Britt, A. | bioRxiv Science

bioRxiv2021

Biased removal and loading of centromeric histone H3 during reproduction underlies uniparental genome elimination

Uniparental genome elimination is a dramatic case of centromeric failure, resulting in the postzygotic loss of a parental chromosome set. Genome partitioning during cell division relies on centromere attachment to spindle fibers through kinetochores. Centromeres are epigenetically specified by CENP-A (CENH3), a conserved centromeric specific histone H3 variant. In Arabidopsis, CENH3 modification results in haploid inducers, whose genome is eliminated frequently when crossed to the wild type. To investigate the underlying mechanism, we dissected the timing and molecular features of genome elimination. In zygotes and early embryos from genome elimination crosses, CENH3 occupied only the centromeres contributed by the wild-type parent. Haploid inducer chromosomes had defective kinetochores and missegregated, often forming micronuclei. This uniparental loss of centromere identity is initiated by the removal of altered CENH3 at fertilization, while wild-type CENH3 persists and maintains strong centromeric identity. Weak centromeres were capable of rebuilding functional kinetochores, but often failed when in competition with normal ones. We induced a similar weak state by mitotic dilution of wild-type CENH3. Furthermore, weakness was suppressed by crosses of haploid inducers to other variants of haploid inducers, and enhanced by mutations in VIM1, a ubiquitin ligase known to modify CENH3 and centromeric DNA methylation.. The differential stability of altered CENH3 during reproduction has important genetic and evolutionary implications.

cell biology↗

bioRxiv2017

A dual sgRNA approach for functional genomics in Arabidopsis thaliana

Reverse genetics uses loss-of-function alleles to interrogate gene function. The advent of CRISPR/Cas9-based gene editing now allows to generate knock-out alleles for any gene and entire gene families. Even in the model plant Arabidopsis thaliana, gene editing is welcomed as T-DNA insertion lines do not always generate null alleles. Here, we show efficient generation of heritable mutations in Arabidopsis using CRISPR/Cas9 with a workload similar to generating overexpression lines. We obtain Cas9 null-segregants with bi-allelic mutations in the T2 generation. Out of seven new mutant alleles we report here, one allele for GRXS17, the ortholog of human GRX3/PICOT, did not phenocopy previously characterized nulls. Notwithstanding, the mutation caused a frameshift and triggered nonsense-mediated decay. We demonstrate that our workflow is also compatible with a dual sgRNA approach in which a gene is targeted by two sgRNAs simultaneously. This paired nuclease method can result in a more reliable loss-of-function alleles that lack a large essential part of the gene. The ease in the CRISPR/Cas9 workflow should help in the eventual generation of true null alleles of every gene in the Arabidopsis genome, which will advance both basic and applied plant research.\n\nOne-sentence summaryWe present a dual sgRNA approach to delete Arabidopsis gene 34 fragments in order to obtain reliable functional knock-outs.

plant biology↗