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Biology subjects

Ajenjo, M.

Publications and source records attributed to Ajenjo, M..

2 recordsLinked to original sources
bioRxiv2020

Edition of complex gene families in tobacco with GoldenBraid 4.0, a multipurpose web-based platform for plant genome engineering

CRISPR/Cas ability to target several loci simultaneously (multiplexing) is a game-changer in plant breeding. Multiplexing not only accelerates trait pyramiding but also can unveil traits hidden by functional redundancy in polyploid crops. Furthermore, multiplexing enhances dCas-based programmable gene expression and enables cascade-like gene regulation. However, multiplex constructs comprising tandemly arrayed gRNAs are difficult to assemble, this hampering more widespread use. Here we present a comprehensive upgrade of the popular cloning platform GoldenBraid (GB), in which, on top of its classical multigene cloning software, we integrate new assembly tools for two-dimensions gRNA multiplexing with both Cas9 and Cas12a, using the gRNA-tRNA-spaced and the gRNA unspaced approaches, respectively. As functional validation, we show, among others, the assembly of up to 17 tandemly-arrayed gRNAs constructs against a subset of the Squamosa-Promoter Binding Protein-Like (SPL) gene family in tobacco. With these constructs we generated a collection of Cas9-free SPL mutants harboring up to 9 biallelic mutations in a single generation. The functionality of GB-assembled dCas9 and dCas12a-based CRISPR activators and repressors using single and multiplexing gRNAs is also validated. With the incorporation of the new CRISPR tools and parts collection, GB4.0 turns an unprecedentedly comprehensive open platform for plant genetic engineering.

molecular biology↗
bioRxiv2018

Strong gene activation with genome-wide specificity using a new orthogonal CRISPR/Cas9-based Programmable Transcriptional Activator.

Synthetic Biology (SynBio) aims at rewiring plant metabolic and developmental programs with orthogonal regulatory circuits. This endeavour requires new molecular tools able to interact with endogenous factors in a potent yet at the same time highly specific manner. A promising new class of SynBio tools that could play this function are the synthetic transcriptional activators based on CRISPR/Cas9 architecture, which combine autonomous activation domains (ADs) capable of recruiting the cells transcription machinery, with the easily customizable DNA-binding activity of nuclease-inactivated Cas9 protein (dCas9), creating so-called Programmable Transcriptional Activators (PTAs). In search for optimized dCas9-PTAs we performed a combinatorial analysis with seven different ADs arranged in four different protein/RNA architectures. This analysis resulted in the selection of a new dCas9-PTA with improved features as compared with previously reported activators. The new synthetic riboprotein, named dCasEV2.1, combines EDLL and VPR ADs using a multiplexable mutated version (v2.1) of the previously described aptamer-containing guide RNA2.0. We show here that dCasEV2.1 is a strong and wide spectrum activator, displaying variable activation levels depending on the basal activity of the target promoter. Maximum activation rates reaching up to 10000 fold were observed when targeting the NbDFR gene. Most remarkably, RNAseq analysis of dCasEV2.1-transformed N. benthamiana leaves revealed that the topmost activation capacity of dCasEV2.1 on target genes is accompanied with strict genome-wide specificity, making dCasEV2.1 an attractive tool for rewiring plant metabolism and regulatory networks.

synthetic biology↗