Anthocyanin-assisted Agrobacterium infiltration for the rapid evaluation of genome editing efficiencies across multiple plant species

CRISPR-based genome editing technologies continue to drive major advances in life sciences.A major challenge for realizing widespread use of genome editing in plants and agriculture is establishing methods that enable the rapid,comprehensive,and precise evaluation of editing technologies using transient methods.Here we report a new and rapid genome editing evaluation method using Agrobacterium infiltration techniques to enable broad-spectrum,simplistic,and precise assessments of genome editing efficiencies.We employed an anthocyanin marker to facilitate visual screenings of genome-edited cells for use in adult strawberry fruits as well as tomato fruits,cotton leaves,and sugar beet leaves.Using this method,we demonstrate the ability to quickly measure genome editing efficiencies mediated by SpCas9,LbCas12a,A3A-PBE,ABE8e,and PPE.This new method will allow researchers to rapidly and easily evaluate genome editing tools across a broad spectrum of plant species,further expediting the development of genome-edited agricultural crops.

Targeted Mutagenesis in Zea mays Using TALENs and the CRISPR/Cas System

Transcription activator-like effector nucleases （TALENs） and clustered regularly interspaced short palindromic repeats （CRISPR）/ CRISPR-associated （Cas） systems have emerged as powerful tools for genome editing in a variety of species. Here, we report, for the first time, targeted mutagenesis in Zea mays using TALENs and the CRISPR/Cas system. We designed five TALENs targeting 4 genes, namely ZmPDS, ZmlPKIA, ZmlPK, ZmMRP4, and obtained targeting efficiencies of up to 23.1% in protoplasts, and about 13.3% to 39.1% of the transgenic plants were somatic mutations. Also, we constructed two gRNAs targeting the ZmlPK gene in maize protoplasts, at frequencies of 16.4% and 19.1%, respectively. In addition, the CRISPR/Cas system induced targeted mutations in Z. mays protoplasts with efficiencies （13.1%） similar to those obtained with TALENs （9.1%）. Our results show that both TALENs and the CRISPR/Cas system can be used for genome modification in maize.

An Uncanonical CCCH-Tandem Zinc-Finger Protein Represses Secondary Wall Synthesis and Controls Mechanical Strength in Rice

Secondary walls, which represent the bulk of biomass, have a large impact on plant growth and adaptation to environments. Secondary wall synthesis is switched and regulated by a sophisticated signaling transduction network. However, there is limited understanding of these regulatory pathways. Here, we report that ILAl-interacting protein 4 （lIP4） can repress secondary wall synthesis, lIP4 is a phosphorylation sub- strate of an Raf-like MAPKKK, but its function is unknown. By generating lip4 mutants and relevant transgenic plants, we found that lesions in lIP4 enhance secondary wall formation. Gene expression and transactivation activity assays revealed that lIP4 negatively regulates the expression of MYB61 and CESAs but does not bind their promoters, lIP4 interacts with NAC29/NAC31, the upstream regulators of secondary wall synthesis, and suppresses the downstream regulatory pathways in plants. Mutagenesis analyses showed that phosphomimic UP4 proteins translocate from the nucleus to the cytoplasm, which releases interacting NACs and attenuates its repression function. Moreover, we revealed that liPs are evolutionarily conserved and share unreported CCCH motifs, referred to as uncanonical CCCH-tandem zinc-finger proteins. Collectively, our study provides mechanistic insights into the control of secondary wall synthesis and presents an opportunity for improving relevant agronomic traits in crops.

Generation of thermosensitive male-sterile maize by targeted knockout of the ZmTMS5 gene

Maize（Zea mays L.）is one of the most important cereal crops,with a global production of 1.02 billion tons in 2013（Baldaufa et al.,2016）.Heterosis is widely used to increase the productivity of maize,and the first commercial hybrid maize was introduced in the 1930s（Duvick,2001）.

Generating broad-spectrum tolerance to ALS-inhibiting herbicides in rice by base editing

Herbicide-tolerant rice varieties generated by genome editing are highly desirable for weed control.We have used a cytosine base editor to create a series of missense mutations in the P171 and/or G628 codons of the acetolactate synthase(ALS)gene to confer herbicide tolerance in rice.The four different missense mutations in the P171 codon,P171S,P171A,P171Yand P171F,exhibited different patterns of tolerance towards five representative herbicides from five chemical families of ALS inhibitors.For example,P171S and P171A had lower levels of tolerance than P171Y and P171F to bispyribac but not to the other herbicides.Interestingly,a novel triple mutant(P171F/G628E/G629S)had the highest tolerance to all five tested herbicides.Field trials showed that both P171F and P171F/G628E/G629S could potentially be used with nicosulfuron.Our work illustrates an effective way of using base editing to generate herbicide tolerance in elite rice varieties.

Transient expression of a TaGRF4-TaGIF1 complex stimulates wheat regeneration and improves genome editing

Genome editing is an unprecedented technological breakthrough but low plant regeneration frequencies and genotype dependence hinder its implementation for crop improvement. Here, we found that transient expression of a complex of the growth regulators TaGRF4 and TaGIF1(TaGRF4-TaGIF1) increased regeneration and genome editing frequency in wheat. When we introduced synonymous mutation in the miR396 target site of TaGRF4, the resulting complex(mTaGRF4-TaGIF1) performed better than original TaGRF4-TaGIF1. Use of m TaGRF4-TaGIF1 together with a cytosine base editor targeting TaALS resulted in 2-9-fold increases in regeneration and transgene-free genome editing in 11 elite common wheat cultivars. Therefore, m TaGRF4-TaGIF1 will undoubtedly be of great value in crop improvement and especially in commercial applications, since it greatly increased the range of cultivars available for transformation.

Shortening the sgRNA-DNA interface enables SpCas9 and eSpCas9(1.1)to nick the target DNA strand

The length of the sgRNA-DNA complementary sequence is a key factor influencing the cleavage activity of Streptococcus pyogenes Cas9(SpCas9)and its variants.The detailed mechanism remains unknown.Here,based on in vitro cleavage assays and base editing analysis,we demonstrate that reducing the length of this complementary region can confer nickase activity on SpCas9 and eSpCas9(1.1).We also show that these nicks are made on the target DNA strand.These properties encouraged us to develop a dual-functional system that simultaneously carries out double-strand DNA cleavage and C-to-T base conversions at separate targets.This system provides a novel tool for achieving trait stacking in plants.

Molecular and Functional Characterization of Sphingosine-1-Phosphate Lyase Homolog from Higher Plants

Sphingosine-1-phosphate lyase （SPL） is involved in degrading the conserved sphingolipid signaling molecule sphingosine-1-phosphate. However, molecular studies on plant SPL have not been reported to date. Here, we present bioinformatic, molecular and functional analyses of putative SPL proteins from Arabidopsis thaliana and rice （designated as AtSPL and OsSPL, respectively）. Amino acid sequence comparison revealed that plant SPL contalned the pyridoxal-dependent decarboxylase domain and the conserved residue that may be involved in substrate catalysis. When expressed in Saccharomyces cerevisiae, AtSPL and OsSPL corrected the hypersensitive phenotype of the yeast dpl1 deletion strain, which is deficient in endogenous SPL activity, to exogenous supplied sphingolipid long chain bases （LCBs）, suggesting that plant SPL protein is functional In vivo in degrading phosphorylated LCBs. In Arabidopsis, AtSPL transcripts were detected in roots, stems, leaves, flowers and siliques. In pAtSPL-AtSPL∷GUS transgenic lines, the AtSPL∷GUS fusion protein was found in a variety of vegetative and reproductive tissues. AtSPL expression level was dynamically regulated during leaf development and senescence, and was steadily and significantly increased in Arabidopsis seedlings treated with the cell death-inducing fungal toxin fumonisin B1. The potential function of SPL in Arabidopsis is discussed.

TALENs:Customizable Molecular DNA Scissors for Genome Engineering of Plants

Precise genome modification with engineered nucleases is a powerful tool for studying basic biology and applied biotechnology. Transcription activator-like effector nucleases（TALENs）,consisting of an engineered specific（TALE） DNA binding domain and a Fok I cleavage domain,are newly developed versatile reagents for genome engineering in different organisms.Because of the simplicity of the DNA recognition code and their modular assembly,TALENs can act as customizable molecular DNA scissors inducing double-strand breaks（DSBs） at given genomic location.Thus,they provide a valuable approach to targeted genome modifications such as mutations, insertions,replacements or chromosome rearrangements.In this article,we review the development of TALENs,and summarize the principles and tools for TALEN-mediated gene targeting in plant cells,as well as current and potential strategies for use in plant research and crop improvement.

Erratum to:Generating broad-spectrum tolerance to ALS-inhibiting herbicides in rice by base editing

This paper contains an error in Figure 2, where P171Y and P171A under treatment of pyroxsulam have a duplicated picture.This picture is for P171Y but not for P171A.We provide the correct picture for P171A as follows. This new picture does not affect the conclusion of this article.

Rapid and Efficient Gene Modification in Rice and Brachypodium Using TALENs

Dear Editor, In the past few years, the use of sequence-specific nucle- ases for efficient targeted mutagenesis has provided plant biologists with a powerful new approach for understanding gene function and developing new traits. These nucleases create DNA double-strand breaks at chromosomal targeted sites that are primarily repaired by the non-homologous end joining （NHEJ） or homologous recombination （HR） pathways. NHEJ is o^en imprecise and can introduce mutations at tar- get sites resulting in the loss of gene function. In contrast, HR uses a homologous DNA template for repair and can be employed to create site-specific sequence modifications or targeted insertions （Moynahan and Jasin, 2010）.

Genome-edited crops: how to move them from laboratory to market

Recent breakthroughs in CRISPR technology allow specific genome manipulation of almost all crops and have initiated a revolution in precision crop breeding.Rationally-based regulation and widespread public acceptance are needed to propel genome-edited crops from laboratory to market and to translate this innovative technology into agricultural productivity.