Editing a Stomatal Developmental Gene in Rice with CRISPR/Cpf1.

CRISPR has arguably been the fastest growing genome editing tool so far. CRISPR/Cas9 (Cas9) has been proved to be efficient and precise in genome editing. However Cas9 has certain limitations. CRISPR/Cpf1 (Cpf1) has been discovered as an alternate approach that can overcome some of those limitations. Cpf1 allows targeting in AT-rich region, creating a staggered cleavage, and cutting at the distal end to the PAM (Protospacer Adjacent Motif) regions. We have successfully tested the efficiency of Cpf1 system in rice using OsEPFL9 which is a developmental gene known to regulate the stomatal density in leaf. Regulation of stomatal density and patterning is an important factor in regulating plant physiology, especially in improving the plant water use efficiency. We targeted the Exon1 of OsEPFL9 and the knockout lines were studied for several generations for establishment of stabilized editing, as well as transmission and segregation of edits through generations. The usage of Cpf1 as a genome editing tool to manipulate stomatal patterning may further help us gain more insight of the physiology of rice in stress conditions.

CRISPR-Cas9 and CRISPR-Cpf1 mediated targeting of a stomatal developmental gene EPFL9 in rice.

KEY MESSAGE: CRISPR-Cas9/Cpf1 system with its unique gene targeting efficiency, could be an important tool for functional study of early developmental genes through the generation of successful knockout plants. The introduction and utilization of systems biology approaches have identified several genes that are involved in early development of a plant and with such knowledge a robust tool is required for the functional validation of putative candidate genes thus obtained. The development of the CRISPR-Cas9/Cpf1 genome editing system has provided a convenient tool for creating loss of function mutants for genes of interest. The present study utilized CRISPR/Cas9 and CRISPR-Cpf1 technology to knock out an early developmental gene EPFL9 (Epidermal Patterning Factor like-9, a positive regulator of stomatal development in Arabidopsis) orthologue in rice. Germ-line mutants that were generated showed edits that were carried forward into the T2 generation when Cas9-free homozygous mutants were obtained. The homozygous mutant plants showed more than an eightfold reduction in stomatal density on the abaxial leaf surface of the edited rice plants. Potential off-target analysis showed no significant off-target effects. This study also utilized the CRISPR-LbCpf1 (Lachnospiracae bacterium Cpf1) to target the same OsEPFL9 gene to test the activity of this class-2 CRISPR system in rice and found that Cpf1 is also capable of genome editing and edits get transmitted through generations with similar phenotypic changes seen with CRISPR-Cas9. This study demonstrates the application of CRISPR-Cas9/Cpf1 to precisely target genomic locations and develop transgene-free homozygous heritable gene edits and confirms that the loss of function analysis of the candidate genes emerging from different systems biology based approaches, could be performed, and therefore, this system adds value in the validation of gene function studies.

Overexpression of HARDY, an AP2/ERF gene from Arabidopsis, improves drought and salt tolerance by reducing transpiration and sodium uptake in transgenic Trifolium alexandrinum L.

Trifolium alexandrinum L. was transformed with the Arabidopsis HARDY gene that belongs to the stress-related AP2/ERF (APETALA2/ethylene responsive element binding factors) superfamily of transcription factors. The fresh weights of the transgenic lines L2 and L3 were improved by 42 and 55% under drought stress and by 38 and 95% under salt stress compared to the wild type, respectively. The dry weights were similarly improved. Overexpression of HARDY improved the instantaneous water use efficiency (WUE) under drought stress by reducing transpiration (E) and under salt stress by improving photosynthesis (A), through reducing Na+ accumulation in leaves, and reducing E. However, HARDY improved the growth of drought-stressed transgenic plants as compared to the wild type by delaying water depletion from soil and preventing rapid decline in A. L2 and L3 had thicker stems and in case of L3, more xylem rows per vascular bundle, which may have made L3 more resistant to lodging in the field. Field performance of L2 and L3 under combined drought and salt stress was significantly better than that of the wild type in terms of fresh and dry weights (40%, 46% and 31%, 40%, respectively). The results provide further evidence for the efficiency of overexpression of a single gene in improving tolerance to abiotic stress under field conditions.

Variation at range margins across multiple spatial scales: environmental temperature, population genetics and metabolomic phenotype.

Range margins are spatially complex, with environmental, genetic and phenotypic variations occurring across a range of spatial scales. We examine variation in temperature, genes and metabolomic profiles within and between populations of the subalpine perennial plant Arabidopsis lyrata ssp. petraea from across its northwest European range. Our surveys cover a gradient of fragmentation from largely continuous populations in Iceland, through more fragmented Scandinavian populations, to increasingly widely scattered populations at the range margin in Scotland, Wales and Ireland. Temperature regimes vary substantially within some populations, but within-population variation represents a larger fraction of genetic and especially metabolomic variances. Both physical distance and temperature differences between sites are found to be associated with genetic profiles, but not metabolomic profiles, and no relationship was found between genetic and metabolomic population structures in any region. Genetic similarity between plants within populations is the highest in the fragmented populations at the range margin, but differentiation across space is the highest there as well, suggesting that regional patterns of genetic diversity may be scale dependent.

Fruit carbohydrate metabolism in an introgression line of tomato with increased fruit soluble solids.

A tomato line (IL9-2-5) of the cultivated species, Lycopersicon esculentum, carrying a 9 cM introgression from the wild species, Lycopersicon pennelli, produces fruit with high soluble solids content (Brix), an important determinant of fruit quality for processing. Two quantitative trait loci (QTLs) relating to fruit soluble solids content have been identified within the introgressed segment. One of these QTLs (PW-9-2-5) is silent under the growth conditions used in this study, while a second (Brix-9-2-5) has been shown to encode a fruit apoplastic invertase (Lin5) with altered kinetic properties. In this study, we have undertaken a detailed biochemical analysis of the introgression line to attempt to gain an understanding of the metabolic changes associated with increased fruit soluble solids. Increased Brix in ripe fruit was shown to be the result of increased sucrose and glucose, with a more minor contribution from aspartate and alanine. The introgression leads to a pronounced increase in apoplastic invertase activity in the columella tissue that extends throughout fruit development. Furthermore, columella tissue from IL9-2-5 fruit has a greater capacity to take up exogenously supplied sucrose, an observation that is consistent with the kinetic properties of the introgressed Lin5 allele. Apart from the increase in mature fruit sugar and increases in some amino acids, metabolite profiling revealed few other metabolic perturbations in fruit from IL9-2-5. The only other major change was a dramatic increase in starch accumulation at earlier stages of fruit metabolism. This occurred without any increase in the activity of the enzymes of sucrose metabolism or starch synthesis and may therefore be driven by increased availability of sucrose. We conclude that the major factor that leads to increased fruit sugar in IL9-2-5 is an increase in the capacity to take up sucrose unloaded from the phloem.