CRISPR-Cas9-based precise engineering of SlHyPRP1 protein towards multi-stress tolerance in tomato.

Recently, CRISPR-Cas9-based genome editing has been widely used for plant breeding. In our previous report, a tomato gene encoding hybrid proline-rich protein 1 (HyPRP1), a negative regulator of salt stress responses, has been edited using a CRISPR-Cas9 multiplexing approach that resulted in precise eliminations of its functional domains, proline-rich domain (PRD) and eight cysteine-motif (8CM). We subsequently demonstrated that eliminating the PRD domain of HyPRP1 in tomatoes conferred the highest level of salinity tolerance. In this study, we characterized the edited lines under several abiotic and biotic stresses to examine the possibility of multiple stress tolerance. Our data reveal that the 8CM removal variants of HK and the KO alleles of both HK and 15T01 cultivars exhibited moderate heat stress tolerance. Similarly, plants carrying either the domains of the PRD removal variant (PR1v1) or 8CM removal variants (PR2v2 and PR2v3) showed better germination under osmosis stress (up to 200 mM mannitol) compared to the WT control. Moreover, the PR1v1 line continuously grew after 5 days of water cutoff. When the edited lines were challenged with pathogenic bacteria of Pseudomonas syringae pv. tomato (Pto) DC3000, the growth of the bacterium was significantly reduced by 2.0- to 2.5-fold compared to that in WT plants. However, the edited alleles enhanced susceptibility against Fusarium oxysporum f. sp. lycopersici, which causes fusarium wilt. CRISPR-Cas9-based precise domain editing of the SlHyPRP1 gene generated multi-stress-tolerant alleles that could be used as genetic materials for tomato breeding.

Improvement of the LbCas12a-crRNA System for Efficient Gene Targeting in Tomato.

Plant gene targeting (GT) can be utilized to precisely replace up to several kilobases of a plant genome. Recent studies using the powerful clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) nucleases significantly improved plant GT efficiency. However, GT for loci without associated selection markers is still inefficient. We previously utilized Lachnospiraceae bacterium Cas12a (LbCas12a) in combination with a replicon for tomato GT and obtained high GT efficiency with some selection markers. In this study, we advance our GT system by inhibiting the cNHEJ pathway with small chemical molecules such as NU7441. Further optimization of the GT is also possible with the treatment of silver nitrate possibly via its pronounced actions in ethylene inhibition and polyamine production. Importantly, the GT efficiency is significantly enhanced with the use of a temperature-tolerant LbCas12a (ttLbCas12a) that is capable of performing target cleavage even at low temperatures. Targeted deep sequencing, as well as conventional methods, are used for the assessment of the editing efficiency at both cell and plant levels. Our work demonstrates the significance of the selection of gene scissors, the appropriate design and number of LbCas12a crRNAs, the use of chemical treatments, and the establishment of favorable experimental conditions for further enhancement of plant HDR to enable efficient GT in tomato.

CRISPR/Cas9-based precise excision of SlHyPRP1 domain(s) to obtain salt stress-tolerant tomato.

KEY MESSAGE: CRISPR/Cas9-based multiplexed editing of SlHyPRP1 resulted in precise deletions of its functional motif(s), thereby resulting in salt stress-tolerant events in cultivated tomato. Crop genetic improvement to address environmental stresses for sustainable food production has been in high demand, especially given the current situation of global climate changes and reduction of the global food production rate/population rate. Recently, the emerging clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas)-based targeted mutagenesis has provided a revolutionary approach to crop improvement. The major application of CRISPR/Cas in plant genome editing has been the generation of indel mutations via error-prone nonhomologous end joining (NHEJ) repair of DNA DSBs. In this study, we examined the power of the CRISPR/Cas9-based novel approach in the precise manipulation of protein domains of tomato hybrid proline-rich protein 1 (HyPRP1), which is a negative regulator of salt stress responses. We revealed that the precise elimination of SlHyPRP1 negative-response domain(s) led to high salinity tolerance at the germination and vegetative stages in our experimental conditions. CRISPR/Cas9-based domain editing may be an efficient tool to engineer multidomain proteins of important food crops to cope with global climate changes for sustainable agriculture and future food security.

CRISPR/Cas-based precision genome editing via microhomology-mediated end joining.

Gene editing and/or allele introgression with absolute precision and control appear to be the ultimate goals of genetic engineering. Precision genome editing in plants has been developed through various approaches, including oligonucleotide-directed mutagenesis (ODM), base editing, prime editing and especially homologous recombination (HR)-based gene targeting. With the advent of CRISPR/Cas for the targeted generation of DNA breaks (single-stranded breaks (SSBs) or double-stranded breaks (DSBs)), a substantial advancement in HR-mediated precise editing frequencies has been achieved. Nonetheless, further research needs to be performed for commercially viable applications of precise genome editing; hence, an alternative innovative method for genome editing may be required. Within this scope, we summarize recent progress regarding precision genome editing mediated by microhomology-mediated end joining (MMEJ) and discuss their potential applications in crop improvement.

Changes in mortality after the recent economic crisis in South Korea.

PURPOSE: To examine the changes in all cause mortality and cause-specific mortality after the economic crisis in South Korea. METHODS: Monthly mortality data for an entire country was used and intervention analysis applied to compare mortality after the crisis with mortality which would have occurred if the trends before the crisis had continued. RESULTS: All cause mortality began to increase about 1 year after the crisis, while cardiovascular increased immediately. Transport accidents decreased significantly during the year following the crisis and then regressed towards the pre-economic crisis level. Suicides increased rapidly and maintained an upward trend but subsequently reduced towards the pre-economic crisis level. CONCLUSIONS: This study has shown an evidence of a relationship between economic crisis and mortality.

Changes in morbidity and medical care utilization after the recent economic crisis in the Republic of Korea.

OBJECTIVE: To examine and quantify the impact of the recent economic crisis on morbidity and medical care utilization in the Republic of Korea. METHODS: 22 675 people from 6791 households and 43 682 people from 12 283 households were questioned for two nationwide surveys that took place in 1995 and 1998, respectively. A separate sample pretest-posttest design was used and we conducted c2 test and logistic regression analysis after controlling for the maturation effect of the morbidity and medical care utilization. FINDINGS: The morbidity rates of chronic disease and acute disease increased significantly by 27.1% and 9.5%, respectively, whereas the utilization rates of outpatient and inpatient services decreased by 15.1% and 5.2%, respectively. In particular, the pace of decline in the utilization rate of outpatient services varied depending on the type of disease: morbidity rates for mental and behavioural disorders were 13.7%; for cardiovascular disease, 7.1%; and for injury, 31.6%. CONCLUSION: After the Republic of Korean economic crisis, the morbidity and medical care utilization rates changed significantly but the degree of change depended on the type of disease or service. The time-dependent relationship between the national economy and the morbidity and medical care utilization rates needs to be further investigated.