Staahl, B. | bioRxiv Science

bioRxiv2021

Phylogeny of the ingoid clade (Caesalpinioideae, Fabaceae), based on nuclear and plastid data

We investigated generic relationships in the ingoid clade (Fabaceae) (sensu Koenen & al. 2020a), with main focus on genera with a taxonomic history in Calliandra s.l. of the tribe Ingeae (i.e. Afrocalliandra, Calliandra s.s., Sanjappa, Thailentadopsis, Viguieranthus, Zapoteca), and three genera of the tribe Acacieae (i.e., Acacia, Acaciella, Senegalia). The nuclear ribosomal ETS and ITS, and the plastid matK, trnL-trnF and ycf1 DNA-regions were analysed for 246 representatives from 36 genera using maximum likelihood as implemented in IQ-tree. The results show an Ingeae-Acacia clade within the ingoid clade, resolved in three major clades. Clade 1 (Calliandra s.s. and Afrocalliandra) is sister to clades 2 and 3. Clade 2 comprises Faidherbia, Sanjappa, Thailentadopsis, Viguieranthus and Zapoteca. Clade 3 comprises the remaining genera of the Ingeae, plus Acacia. The ingoid genus Senegalia is excluded from the Ingeae-Acacia clade. Acaciella is sister to the remaining ingoid clade when nuclear ribosomal data is included in the analyses, but included in the Ingeae-Acacia clade based on plastid data. Acacia and perhaps also Acaciella are thus nested within Ingeae. Species traditionally referred to Calliandra (Calliandra s.l.) are resolved in two clades, and the "Calliandra-pod" has apparently evolved independently several times.

evolutionary biology↗

bioRxiv2020

Comprehensive deletion landscape of CRISPR-Cas9 identifies minimal RNA-guided DNA-binding modules

Proteins evolve through the modular rearrangement of elements known as domains. It is hypothesized that extant, multidomain proteins are the result of domain accretion, but there has been limited experimental validation of this idea. Here, we introduce a technique for genetic minimization by iterative size-exclusion and recombination (MISER) that comprehensively assays all possible deletions of a protein. Using MISER, we generated a deletion landscape for the CRISPR protein Cas9. We found that Cas9 can tolerate large single deletions to the REC2, REC3, HNH, and RuvC domains, while still functioning in vitro and in vivo, and that these deletions can be stacked together to engineer minimal, DNA-binding effector proteins. In total, our results demonstrate that extant proteins retain significant modularity from the accretion process and, as genetic size is a major limitation for viral delivery systems, establish a general technique to improve genome editing and gene therapy-based therapeutics.

molecular biology↗