Similar Seed Composition Phenotypes Are Observed From CRISPR-Generated In-Frame and Knockout Alleles of a Soybean KASI Ortholog.

The ß-ketoacyl-[acyl carrier protein] synthase 1 (KASI) gene has been shown in model plant systems to be critical for the conversion of sucrose to oil. A previous study characterized the morphological and seed composition phenotypes associated with a reciprocal chromosomal translocation that disrupted one of the KASI genes in soybean. The principle findings of this work included a wrinkled seed phenotype, an increase in seed sucrose, a decrease in seed oil, and a low frequency of transmission of the translocation. However, it remained unclear which, if any, of these phenotypes were directly caused by the loss of KASI gene function, as opposed to the chromosomal translocation or other associated factors. In this study, CRISPR/Cas9 mutagenesis was used to generate multiple knockout alleles for this gene, and also one in-frame allele. These soybean plants were evaluated for morphology, seed composition traits, and genetic transmission. Our results indicate that the CRISPR/Cas9 mutants exhibited the same phenotypes as the chromosomal translocation mutant, validating that the observed phenotypes are caused by the loss of gene function. Furthermore, the plants harboring homozygous in-frame mutations exhibited similar phenotypes compared to the plants harboring homozygous knockout mutations. This result indicates that the amino acids lost in the in-frame mutant are essential for proper gene function. In-frame edits for this gene may need to target less essential and/or evolutionarily conserved domains in order to generate novel seed composition phenotypes.

Integration, abundance, and transmission of mutations and transgenes in a series of CRISPR/Cas9 soybean lines.

BACKGROUND: As with many plant species, current genome editing strategies in soybean are initiated by stably transforming a gene that encodes an engineered nuclease into the genome. Expression of the transgene results in a double-stranded break and repair at the targeted locus, oftentimes resulting in mutation(s) at the intended site. As soybean is a self-pollinating species with 20 chromosome pairs, the transgene(s) in the T0 plant are generally expected to be unlinked to the targeted mutation(s), and the transgene(s)/mutation(s) should independently assort into the T1 generation, resulting in Mendellian combinations of transgene presence/absence and allelic states within the segregating family. This prediction, however, is not always consistent with observed results. RESULTS: In this study, we investigated inheritance patterns among three different CRISPR/Cas9 transgenes and their respective induced mutations in segregating soybean families. Next-generation resequencing of four T0 plants and four T1 progeny plants, followed by broader assessments of the segregating families, revealed both expected and unexpected patterns of inheritance among the different lineages. These unexpected patterns included: (1) A family in which T0 transgenes and mutations were not transmitted to progeny; (2) A family with four unlinked transgene insertions, including two respectively located at paralogous CRISPR target break sites; (3) A family in which mutations were observed and transmitted, but without evidence of transgene integration nor transmission. CONCLUSIONS: Genome resequencing provides high-resolution of transgene integration structures and gene editing events. Segregation patterns of these events can be complicated by several potential mechanisms. This includes, but is not limited to, plant chimeras, multiple unlinked transgene integrations, editing of intended and paralogous targets, linkage between the transgene integration and target site, and transient expression of the editing reagents without transgene integration into the host genome.

CRISPR/Cas9 and TALENs generate heritable mutations for genes involved in small RNA processing of Glycine max and Medicago truncatula.

Processing of double-stranded RNA precursors into small RNAs is an essential regulator of gene expression in plant development and stress response. Small RNA processing requires the combined activity of a functionally diverse group of molecular components. However, in most of the plant species, there are insufficient mutant resources to functionally characterize each encoding gene. Here, mutations in loci encoding protein machinery involved in small RNA processing in soya bean and Medicago truncatula were generated using the CRISPR/Cas9 and TAL-effector nuclease (TALEN) mutagenesis platforms. An efficient CRISPR/Cas9 reagent was used to create a bi-allelic double mutant for the two soya bean paralogous Double-stranded RNA-binding2 (GmDrb2a and GmDrb2b) genes. These mutations, along with a CRISPR/Cas9-generated mutation of the M. truncatula Hua enhancer1 (MtHen1) gene, were determined to be germ-line transmissible. Furthermore, TALENs were used to generate a mutation within the soya bean Dicer-like2 gene. CRISPR/Cas9 mutagenesis of the soya bean Dicer-like3 gene and the GmHen1a gene was observed in the T0 generation, but these mutations failed to transmit to the T1 generation. The irregular transmission of induced mutations and the corresponding transgenes was investigated by whole-genome sequencing to reveal a spectrum of non-germ-line-targeted mutations and multiple transgene insertion events. Finally, a suite of combinatorial mutant plants were generated by combining the previously reported Gmdcl1a, Gmdcl1b and Gmdcl4b mutants with the Gmdrb2ab double mutant. Altogether, this study demonstrates the synergistic use of different genome engineering platforms to generate a collection of useful mutant plant lines for future study of small RNA processing in legume crops.

Colorectal cancer educational intervention targeting latino patients attending a community health center.

OBJECTIVE: Colorectal cancer (CRC) is the third leading cause of cancer death for Latino men and women; and Latinos are more likely to be diagnosed at a later stage, which is most likely due to underutilization of CRC preventive screening. The purpose of this study was to determine whether a brief, clinic-based intervention by a community health advisor (CHA) would increase CRC knowledge compared with traditional educational methodologies (eg, use of print materials). METHODS: Latino adults 50 years and older attending a San Diego community health center were recruited while waiting for their primary care provider routine visit and were randomly assigned to receive 1 of 3 CRC educational interventions: community health advisor (CHA) plus CRC educational brochure (CHA intervention group), CRC educational brochure (minimal intervention group), or 5-a-day nutrition brochure (usual care). CRC knowledge was assessed before and after the primary care provider visit for 130 participants. RESULTS: Results demonstrate that the CRC educational brochure (minimal intervention group) was effective at increasing CRC screening knowledge as compared to usual care. CONCLUSIONS: Future research is needed to explore innovative health education strategies that improve knowledge and subsequent CRC screening behaviors among low-income, low-literacy, unacculturated Latinos.

Truncal adiposity and lung function in older black women.

The increase in adiposity associated with aging is a concern in older adults, especially as it relates to the risk for ventilatory complications. Therefore, the specific aim of this study was to determine the association of various measures of abdominal adiposity with lung function in a sample of older healthy black women. Participants (n=27) had no history of diabetes or respiratory disease. The mean age was 67 years. Lung function was measured by spirometry using percent of predicted values for forced vital capacity (FVC) and forced expiratory volume in 1 second (FEV1). Body fat was measured using a three-dimensional photonic scanner and dual energy X-ray absorptiometry (DXA). Correlation analyses show that percent body fat in the trunk (%TF) is significantly associated with percent predicted FVC (r=-0.38; p<0.05). No association was observed between anthropometric indices of truncal adiposity and lung function. Results of this study show that truncal fat mass measured by DXA is more strongly associated with lung function than anthropometric indices of truncal adiposity in this sample of women.

Our lives were healthier before: focus groups with African American, American Indian, Hispanic/Latino, and Hmong people with diabetes.

Focus groups were conducted to explore health-related beliefs and experiences of African American, Hispanic/Latino, American Indian, and Hmong people with diabetes and engage community members in improving diabetes care and education for these populations. Eighty participants attended 12 focus groups, 3 per population. Major themes were loss of health attributed to modern American lifestyles, lack of confidence in the medical system, and the importance of spirituality. Participants recommended improvements in the areas of health care, diabetes education, social support, and community action. Their recommendations emphasize the importance of respectful, knowledgeable health care providers; culturally responsive diabetes education for people with diabetes and their families; and broad-based community action. These recommendations align with current public health priorities and medical knowledge. It is proposed that healthy traditions from diverse populations can be leveraged to improve the health of all people with diabetes.