of producing a genetically modified cell, and a kit comprising the gene editing system.

Shengxing Wang

Papers

Prime genome editing in rice and wheat

Precise, predictable multi-nucleotide deletions in rice and wheat using APOBEC-Cas9.

Antagonistic control of seed dormancy in rice by two bHLH transcription factors

A precise and efficient adenine base editor.

Improved gene editing system