A method for the production and expedient screening of CRISPR/Cas9-mediated non-transgenic mutant plants

Developing CRISPR/Cas9-mediated non-transgenic mutants in asexually propagated perennial crop plants is challenging but highly desirable.Here,we report a highly useful method using an Agrobacterium-mediated transient CRISPR/Cas9 gene expression system to create non-transgenic mutant plants without the need for sexual segregation.We have also developed a rapid,cost-effective,and high-throughput mutant screening protocol based on Illumina sequencing followed by high-resolution melting(HRM)analysis.Using tetraploid tobacco as a model species and the phytoene desaturase(PDS)gene as a target,we successfully created and expediently identified mutant plants,which were verified as tetra-allelic mutants.We produced pds mutant shoots at a rate of 47.5%from tobacco leaf explants,without the use of antibiotic selection.Among these pds plants,17.2%were confirmed to be non-transgenic,for an overall non-transgenic mutation rate of 8.2%.Our method is reliable and effective in creating non-transgenic mutant plants without the need to segregate out transgenes through sexual reproduction.This method should be applicable to many economically important,heterozygous,perennial crop species that are more difficult to regenerate.

A reporter for noninvasively monitoring gene expression and plant transformation

Reporters have been widely used to visualize gene expression,protein localization,and other cellular activities,but the commonly used reporters require special equipment,expensive chemicals,or invasive treatments.Here,we construct a new reporter RUBY that converts tyrosine to vividly red betalain,which is clearly visible to naked eyes without the need of using special equipment or chemical treatments.We show that RUBY can be used to noninvasively monitor gene expression in plants.Furthermore,we show that RUBY is an effective selection marker for transformation events in both rice and Arabidopsis.The new reporter will be especially useful for monitoring cellular activities in large crop plants such as a fruit tree under field conditions and for observing transformation and gene expression in tissue culture under sterile conditions.

Synergistic roles of LAX1 and FZP in the development of rice sterile lemma

Rice florets are subtended by two sterile lemmas,whose origin and biological functions have not been studied extensively.Here we demonstrate that two putative transcription factors,LAX PANICLE1(LAX1)and FRIZZY PANICLE(FZP),synergistically control the development of sterile lemmas.Both LAX1 and FZP are previously known for their roles in panicle and floret development.Disruption of either LAX1 or FZP greatly reduces the number of floret development.We generated new lax1 mutants(lax1-c)using CRISPR/Cas9 gene editing technology.In addition to the expected lax panicle phenotypes,we noticed that a significant number of spikelets of lax1-c developed elongated sterile lemmas.Moreover,our characterization of lax1-RNAi plants also revealed sterile lemma phenotypes similar to lax1-c mutants.We isolated a weak allele of fzp(fzp-14)in a genetic screen for lax1–1 enhancers.The fzp-14 lax1–1 double mutants completely eliminated flower development.Interestingly,the isolated fzp-14 produced spikelets with elongated sterile lemmas.Furthermore,fzp-14 was haploid-insufficient in the lax1–1 background whereas fzp-14 heterozygous plants were indistinguishable from wild type plants.The lax1–1 fzp-14+/−also developed elongated sterile lemma as observed in lax1-c,lax1-RNAi,and fzp-14,suggesting that LAX1 and FZP synergistically control sterile lemma development.

Self-processing of ribozyme-flanked RNAs into guide RNAs in vitro and in vivo for CRISPR-mediated genome editing

CRISPR/Cas9 uses a guide RNA （gRNA） molecule to execute sequence-specific DNA cleavage and it has been widely used for genome editing in many organisms. Modifications at either end of the gRNAs often render Cas9/gRNA inactive. So far, production of gRNA in vivo has only been achieved by using the U6 and U3 snRNA promoters. However, the U6 and U3 promoters have major limitations such as a lack of cell specificity and unsuitability for in vitro transcription. Here, we present a versatile method for efficiently producing gRNAs both in vitro and in vivo. We design an artificial gene named RGR that, once transcribed, generates an RNA molecule with ribozyme sequences at both ends of the designed gRNA. We show that the primary transcripts of RGR undergo self-catalyzed cleavage to generate the desired gRNA, which can efficiently guide sequence-specific cleavage of DNA targets both in vitro and in yeast. RGR can be transcribed from any promoters and thus allows for cell- and tissue-specific genome editing if appropriate promoters are chosen. Detecting mutations generated by CRISPR is often achieved by enzyme digestions, which are not very compatible with high-throughput analysis. Our system allows for the use of universal primers to produce any gRNAs in vitro, which can then be used with Cas9 protein to detect mutations caused by the gRNAs/CRISPR. In conclusion, we provide a versatile method for generating targeted mutations in specific cells and tissues, and for efficiently detecting the mutations generated.

Auxin Biosynthesis： A Simple Two-Step Pathway Converts Tryptophan to Indole-3-Acetic Acid in Plants

Indole-3-acetic acid （IAA）, the main naturally occurring auxin, is essential for almost every aspect of plant growth and development. However, only recently have studies finally established the first complete auxin biosynthesis pathway that converts tryptophan （Trp） to IAA in plants. Trp is first converted to indole-3-pyruvate （IPA） by the TAA family of amino transferases and subsequently IAA is produced from IPA by the YUC family of flavin monooxygenases. The two- step conversion of Trp to IAA is the main auxin biosynthesis pathway that plays an essential role in many developmental processes.

Efficient allelic replacement in rice by gene editing： A case study of the NRT1.1B gene

Summary Precise replacement of an existing allele in commercial cultivars with an elite allele is a major goal in crop breeding. A single nucleotide polymorphism in the NRT1.1B gene between japonica and indica rice is responsible for the improved nitrogen use efficiency in indica rice. Herein, we precisely replaced the japonica NRT1.1B allele with the indica allele, in just one generation, using CRISPR/Cas9 gene-editing technology. No additional selective pressure was needed to enrich the precise replacement events.

NPY Genes Play an Essential Role in Root Gravitropic Responses in Arabidopsis

Plants can sense the direction of gravity and orient their growth to ensure that roots are anchored in soil and that shoots grow upward. Gravitropism has been studied extensively using Arabidopsis genetics, but the exact mecha- nisms for gravitropism are not fully understood. Here, we demonstrate that five NPY genes play a key role in Arabidopsis root gravitropism. NPYgenes were previously identified as regulators of auxin-mediated organogenesis in a genetic pathway with the AGC kinases PID, PID2, WAG1, and WAG2. We show that all five NPYgenes are highly expressed in primary root tips. The single npy mutants do not display obvious gravitropism defects, but the npyl npy2 npy3 npy4 npy5 quin- tuple mutants show dramatic gravitropic phenotypes. Systematic analysis of all the npy double, triple, and quadruple combinations demonstrates that the five NPY genes all contribute to gravitropism. Our work indicates that gravitropism, phototropism, and organogenesis use analogous mechanisms in which at least one AGC kinase, one NPH3/NPY gene, and one ARF are required.

Plant genome editing using xCas9 with expanded PAM compatibility

CRISPR/Cas enables robust genome editing and has revolution-ized both functional genomics and crop breeding.The specificity of Cas-directed DNA cleavage is strictly determined by a chimeric single guide RNA(SgRNA)and a short protospacer adjacent motif(PAM)in the genome(Cong et al,2013;Zetsche et al,2015).The widely used Cas9 from Streptococcus pyogenes(SpCas9)generally recognizes the canonical NGG PAM(where N indicates any nucleicacid base)(Miao et al.,2013;Ma et al,2015),making many regionsuntargetable by Cas9.SpCas9 VQR and VRER variants,which recog-nize the non-canonical PAM sequences of NGA and NGCG,respectively,have been used to expand targetable sequences in plants(Hu et al.,2016).In addition,the applications of other Cas endonucleases such as SaCas9(Staphylococcus aureus Cas9)(Ran et al..2015).

Self-cleaving ribozymes enable the production of guide RNAs from unlimited choices of promoters for CRISPR/Cas9 mediated genome editing

Development of tools for targeted modifications of specific DNA sequences in plants is of great importance to basic plant biology research as well as crop improvement.The ability to cut DNA at specific locations in the genome to generate doublestrand breaks（DSBs）in vivo is a prerequisite for any genome editing efforts.

ESCRT-dependent vacuolar sorting and degradation of the auxin biosynthetic enzyme YUC1 flavin monooxygenase

YUC flavin monooxygenases catalyze the ratelimiting step of auxin biosynthesis. Here we report the vacuolar targeting and degradation of GFP-YUC1. GFP-YUC1 fusion expressed in Arabidopsis protoplasts or transgenic plants was primarily localized in vacuoles. Surprisingly, we established that GFP-YUC1, a soluble protein, was sorted to vacuoles through the ESCRT pathway, which has long been recognized for sorting and targeting integral membrane proteins. We further show that GFP-YUC1 was ubiquitinated and in this form GFP-YUC1 was targeted for degradation, a process that was also stimulated by elevated auxin levels. Our findings revealed a molecular mechanism of GFP-YUC1 degradation and demonstrate that the ESCRT pathway can recognize both soluble and integral membrane proteins as cargoes.

Fast-Suppressor Screening for New Components in Protein Trafficking,Organelle Biogenesis and Silencing Pathway in Arabidopsis thaliana Using DEX-Inducible FREE1-RNAi Plants

Membrane trafficking is essential for plant growth and responses to external signals.The plant unique FYVE domain-containing protein FREE1 is a component of the ESCRT complex（endosomal sorting complex required for transport）.FREE1 plays multiple roles in regulating protein trafficking and organelle biogenesis including the formation of intraluminal vesicles of multivesicular body（MVB）,vacuolar protein transport and vacuole biogenesis,and autophagic degradation.FREE1 knockout plants show defective MVB formation,abnormal vacuolar transport,fragmented vacuoles,accumulated autophagosomes,and seedling lethality.To further uncover the underlying mechanisms of FREE1 function in plants,we performed a forward genetic screen for mutants that suppressed the seedling lethal phenotype of FREE1-RNAi transgenic plants.The obtained mutants are termed as suppressors of free1（sof）.To date,229 putative sof mutants have been identified.Barely detecting of FREE1 protein with M3 plants further identified 84 FREE1-related suppressors.Also145 mutants showing no reduction of FREE1 protein were termed as RNAi-related mutants.Through next-generation sequencing（NGS）of bulked DNA from F2 mapping population of two RNAi-related sof mutants,FREE1-RNAi T-DNA inserted on chromosome 1 was identified and the causal mutation of putative sof mutant is being identified similarly.These FREE1-and RNAi-related sof mutants will be useful tools and resources for illustrating the underlying mechanisms of FREE1 function in intracellular trafficking and organelle biogenesis,as well as for uncovering the new components involved in the regulation of silencing pathways in plants.

Technological breakthroughs in generating transgene-free and genetically stable CRISPR-edited plants

CRISPR/Cas9 gene-editing technologies have been very effective in editing target genes in all major crop plants and offer unprecedented potentials in crop improvement.A major challenge in using CRISPR gene-editing technology for agricultural applications is that the target gene-edited crop plants need to be transgene free to maintain trait stability and to gain regulatory approval for commercial production.In this article,we present various strategies for generating transgene-free and target geneedited crop plants.The CRISPR transgenes can be removed by genetic segregation if the crop plants are reproduced sexually.Marker-assisted tracking and eliminating transgenes greatly decrease the time and labor needed for identifying the ideal transgene-free plants.Transgenes can be programed to undergo self-elimination when CRISPR genes and suicide genes are sequentially activated,greatly accelerating the isolation of transgene-free and target gene-edited plants.Transgene-free plants can also be generated using approaches that are considered non-transgenic such as ribonucleoprotein transfection,transient expression of transgenes without DNA integration,and nano-biotechnology.Here,we discuss the advantages and disadvantages of the various strategies in generating transgene-free plants and provide guidance for adopting the best strategies in editing a crop plant.

Overexpression of the bacterial tryptophan oxidase RebO affects auxin biosynthesis and Arabidopsis development

Both tryptophan(Trp) and auxin are essential for plant growth and Trp is a precursor for auxin biosynthesis.Concentrations of Trp and auxin need to be tightly controlled to ensure optimal growth and development. It has been very difficult to study the homeostasis of these two essential and inter-dependent compounds. Auxin is mainly synthesized from Trp via a two-step pathway using indole-3-pyruvate(IPA) as the intermediate. Here we used a bacterial Trp oxidase Reb O, which does not exist in Arabidopsis and which converts Trp to the imine form of IPA,to modulate IPA levels in Arabidopsis. Our results demonstrate that Arabidopsis plants use two strategies to ensure that no excess IPA is made from Trp. IPA is made from Trp by the TAA family of aminotransferases, which we show catalyzes the reverse reaction when IPA level is high. Moreover, excess IPA is converted back to Trp by the VAS1 aminotransferase. We show that the VAS1-catalyzed reaction is very important for Trp homeostasis. This work not only elucidates the intricate biochemical mechanisms that control the homeostasis of Trp, IPA, and auxin,but also provides novel tools for further biochemical studies on Trp metabolism and auxin biosynthesis in plants.

Generation of Targeted Point Mutations in Rice by a Modified CRISPR/Cas9 System

CRISPR/Cas9 （Clustered Regularly Interspaced Short Palin- dromic Repeats/CRISPR-associated Cas9 endonuclease）- mediated genome editing has revolutionized biological research and crop improvement because of its specificity, simplicity, and versatility （reviewed in Komor et al., 2016a）.

Programmed Self-Elimination of the CRISPR/Cas9 Construct Greatly Accelerates the Isolation of Edited and Transgene-Free Rice Plants

Dear Editor CRISPR gene-editing technology has successfully generated targeted mutations in rice and many other plant species （Ma et al., 2015）. Assessment of heritability and phenotypic stability of CRISPR-edited plants requires the elimination of the CRISPR construct. The presence of the CRISPR construct makes it difficult to distinguish the mutations transmitted from the previous generation from newly generated mutations by the CRISPR construct at the current generation. The existence of the CRISPR construct also greatly increases the risk of off-target effects.

Positional effects on efficiency of CRISPR/Cas9-based transcriptional activation in rice plants

The nuclease-dead Cas9(dCas9)has been reprogrammed for transcriptional activation by fusing dCas9 to a transcriptional activation domain.In the presence of a guide RNA(gRNA),the dCas9 fusions specifically bind to regions of a promoter to activate transcription.Significant amount of effort has been directed toward the identification and optimization of the fusions of dCas9-activation domain,but very little is known about the impact of gRNA target positions within a promoter in plants on transcriptional activation efficiency.The dCas9–6TAL–VP128 system(dCas9-TV)has been optimized to activate transcription in plants.Here we use the dCas9-TV to activate transcription of OsWOX11 and OsYUC1,two genes that cause dramatic developmental phenotypes when overexpressed.We designed a series of gRNAs targeting the promoters of the two genes.We show that gRNAs that target regions within 350 bp upstream of the transcription start site were most effective in transcriptional activation.Moreover,we show that using two gRNAs that simultaneously target two discrete sites in a promoter can further enhance transcription.This work provides guidelines for designed transcriptional activation through CRISPR/dCas9 systems.

Expanding the Scope of CRISPR/Cpfl-Mediatec Senome Editing in Rice

Dear Editor The CRISPR/Cas gene editing system offers great potential for functional genomics in plants and crop improvement. The spec- ificity of Cas-directed DNA cleavage is strictly determined by a chimeric single guide RNA （sgRNA） and a short protospacer adjacent motif （PAM） in the genome （Cong et al., 2013; Zetsche et al., 2015）. The widely used SpCas9 and its variants have been shown to recognize PAM sequences in the canonical form NGG and non-canonical NGA, NAG, or NGCG in plants （Miao et al., 2013; Ma et al., 2015; Hu et al., 2016）. CRISPR/Cpfl, a new class 2 CRISPR/Cas system, was recently exploited as an alternative tool for genome editing in various organisms, including plants （Zetsche et al., 2015; Kim et al., 2017; Tang et at., 2017; Wang et al., 2017; Xu et al., 2017）. Cpfl utilizes a thymidine-rich PAM site, TTTN, and is guided by a single CRISPR RNA （crRNA） （Zetsche et al., 2015）.

Allelic Analyses of the Arabidopsis YUC1 Locus Reveal Residues and Domains Essential for the Functions of YUC Family of Flavin Monooxygenases

Flavin monooxygenases （FMOs） play critical roles in plant growth and development by synthesizing auxin and other signaling molecules. However, the structure and function relationship within plant FMOs is not understood. Here we defined the important residues and domains of the Arabidopsis YUC1 FMO, a key enzyme in auxin biosynthesis. We previously showed that simultaneous inactivation of YUC1 and its homologue YUC4 caused severe defects in vascular and floral development. We mutagenized the yuc4 mutant and screened for mutants with phenotypes similar to those of yucl yuc4 double mutants. Among the isolated mutants, five of them contained mutations in the YUC1 gene. Interestingly, the mutations identified in the new yucl alleles were concentrated in the two GXGXXG motifs that are highly conserved among the plant FMOs. One such motif presumably binds to flavin adenine dinucleotide （FAD） cofactor and the other binds to nicotinamide adenine dinucleotide phosphate （NADPH）. We also identified the Ser139 to Phe conversion in yucl, a mutation that is located between the two nucleotide-binding sites. By analyzing a series of yucl mutants, we identified key residues and motifs essential for the functions of YUC1 FMO.

Two homologous INDOLE-3-ACETAIVHDE (IAM) HYDROLASE genes are required for the auxin effects of IAM in Arabidopsis

Indole-3-acetamide(IAM)is the first confirmed auxin biosynthetic intermediate in some plant pathogenic bacteria.Exogenously applied IAM or production of IAM by overexpressing the bacterial iaaM gene in Arabidopsis causes auxin overproduction phenotypes.However,it is still inconclusive whether plants use IAM as a key precursor for auxin biosynthesis.Herein,we reported the isolation IAM HYDROLASE 1(IAMH1)gene in Arabidopsis from a forward genetic screen for IAM-insensitive mutants that display normal auxin sensitivities.IAMH1 has a close homolog named IAMH2 that is located right next to IAMH1on chromosomeⅣin Arabidopsis.We generated iamh1 iamh2 double mutants using our CRISPR/Cas9gene editing technology.We showed that disruption of the IAMH genes rendered Arabidopsis plants resistant to IAM treatments and also suppressed the iaaM overexpression phenotypes,suggesting that IAMH1 and IAMH2 are the main enzymes responsible for converting IAM into indole-3-acetic acid(IAA)in Arabidopsis.The iamh double mutants did not display obvious developmental defects,indicating that IAM does not play a major role in auxin biosynthesis under normal growth conditions.Our findings provide a solid foundation for clarifying the roles of IAM in auxin biosynthesis and plant development.