Source: UNIV OF MARYLAND submitted to
Sponsoring Institution
State Agricultural Experiment Station
Project Status
Funding Source
Reporting Frequency
Accession No.
Grant No.
Project No.
Proposal No.
Multistate No.
Program Code
Project Start Date
Feb 3, 2015
Project End Date
Jun 30, 2016
Grant Year
Project Director
Liu, ZH.
Recipient Organization
Performing Department
Cell Biology & Molecular Genetics
Non Technical Summary
The wild strawberry Fragaria vesca has recently emerged as an excellent model for investigating fundamental questions in economically important fruit crops. This proposal consists of two specific goals. One is to establish and optimize the CRISPR-based gene-editing for F. vesca by targeting two screenable marker genes, Phytoene desaturase (PDS) and Myb10. Adding CRISPR to the tool kit of this important model crop will not only revolutionize our ability to identify gene function in F. vesca but also make it possible to produce transgene-free fruit crops with engineered traits. This is because once gene-editing is accomplished, the CRIPSR-transgenes can be easily removed by segregation in the next generation. The second goal is to investigate the function of a novel strawberry miRNA and its regulated network via CRISPR. miRN39 was recently identified and shown to be preferentially expressed in the developing receptacle fruit by us. We found that miRN39 triggers 21 nt phased siRNA (phasiRNA) production by cleaving six F-box (FBX) genes. The resulting phasiRNAs cleave additional FBX genes, thus affecting the activity of a network of FBX genes. We propose that this miRNAs-phasiRNA network may evolve to regulate recently expanded FBX gene family to control fruit-specific processes.
Animal Health Component
Research Effort Categories

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
Goals / Objectives
Goal #1: Establish the CRISPR-based gene editing technology in strawberry.Goal #2: Investigate the biological function of a recently discovered novel miRNA-phasiRNA network.
Project Methods
AIM1. Develop CRISPR in F. vesca to facilitate reverse genetics and functional genomicsThe type II CRISPR/Cas RNA-guided genome editing is the most recent addition to the tool kit of reverse genetics with additional advantages of low cost, easy to use, high efficiency, and the possibility of GMO-free plants after the CAS9 construct is segregated away upon finishing its editing task (Jiang et al. 2013; Zhou et al. 2015). Although this technology has been demonstrated to work in Arabidopsis, Tabacco, and rice, it is not yet demonstrated in strawberry. The potential of this tool for targeted gene editing combined with the ability to obtain transgene-free plants in subsequent generations makes this technique extremely powerful and practical. We are very interested in adopting this technology for strawberry as it will empower us in basic research for gene function identification as well as allow us to more easily translate the research results into garden strawberry.We obtained pCAMBIA CAS9 AtU6-gRNA vector from Donald Weeks lab (Jiang et al., 2013). In this vector, the CAS9 nuclease gene is driven by 35S promoter, but the guide-RNA (gRNA) is driven by the Arabidopsis U6 promoter and terminator, which has little sequence homology to the strawberry U6 sequence. We will isolate the strawberry U6 gene to replace the Arabidopsis U6 promoter and the terminator and then test the efficiency of CRISPR in strawberry. Two strawberry target genes will be tested. One is the Phytoene Desaturase (PDS) gene which is frequently used as a marker in Virus-Induced Gene Silensing (VIGS) (Velasquez et al. 2009). Knockdown of PDS results in white sectors on green tissues indicating successful reductions of PDS gene function. By Blast search of F. vesca genome, a single PDS gene has been identified. gRNA targeting the F. vesca PDS gene will be designed and cloned into the F. vesca U6 cassette. A second target gene is the F. vesca Myb10 (fve-Myb10). This gene has been shown to control garden strawberry red fruit color (Lin-Wang et al. 2010; Medina-Puche et al. 2014). gRNA will be designed against the fve-Myb10, and the transgenic plants with compromised fve-Myb10 function should develop white fruit.Several gRNA designing tools are available online ( The key is to design the 20 bp gRNA homologous to a region immediately upstream of a PAM site (NGG) at the targeted gene. The gRNA fragment will be inserted into the U6 cassette using a two step PCR with specifically designed primers harboring the target-homology sequence. The resulting U6-gRNA fragment will then be cloned into pCAMBIA CAS9 binary vetor for transformation (Jiang et al., 2013).Our lab has successfully established strawberry transformation procedure (Kang et al., 2013). Unlike Arabidopsis transformation, this is a labor-intensive tissue-culture-based procedure requiring 8 months of time from agrobacterium infection of leaves to rooted transgenic plants in soil. Using the Fve-PDS and Fve-Myb10 constructs, we can more easily detect successful gene knockout and determine its efficiency. If CRISPR is as efficient as reported for other plants, we should be able to observe white sectors (knockouts of both parental copies of PDS) in the first generation transgenic plants. We can also use PCR-sequencing to detect mutations in targeted genes. Because of the ease of scoring mutated target genes, we may optimize the technology by experimenting with medium composition, culturing lights, antibiotic concentrations, and promoters used to drive the gRNA.AIM2. Identify functiom of a novel miRNA-phasiRNA networkPreviously we used RNA-Seq to identify novel small RNAs, in particular miRNAs, from F. vesca (Ye et al., In prep). We identified 41 novel miRNAs, many of which are likely to be specific to strawberry. Subsequently, we employed Parallel Analyses of RNA ends (PARE) (German et al. 2008) to verify miRNA-guided cleavage events and identify corresponding target genes. The most exciting finding of this work is the discovery of a 22 nt novel miRNA, miRN39, which is derived from an 8 kb genomic region containing 8 novel miRNAs. miRN39 is capable of cleaving at least 6 F-box (FBX) genes to initiate progressive and phased cleavage of 21 nt siRNAs, ie. phasiRNAs . These phased siRNAs from the FBX genes are called phasiFBXs. They are capable of cleaving additional FBX genes in trans or in cis. Hence, a single miRN39 controls the expression and function of at least 13 FBX genes (Xia et al., In prep), forming a layered regulatory network.FBX proteins are subunits of the Skp1-Cullin F-box (SCF) ubiquitin-ligase complex with F-box proteins acting to target specific substrates for ubiquitination (Kipreos and Pagano 2000; Bai et al. 1996). Ubiquitinated substrates are subsequently degraded by the 26S proteasome. Since miRN39 is more highly expressed in the receptacle fruit at 10 day post anthesis, miRN39 could be involved in the strawberry receptacle fruit development. Most of the 13 FBX genes in the miRN39-phasiFBX network are homologous to the Arabidopsis CPR1/CPR30 (At4g12560) gene, which were shown to negatively regulate the accumulation of an R protein SNC1 via the SCF complex (Gou et al. 2012). By cleaving CPR1/CPR30-like FBX transcripts, miRN39 may act to promote R protein stability and accumulation and enhance disease resistance in the developing receptacle.To investigate the function of this miRN39-phasiFBX network, we will over-expressing as well as knockout miRN39. We have identified an EST clone which is the precursor transcript of miRN39. We cloned this miRN39 precursor into pMDC103 to over-express the miRN39 precursor transcript. Agrobacterium containing this construct has been made and will be transformed into F. vesca. Second, we will use CRISPR established in AIM1 to knockout miRN39. Two different CRISPR constructs will be made. In one design, the CRISPR gRNA will pair and aim to cleave the miRN39 to create small deletions in the middle of miRN39. In the second design, we will design two different gRNAs flanking the miRN39. The double gRNAs in the same vector are intended to create a deletion removing the entire miRN39. The above constructs will be transformed into F. vesca using agrobacterium-mediated, tissue-culture method described in AIM1. We aim to generate at least 10 transgenic lines for each construct. PCR and sequencing of PCR products will be carried out in the first generation transgenic plants to identify deleterious mutations. Second generation plants homozygous for the deleterious mutations will be characterized for mutant phenotypes. If no obvious morphological phenotype is observed, we will challenge the fruit with bacterium and fungal infection. Julie Caruana, a postdoc in our lab, did her PhD thesis on pseudomonas syringae resistant Arabidopsis plants and will be carrying out the test of pathogen sensitivity and resistance. We will seek advice from Dr. Shunyuan Xiao in testing fungal resistance.The potential of manipulating the genetics of a single miRNA to regulate a network of genes in plant disease resistance is very exciting. It should be easily applicable to the garden strawberry. In addition, if new delivery methods of small RNAs could be devised for plants, crop plants could be manipulated via non-transgenic approaches such as a simple spray of synthetic miRNAs or phasiRNAs to achieve beneficial effects.

Progress 02/03/15 to 06/30/16

Target Audience:Strawberry research community and strawberry industry will benefit from a novel gene knockout approach developed during this project. Specifically, we have constructed several vectors that express the CRISPR/Cas9 system in the wild strawberry Fragaria vesca in order to specifically knockout target gene function. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The projet provided training to graduate student Fuxi Wang. She learned how to transform strawberry, how to make plasmid constructs, how to design experiments, and how to utilize CRISPR and "Small Tandem Target Mimic (STTM)" technlogies. How have the results been disseminated to communities of interest?We plan to publish the CRISPR work in the near future. PI Zhongchi Liu will give a talk at the 8th International Rosaceae Genomics Conference in June 21, 2016. She plans to talk about our work on creating CRISPR-knockout strawberries. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

What was accomplished under these goals? Goal#1: We have designed and constructed several CRISPR/Cas9 vectors that allow easy cloning of sgRNAs that target genes of interests. We have incorporated different promoters to drive the CAS9 nuclease in the vectors in order to improve the efficiency gene knockout. We have designed an sgRNA that targets PDS (Phytoene Desaturase) and have obtained transgenic strawberry plants that show white sectors on leaves indicating successful knockout of the PDS gene. Sequencing results confirmed that the white sectors are due to single base deletions in the PDS gene. Goal#2: We aim to investiage the function a set of two miRNAs that are predicted to target over 20 F-BOX genes during strawberry fruit development. We have so far made over-expression construct of the two miRNAs of interest. Transgenic plants are being generated and the transgenic plants will be characetrized in the near future. Second, we could not design sgRNA constructs that knockout these two miRNAs because the designed sgRNAs have too many "off target" sites in the genome. We recently decided to use an alternative method called "Small Tandem Target Mimic (STTM)" that can lead to degradation of target miRNAs. We have only recently initiated this approach.