Qiupeng Lin

Bio: Qiupeng Lin is an academic researcher from Chinese Academy of Sciences. The author has contributed to research in topics: Genome & Genome editing. The author has an hindex of 8, co-authored 10 publications receiving 413 citations.

Prime genome editing in rice and wheat

Abstract: 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%.

High-efficiency prime editing with optimized, paired pegRNAs in plants.

Abstract: Prime editing (PE) applications are limited by low editing efficiency. Here we show that designing prime binding sites with a melting temperature of 30 °C leads to optimal performance in rice and that using two prime editing guide (peg) RNAs in trans encoding the same edits substantially enhances PE efficiency. Together, these approaches boost PE efficiency from 2.9-fold to 17.4-fold. Optimal pegRNAs or pegRNA pairs can be designed with our web application, PlantPegDesigner. Improved guide RNAs enhance the efficiency of prime editing.

Prime editing efficiently generates W542L and S621I double mutations in two ALS genes in maize.

Abstract: Prime editing is a novel and universal CRISPR/Cas-derived precision genome-editing technology that has been recently developed. However, low efficiency of prime editing has been shown in transgenic rice lines. We hypothesize that enhancing pegRNA expression could improve prime-editing efficiency. In this report, we describe two strategies for enhancing pegRNA expression. We construct a prime editing vector harboring two pegRNA variants for W542L and S621I double mutations in ZmALS1 and ZmALS2. Compared with previous reports in rice, we achieve much higher prime-editing efficiency in maize. Our results are inspiring and provide a direction for the optimization of plant prime editors.

Fine-tuning the amylose content of rice by precise base editing of the Wx gene.

Abstract: The genetic diversity and phenotypic variability of crop agronomic traits is valued by breeders for their benefits in crop breeding but are limited for most target traits.

Genome-wide specificity of prime editors in plants.

Abstract: Although prime editors (PEs) have the potential to facilitate precise genome editing in therapeutic, agricultural and research applications, their specificity has not been comprehensively evaluated. To provide a systematic assessment in plants, we first examined the mismatch tolerance of PEs in plant cells and found that the editing frequency was influenced by the number and location of mismatches in the primer binding site and spacer of the prime editing guide RNA (pegRNA). Assessing the activity of 12 pegRNAs at 179 predicted off-target sites, we detected only low frequencies of off-target edits (0.00~0.23%). Whole-genome sequencing of 29 PE-treated rice plants confirmed that PEs do not induce genome-wide pegRNA-independent off-target single-nucleotide variants or small insertions/deletions. We also show that ectopic expression of the Moloney murine leukemia virus reverse transcriptase as part of the PE does not change retrotransposon copy number or telomere structure or cause insertion of pegRNA or messenger RNA sequences into the genome.

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

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.

Applications of CRISPR–Cas in agriculture and plant biotechnology

Abstract: 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.

The emerging and uncultivated potential of CRISPR technology in plant science.

Abstract: The application of clustered regularly interspaced short palindromic repeats (CRISPR) for genetic manipulation has revolutionized life science over the past few years. CRISPR was first discovered as an adaptive immune system in bacteria and archaea, and then engineered to generate targeted DNA breaks in living cells and organisms. During the cellular DNA repair process, various DNA changes can be introduced. The diverse and expanding CRISPR toolbox allows programmable genome editing, epigenome editing and transcriptome regulation in plants. However, challenges in plant genome editing need to be fully appreciated and solutions explored. This Review intends to provide an informative summary of the latest developments and breakthroughs of CRISPR technology, with a focus on achievements and potential utility in plant biology. Ultimately, CRISPR will not only facilitate basic research, but also accelerate plant breeding and germplasm development. The application of CRISPR to improve germplasm is particularly important in the context of global climate change as well as in the face of current agricultural, environmental and ecological challenges.