Precision genome editing in plants : state-of- the-art in CRISPR/Cas9-based genome engineering

Traditionally, generation of new plants with improved or desirable features has relied on laborious and time-consuming breeding techniques. Genome-editing technologies have led to a new era of genome engineering, enabling an effective, precise, and rapid engineering of the plant genomes. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (CRISPR/Cas9) has emerged as a new genome-editing tool, extensively applied in various organisms, including plants. The use of CRISPR/Cas9 allows generating transgene-free genome-edited plants (“null segregants”) in a short period of time. In this review, we provide a critical overview of the recent advances in CRISPR/Cas9 derived technologies for inducing mutations at target sites in the genome and controlling the expression of target genes. We highlight the major breakthroughs in applying CRISPR/Cas9 to plant engineering, and challenges toward the production of null segregants. We also provide an update on the efforts of engineering Cas9 proteins, newly discovered Cas9 variants, and novel CRISPR/Cas systems for use in plants. The application of CRISPR/Cas9 and related technologies in plant engineering will not only facilitate molecular breeding of crop plants but also accelerate progress in basic research

Multiplex Genome Editing in Tomato

Several expression systems for multiple guide RNA (gRNAs) have been developed in the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9) system to induce multiple-gene modifications in plants. Here, we evaluated mutation efficiencies in the tomato genome using multiplex CRISPR/Cas9 vectors consisting of various Cas9 expression promoters with multiple gRNA expression combinations. In transgenic tomato calli induced with these vectors, mutation patterns varied depending on the promoters used to express Cas9. By using the tomato ELONGATION FACTOR-1α (SlEF1α) promoter to drive Cas9, occurrence of various types of mutations with high efficiency was detected in the tomato genome. Furthermore, sequence analysis showed that the majority of mutations using the multiplex system with the SlEF1α promoter corresponded to specific mutation pattern of deletions produced by self-ligation at two target sites of CRISPR/Cas9 with low mosaic mutations. These results suggest that optimizing the Cas9 expression promoter used in CRISPR/Cas9-mediated mutation improves multiplex genome editing, and could be used effectively to disrupt functional domains precisely in the tomato genome

SlIAA9 Controls Tomato Elongation

Tomato INDOLE-3-ACETIC ACID9 (SlIAA9) is a transcriptional repressor in auxin signal transduction, and SlIAA9 knockout tomato plants develop parthenocarpic fruits without fertilization. We generated sliaa9 mutants with parthenocarpy in several commercial tomato cultivars (Moneymaker, Rio Grande, and Ailsa Craig) using CRISPR-Cas9, and null-segregant lines in the T1 generation were isolated by self-pollination, which was confirmed by PCR and Southern blot analysis. We then estimated shoot growth phenotypes of the mutant plants under different light (low and normal) conditions. The shoot length of sliaa9 plants in Moneymaker and Rio Grande was smaller than those of wild-type cultivars in low light conditions, whereas there was not clear difference between the mutant of Ailsa Craig and the wild-type under both light conditions. Furthermore, young seedlings in Rio Grande exhibited shade avoidance response in hypocotyl growth, in which the hypocotyl lengths were increased in low light conditions, and sliaa9 mutant seedlings of Ailsa Craig exhibited enhanced responses in this phenotype. Fruit production and growth rates were similar among the sliaa9 mutant tomato cultivars. These results suggest that control mechanisms involved in the interaction of AUX/IAA9 and lights condition in elongation growth differ among commercial tomato cultivars

Genome editing in plants using CRISPR type I-D nuclease

Genome editing in plants has advanced greatly by applying the clustered regularly interspaced short palindromic repeats (CRISPRs)-Cas system, especially CRISPR-Cas9. However, CRISPR type I—the most abundant CRISPR system in bacteria—has not been exploited for plant genome modification. In type I CRISPR-Cas systems, e.g., type I-E, Cas3 nucleases degrade the target DNA in mammals. Here, we present a type I-D (TiD) CRISPR-Cas genome editing system in plants. TiD lacks the Cas3 nuclease domain; instead, Cas10d is the functional nuclease in vivo. TiD was active in targeted mutagenesis of tomato genomic DNA. The mutations generated by TiD differed from those of CRISPR/Cas9; both bi-directional long-range deletions and short indels mutations were detected in tomato cells. Furthermore, TiD can be used to efficiently generate bi-allelic mutant plants in the first generation. These findings indicate that TiD is a unique CRISPR system that can be used for genome engineering in plants

Optimization of CRISPR/Cas9 genome editing to modify abiotic stress responses in plants.

Genome editing using the CRISPR/Cas9 system can be used to modify plant genomes, however, improvements in specificity and applicability are still needed in order for the editing technique to be useful in various plant species. Here, using genome editing mediated by a truncated gRNA (tru-gRNA)/Cas9 combination, we generated new alleles for OST2, a proton pump in Arabidopsis, with no off-target effects. By following expression of Cas9 and the tru-gRNAs, newly generated mutations in CRIPSR/Cas9 transgenic plants were detected with high average mutation rates of up to 32.8% and no off-target effects using constitutive promoter. Reducing nuclear localization signals in Cas9 decreased the mutation rate. In contrast, tru-gRNA Cas9 cassettes driven by meristematic- and reproductive-tissue-specific promoters increased the heritable mutation rate in Arabidopsis, showing that high expression in the germ line can produce bi-allelic mutations. Finally, the new mutant alleles obtained for OST2 exhibited altered stomatal closing in response to environmental conditions. These results suggest further applications in molecular breeding to improve plant function using optimized plant CRISPR/Cas9 systems

Rapid breeding of parthenocarpic tomato plants using CRISPR/Cas9

Parthenocarpy in horticultural crop plants is an important trait with agricultural value for various industrial purposes as well as direct eating quality. Here, we demonstrate a breeding strategy to generate parthenocarpic tomato plants using the CRISPR/Cas9 system. We optimized the CRISPR/Cas9 system to introduce somatic mutations effectively into SlIAA9—a key gene controlling parthenocarpy— with mutation rates of up to 100% in the T0 generation. Furthermore, analysis of off-target mutations using deep sequencing indicated that our customized gRNAs induced no additional mutations in the host genome. Regenerated mutants exhibited morphological changes in leaf shape and seedless fruit—a characteristic of parthenocarpic tomato. And the segregated next generation (T1) also showed a severe phenotype associated with the homozygous mutated genome. The system developed here could be applied to produce parthenocarpic tomato in a wide variety of cultivars, as well as other major horticultural crops, using this precise and rapid breeding technique

High Serum Advanced Glycation End Products Are Associated with Decreased Insulin Secretion in Patients with Type 2 Diabetes: A Brief Report

Advanced glycation end products (AGEs) are important in the pathophysiology of type 2 diabetes mellitus (T2DM). They directly cause insulin secretory defects in animal and cell culture models and may promote insulin resistance in nondiabetic subjects. We have developed a highly sensitive liquid chromatography-tandem mass spectrometry method for measuring AGEs in human serum. Here, we use this method to investigate the relationship between AGEs and insulin secretion and resistance in patients with T2DM. Methods. Our study involved 15 participants with T2DM not on medication and 20 nondiabetic healthy participants. We measured the AGE carboxyethyllysine (CEL), carboxymethyllysine (CML), and methyl-glyoxal-hydro-imidazolone (MG-H1). Plasma glucose and insulin were measured in these participants during a meal tolerance test, and the glucose disposal rate was measured during a euglycemic-hyperinsulinemic clamp. Results. CML and CEL levels were significantly higher in T2DM than non-DM participants. CML showed a significant negative correlation with insulin secretion, HOMA-%B, and a significant positive correlation with the insulin sensitivity index in T2DM participants. There was no correlation between any of the AGEs measured and glucose disposal rate. Conclusions. These results suggest that AGE might play a role in the development or prediction of insulin secretory defects in type 2 diabetes

Body mass index >= 23 is a risk factor for insulin resistance and diabetes in Japanese people: A brief report

Background: Screening for undiagnosed type 2 diabetes mellitus is recommended for Asian Americans with a body mass index ≥23. However, the optimal body mass index cut-off score for predicting the risk of diabetes mellitus in Japanese people is not well known. The aim of this study was to determine the best body mass index cut-off score for predicting insulin resistance and diabetes mellitus in the Japanese population. Methods: This study had two parts, a clinical investigation and a retrospective observational investigation. In the clinical part of the study, 58 participants (26 with type 2 diabetes mellitus and 32 non-diabetics) underwent a hyperinsulinemic-euglycemic clamp from which their glucose disposal rate was measured. For the retrospective part of the study, medical check-up data from 88,305 people in the Tottori Prefecture were analyzed for clinical evidence of diabetes mellitus. The optimal BMI cut-off scores for prediction of insulin resistance and diabetes mellitus were determined. Results: In the clamp study, the optimal body mass index cut-off score to predict insulin resistance in non-diabetic patients was 22.7. All participants with type 2 diabetes mellitus were insulin resistant, and the optimal body mass index cut-off score for prediction of severe insulin resistance was 26.2. When the data from the type 2 diabetic and the non-diabetic participants were combined, the optimal body mass index cut-off score for prediction of insulin resistance was 23.5. Analysis of 88,305 medical check-up records yielded an optimal body mass index cut-off score for prediction of diabetes mellitus of 23.6. Conclusions: These results suggest that having a body mass index ≥23 is a risk factor for insulin resistance and diabetes mellitus in the Japanese population

Rapid breeding of parthenocarpic tomato plants using CRISPR/Cas9

Parthenocarpy in horticultural crop plants is an important trait with agricultural value for various industrial purposes as well as direct eating quality. Here, we demonstrate a breeding strategy to generate parthenocarpic tomato plants using the CRISPR/Cas9 system. We optimized the CRISPR/Cas9 system to introduce somatic mutations effectively into SlIAA9—a key gene controlling parthenocarpy—with mutation rates of up to 100% in the T0 generation. Furthermore, analysis of off-target mutations using deep sequencing indicated that our customized gRNAs induced no additional mutations in the host genome. Regenerated mutants exhibited morphological changes in leaf shape and seedless fruit—a characteristic of parthenocarpic tomato. And the segregated next generation (T1) also showed a severe phenotype associated with the homozygous mutated genome. The system developed here could be applied to produce parthenocarpic tomato in a wide variety of cultivars, as well as other major horticultural crops, using this precise and rapid breeding technique

CPR-IR is an insulin resistance index that is minimally affected by hepatic insulin clearance-A preliminary research

Background: Increased hepatic insulin clearance (HIC) is important in the pathophysiology of type 2 diabetes mellitus (T2DM). The aim of this study is to analyze an effective insulin resistance (IR) index that is minimally affected by HIC. Methods: Our study involved 20 participants with T2DM and 21 healthy participants without diabetes (Non-DM). Participants underwent a meal tolerance test from which plasma glucose, insulin and serum C-peptide immunoreactivity (CPR) were measured, and HOMA-IR and HIC were calculated. Participants then underwent a hyperinsulinemic-euglycemic clamp from which the glucose disposal rate (GDR) was measured. Results: The index CPR-IR = 20/(fasting CPR × fasting plasma glucose) was correlated more strongly with GDR, than was HOMA-IR, and CPR-IR could be used to estimate GDR. In T2DM participants with HIC below the median, HOMA-IR and CPR-IR were equally well correlated with GDR. In T2DM with high HIC, CPR-IR correlated with GDR while HOMA-IR did not. In Non-DM, CPR-IR and HOMA-IR were equally well correlated with GDR regardless of HIC. The mean HIC value in T2DM was significantly higher than that of Non-DM. Conclusions: CPR-IR could be a simple and effective index of insulin resistance for patients with type 2 diabetes that is minimally affected by HIC