A variety of changes, including CRISPR/Cas9-mediated deletions, in CENH3 lead to haploid induction on outcrossing.

Creating true-breeding lines is a critical step in plant breeding. Novel, completely homozygous true-breeding lines can be generated by doubled haploid technology in single generation. Haploid induction through modification of the centromere-specific histone 3 variant (CENH3), including chimeric proteins, expression of non-native CENH3 and single amino acid substitutions, has been shown to induce, on outcrossing to wild type, haploid progeny possessing only the genome of the wild-type parent, in Arabidopsis thaliana. Here, we report the characterization of 31 additional EMS-inducible amino acid substitutions in CENH3 for their ability to complement a knockout in the endogenous CENH3 gene and induce haploid progeny when pollinated by the wild type. We also tested the effect of double amino acid changes, which might be generated through a second round of EMS mutagenesis. Finally, we report on the effects of CRISPR/Cas9-mediated in-frame deletions in the αN helix of the CENH3 histone fold domain. Remarkably, we found that complete deletion of the αN helix, which is conserved throughout angiosperms, results in plants which exhibit normal growth and fertility while acting as excellent haploid inducers when pollinated by wild-type pollen. Both of these technologies, CRISPR mutagenesis and EMS mutagenesis, represent non-transgenic approaches to the generation of haploid inducers.

Increased Hospital Encounters in Individuals With Spina Bifida Compared to the General Population: Statewide Healthcare Utilization in California From 1995-2017.

PURPOSE: To understand healthcare utilization by comparing hospital encounters among individuals with Spina Bifida and the general population and to identify the factors associated with utilization. METHOD: Using the Department of Health Care Access and Information database (1995-2017), individuals with Spina Bifida were identified and matched to controls by birth year. The primary outcome measures were the number of hospital encounters (stratified as ≤ 2 vs ≥ 3 encounters) and the time between the first and second encounters. Univariate, multivariate and sub-group analyses were performed to identify factors associated with ≥ 3 encounters. RESULTS: When compared to controls, individuals with Spina Bifida had more ≥ 3 hospital encounters (69% vs 29%), spent more days in the hospital (58 days vs 13 days), and had a higher average charge per hospital stay ($433,537 vs $99,975) (P < .001 for all). After adjusting for covariates, we found that having Spina Bifida was associated with increased hospital encounters compared to controls (OR = 3.95, 95% CI = 3.77, 4.14, P < .001). Individuals with Spina Bifida were found to have less time between their first and second encounters (2.5 vs 3.3 years, P < .001). Within the Spina Bifida population, sex, race, ethnicity, comorbidities, and non-private insurance were associated with ≥ 3 encounters. CONCLUSIONS: Spina Bifida is associated with more hospital encounters and fewer days between first and second encounters compared to the general population. These findings highlight factors driving increased utilization of resources thereby empowering providers to better support this vulnerable population.