Potential high-frequency off-target mutagenesis induced by CRISPR/Cas9 in Arabidopsis and its prevention.

KEY MESSAGE: We present novel observations of high-specificity SpCas9 variants, sgRNA expression strategies based on mutant sgRNA scaffold and tRNA processing system, and CRISPR/Cas9-mediated T-DNA integrations. Specificity of CRISPR/Cas9 tools has been a major concern along with the reports of their successful applications. We report unexpected observations of high frequency off-target mutagenesis induced by CRISPR/Cas9 in T1 Arabidopsis mutants although the sgRNA was predicted to have a high specificity score. We also present evidence that the off-target effects were further exacerbated in the T2 progeny. To prevent the off-target effects, we tested and optimized two strategies in Arabidopsis, including introduction of a mCherry cassette for a simple and reliable isolation of Cas9-free mutants and the use of highly specific mutant SpCas9 variants. Optimization of the mCherry vectors and subsequent validation found that fusion of tRNA with the mutant rather than the original sgRNA scaffold significantly improves editing efficiency. We then examined the editing efficiency of eight high-specificity SpCas9 variants in combination with the improved tRNA-sgRNA fusion strategy. Our results suggest that highly specific SpCas9 variants require a higher level of expression than their wild-type counterpart to maintain high editing efficiency. Additionally, we demonstrate that T-DNA can be inserted into the cleavage sites of CRISPR/Cas9 targets with high frequency. Altogether, our results suggest that in plants, continuous attention should be paid to off-target effects induced by CRISPR/Cas9 in current and subsequent generations, and that the tools optimized in this report will be useful in improving genome editing efficiency and specificity in plants and other organisms.

MISSA 2.0: an updated synthetic biology toolbox for assembly of orthogonal CRISPR/Cas systems.

Efficient generation of plants carrying mutations in multiple genes remains a challenge. Using two or more orthogonal CRISPR/Cas systems can generate plants with multi-gene mutations, but assembly of these systems requires a robust, high-capacity toolkit. Here, we describe MISSA 2.0 (multiple-round in vivo site-specific assembly 2.0), an extensively updated toolkit for assembly of two or more CRISPR/Cas systems. We developed a novel suicide donor vector system based on plasmid RK2, which has much higher cloning capacity than the original, plasmid R6K-based system. We validated the utility of MISSA 2.0 by assembling multiple DNA fragments into the E. coli chromosome, and by creating transgenic Arabidopsis thaliana that constitutively or inducibly overexpress multiple genes. We then demonstrated that the higher cloning capacity of the RK2-derived MISSA 2.0 donor vectors facilitated the assembly of two orthogonal CRISPR/Cas systems including SpCas9 and SaCas9, and thus facilitated the creation of transgenic lines harboring these systems. We anticipate that MISSA 2.0 will enable substantial advancements in multiplex genome editing based on two or more orthogonal CRISPR/Cas9 systems, as well as in plant synthetic biology.

Egg cell-specific promoter-controlled CRISPR/Cas9 efficiently generates homozygous mutants for multiple target genes in Arabidopsis in a single generation.

Arabidopsis mutants produced by constitutive overexpression of the CRISPR/Cas9 genome editing system are usually mosaics in the T1 generation. In this study, we used egg cell-specific promoters to drive the expression of Cas9 and obtained non-mosaic T1 mutants for multiple target genes with high efficiency. Comparisons of 12 combinations of eight promoters and two terminators found that the efficiency of the egg cell-specific promoter-controlled CRISPR/Cas9 system depended on the presence of a suitable terminator, and the composite promoter generated by fusing two egg cell-specific promoters resulted in much higher efficiency of mutation in the T1 generation compared with the single promoters.

A CRISPR/Cas9 toolkit for multiplex genome editing in plants.

BACKGROUND: To accelerate the application of the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/ CRISPR-associated protein 9) system to a variety of plant species, a toolkit with additional plant selectable markers, more gRNA modules, and easier methods for the assembly of one or more gRNA expression cassettes is required. RESULTS: We developed a CRISPR/Cas9 binary vector set based on the pGreen or pCAMBIA backbone, as well as a gRNA (guide RNA) module vector set, as a toolkit for multiplex genome editing in plants. This toolkit requires no restriction enzymes besides BsaI to generate final constructs harboring maize-codon optimized Cas9 and one or more gRNAs with high efficiency in as little as one cloning step. The toolkit was validated using maize protoplasts, transgenic maize lines, and transgenic Arabidopsis lines and was shown to exhibit high efficiency and specificity. More importantly, using this toolkit, targeted mutations of three Arabidopsis genes were detected in transgenic seedlings of the T1 generation. Moreover, the multiple-gene mutations could be inherited by the next generation. CONCLUSIONS: We developed a toolkit that facilitates transient or stable expression of the CRISPR/Cas9 system in a variety of plant species, which will facilitate plant research, as it enables high efficiency generation of mutants bearing multiple gene mutations.

Relationship between thyroid-stimulating hormone and blood pressure in the middle-aged and elderly population.

INTRODUCTION: Hypothyroidism and subclinical hypothyroidism may be associated with hypertension and metabolic syndrome. The aim of this study was to investigate the relationship between thyroid-stimulating hormone (TSH) and blood pressure, as well as the relationship between thyroid function and insulin resistance in middle-aged and elderly Chinese. METHODS: This was a cross-sectional, community-based study. Serum TSH, fasting glucose and insulin were measured in 2,988 subjects aged 35-80 years. Logistic regression analysis was used to identify the risk factors for hypertension. Analysis of variance and multiple linear regression analysis were performed to characterise the relationship among TSH, insulin resistance and blood pressure. RESULTS: Higher serum TSH concentration was found to be an independent risk factor for hypertension in females (odds ratio 1.4, 95% confidence interval 1.02-1.93; p-value = 0.039). The female group with subclinical hypothyroidism and high normal TSH (2.5-4.8 mIU/L) were more susceptible to high blood pressure than those with low normal TSH (0.3-2.5 mIU/L) (p-value < 0.05). After adjustment for waist-hip ratio and body mass index, neither the correlation between blood pressure and homeostasis model assessment of insulin resistance (HOMA-IR) nor the correlation between TSH and HOMA-IR were found to be significant in this study. CONCLUSION: This study provides evidence that both subclinical hypothyroidism and high normal TSH are independent risk factors for hypertension in middle-aged and elderly Chinese women.

Prevalence and awareness of hypertension in Gansu Gannan Tibetan adult population / 中华心血管病杂志

<p><b>OBJECTIVE</b>To investigate the prevalence and awareness status of hypertension in adult Gansu Gannan Tibetan population.</p><p><b>METHODS</b>A total of 6948 adult (aged 18 and over) Tibetan residents were selected by random cluster multistage method. Participants were divided into city, rural dwellers and rural nomads groups. Questionnaires were collected and blood pressure measured from all participants.</p><p><b>RESULTS</b>Prevalence of hypertension in this district was 24.6% (1708/6948), of which city, rural dwellers and nomadic areas were 25.3% (848/3340), 22.7% (628/2765), 27.5% (232/843), respectively. Prevalence of hypertension was significantly higher in city than in rural dwellers group (P<0.05), significantly higher in rural nomads group than in city and rural dwellers groups (all P<0.05). Awareness, treatment and control rates of hypertension in city group were 35.7% (303/848), 25.1% (213/848), 6.9% (59/848), respectively; in rural dwellers group were 28.3% (178/628), 19.3% (121/628), 4.7% (30/628), respectively; in rural nomads group were 16.8% (39/232), 8.6% (20/232), 2.2% (5232), respectively. Rates of awareness, treatment and control were significantly lower in rural dwellers than in city group (all P<0.01), and significantly lower in rural nomads group than in city and rural dwellers groups (all P<0.01).</p><p><b>CONCLUSIONS</b>Prevalence of hypertension in Gansu Gannan Tibetan population aged 18 and over is high. Awareness, treatment and control rates of hypertension are low, especially in rural dwellers and rural nomads residents.</p>

Prevalence and risk factors of peripheral arterial disease in diabetic patients over 50 years old in China.

OBJECTIVE: To investigate the prevalence of peripheral arterial disease (PAD) in China type 2 diabetic patients and to demonstrate the relationships between putative risk factors and PAD. METHODS: In total 1,397 type 2 diabetic patients aged 50 years and older were enrolled and determined ankle-brachial index (ABI) and brachial-ankle pulse wave velocity (baPWV) in 15 Class III Grade A hospitals in 7 major cities of China. RESULTS: Mean patient age was 63.7 +/- 8.2 years and mean duration of diabetes mellitus was 9.39 +/- 7.4 years. Two hundreds and seventy-two (19.47%) patients were diagnosed as PAD by ABI < 0.9, 122 (18.37%) in male and 150 (20.46%) in female. PAD patients had a significantly longer duration of diabetes mellitus, higher hemoglobin A1c, and a significantly lower mean body mass index than non-PAD ones. Aging, smoking, and systolic blood pressure were found to be positively related with the prevalence of PAD. In terms of lipid profiles, no variable was found to relate with PAD. Notably, baPWV showed as the same significant guiding index for PAD, almost matched with ABI. CONCLUSIONS: PAD is a common complication in China type 2 diabetic patients. Therefore, PAD screening and treatment should be emphasized for diabetic patients with high risk factors.

[Analysis of peripheral arterial obstructive disease related factors among diabetic population aged > or = 50].

OBJECTIVE: To analyze the peripheral arterial obstructive disease (PAD) related factors among diabetic population aged > or = 50 in China. METHODS: The clinical data of 1397 diabetic patients aged > or = 50 with at least one of the following risk factors: smoking, hypertension, and hyperlipidemia, from 15 Class III Grade A hospitals in 7 major cities of China were collected. Diagnosis of PAD was based on the ankle brachial index (ABI) < 0.9, and diagnosis of arteriosclerosis was based on pulse wave velocity (PWV) > 1400 cm/s. Regression studies were made to analyze the relations among PAD and various risk factors: age, sex, body mass index (BMI), smoking, hypertension, hyperlipidemia, history of cerebral vascular disease (CVD), history of ischemia heart disease (IHD) etc. RESULTS: The current prevalence rate of PAD was 19.47% among the 1397 patients, 18.3% (122/664) among the male patients, and 20.4% (150/733) among the female patients. The prevalence of PAD in the patients aged > or = 70 was as high as 31.9%. The duration of diabetes course was positively correlated with the prevalence of PAD (chi2 = 11.9, P = 0.0026). The ABI abnormality rate was 15.78% among those with a diabetic course of 5 years and was 23.84% among those with a diabetic course of 10 years. The abnormal ABI rate of the patients with CVD was 30.57%, significantly higher than that of hose without CVD (17.29%, chi2 = 21.49, P < 0.0001). The abnormal ABI rate of the patients with IHD was 24.64%, significantly higher than that of the patients without IHD (18.20%, chi2 = 5.85, P = 0.0155). The HbA1c value of the PAD patients was significantly higher than that of the patients without PAD (chi2 = 5.10, P = 0.0239) Odd risk analysis showed that age increase of 10 years increased the PAD risk by 1.64 times (OR = 1.6444, P = 0.0001). The PAD risk of the smokers was 1.68 times higher than that of the non-smokers (OR = 1.6852, P = 0.0001). Increase of 10 mm Hg in systolic blood pressure (SBP) increased the PAD risk by 1.19 times (OR = 1.1926, P = 0.01). The PAD risk of the patients with abnormal HbAlc was 2.44 times higher than that of the patients with normal HbA1c (OR = 2.4473, P = 0.0001). One-year's increase of the hypertension course increased the PAD risk by 1.02 times (OR = 1.0194, P = 0.03). Logistic analysis indicated that the relations among PWV and the risk factors were almost the same among ABI abnormality and the risk factors. CONCLUSION: Approximately one fifth of diabetic patients aged > or = 50 in China suffer from PAD. Age, course of diabetes, blood glucose level, SBP, IHD, and CVD are risk factors for PAD. Early intervention and treatment of hypertension and hyperglycemia, and quitting smoking are important in reducing the occurrence of PAD. ABI and PWV are not only diagnostic means for PAD, but also alarm guide indexes for cerebral vascular disease (CVD).

[Multicenter, randomized, positive-controlled clinical study for the effects of nerotropin on diabetic neuropathy].

OBJECTIVE: To estimate the effects of Neurotropin in the treatment of neuralgia and numbness in type 2 diabetes mellitus patients with diabetic peripheral neuropathy (DPN), and compare with Methycobal. METHODS: Ninety-five patients with DPN from 4 hospitals in shanghai were randomly assigned to two groups: 49 cases in Neurotropin group (6 ml, IV, QD for 2 weeks, then, 2 tablets, BID for another 2 weeks). 46 cases in Methycobal group (1 ml, IM, QD for 2 weeks, then, 1 tablet TID for another 2 weeks. RESULTS: Neurotropin significantly improved neuralgia in type 2 diabetes mellitus with DPN, the efficacy rate was 67.3% in the first week, and 87.0% in the 4th week, comparing with 34.8% and 68.5% in the control group. The efficacy rate for numbness was 58.7% in the study group after 4 weeks therapy, and 69.5% in control group. In the whole 4 weeks therapeutic course, neuralgia and numbness were improved in the two groups, and the improved rate in Neurotropin group is high than in the control group (P < 0.01). CONCLUSION: Neurotropin had good effects in the treatment of diabetic neuralgia and numbness, the efficacy of Neurotropin in improving the senses of neuralgia is better than Methecobal., and similar with Methecobal in treating numbness.