Paulraj, S. | bioRxiv Science

bioRxiv2020

Chromatin Regulates Bipartite-Classified Small RNA Expression to Maintain Epigenome Homeostasis in Arabidopsis

Eukaryotic genomes are partitioned into euchromatic and heterochromatic domains to regulate gene expression and other fundamental cellular processes. However, chromatin is dynamic during growth and development, and must be properly re-established after its decondensation. Small interfering RNAs (siRNAs) promote heterochromatin formation in eukaryotes, but little is known about how chromatin regulates siRNA transcription. We demonstrated that thousands of transposable elements (TEs) produce exceptionally high levels of siRNAs in Arabidopsis thaliana embryos. Depending on whether they are located in euchromatic or heterochromatic regions of the genome, bipartite-classified TEs generate siRNAs throughout embryogenesis according to two distinct patterns. siRNAs are transcribed in embryos and required to direct the re-establishment of DNA methylation on TEs from which they are derived in the new generation. Decondensed chromatin also permits the production of 24-nt siRNAs from heterochromatic TEs during post-embryogenesis, and siRNA production from bipartite-classified TEs is controlled by their chromatin states. Decondensation of heterochromatin in response to developmental, and perhaps environmental, cues promotes the transcription and function of siRNAs in plants. Our results indicate that chromatin-mediated siRNA transcription provides a cell-autonomous homeostatic control mechanism to reconstitute pre-existing chromatin states during growth and development including those that ensure silencing of TEs in the future germ line.

genomics↗

bioRxiv2020

Biochemically diverse CRISPR-Cas9 orthologs

CRISPR-Cas9 nucleases are abundant in microbes. To explore this largely uncharacterized diversity, we applied cell-free biochemical screens to rapidly assess the protospacer adjacent motif (PAM) and guide RNA (gRNA) requirements of novel Cas9 proteins. This approach permitted the characterization of 79 Cas9 orthologs with at least 7 distinct classes of gRNAs and 50 different PAM sequence requirements. PAM recognition spanned the entire spectrum of T-, A-, C-, and G-rich nucleotides ranging from simple di-nucleotide recognition to complex sequence strings longer than 4. Computational analyses indicated that most of this diversity came from 4 groups of interrelated sequences providing new insight into Cas9 evolution and efforts to engineer PAM recognition. A subset of Cas9 orthologs were purified and their activities examined further exposing additional biochemical diversity. This constituted both narrow and broad ranges of temperature dependence, staggered-end DNA target cleavage, and a requirement for longer stretches of homology between gRNA and DNA target to function robustly. In all, the diverse collection of Cas9 orthologs presented here sheds light on Cas9 evolution and provides a rich source of PAM recognition and other potentially desirable properties that may be mined to expand the genome editing toolbox with new RNA-programmable nucleases.

biochemistry↗