Biology subjects

Minguet, E. G.

Publications and source records attributed to Minguet, E. G..

3 recordsLinked to original sources

bioRxiv2020

Ares-GT: design of guide RNAs targeting multiple genes for CRISPR-Cas experiments

MotivationThere is a lack of tools to design guide RNA for CRISPR genome editing of gene families and usually good candidate sgRNAs are tagged with low scores precisely because they match several locations in the genome, thus time-consuming manual evaluation of targets is required. Moreover, online tools are limited to a restricted list of reference genome and lack the flexibility to incorporate unpublished genomes or contemplate genomes of populations with allelic variants. ResultsTo address these issues, I have developed the ARES-GT, a local command line tool in Python software. ARES-GT allows the selection of candidate sgRNAs that match multiple input query sequences, in addition of candidate sgRNAs that specifically match each query sequence. It also contemplates the use of unmapped contigs apart from complete genomes thus allowing the use of any genome provided by user and being able to handle intraspecies allelic variability and individual polymorphisms. AvailabilityARES-GT is available at GitHub (https://github.com/eugomin/ARES-GT.git).

bioinformatics↗

bioRxiv2020

COP1 destabilizes DELLA proteins in Arabidopsis

DELLA transcriptional regulators are central components in the control of plant body form in response to the environment. This is considered to be mediated by changes in the metabolism of the hormones gibberellins (GAs), which promote the degradation of DELLAs. However, here we show that warm temperature or shade reduced the stability of a GA-insensitive DELLA allele in Arabidopsis. Furthermore, the degradation of DELLA induced by the warmth anticipated changes in GA levels and depended on the E3 ubiquitin ligase CONSTITUTIVELY PHOTOMORPHOGENIC1 (COP1). COP1 enhanced the degradation of normal and GA-insensitive DELLA alleles when co-expressed in N. benthamiana. DELLA proteins physically interacted with COP1 in yeast, mammalian and plant cells. This interaction was enhanced by the COP1 complex partner SUPRESSOR OF phyA-105 1 (SPA1). The level of ubiquitination of DELLA was enhanced by COP1 and COP1 ubiquitinated DELLA proteins in vitro. We propose that DELLAs are destabilized not only by the canonical GA-dependent pathway but also by COP1 and that this control is relevant for growth responses to shade and warm temperature. SignificanceDELLA proteins are plant-specific transcriptional regulators that act as signaling hubs at the interface between the environment and the transcriptional networks that control growth. DELLAs are destabilized by the growth-promoting hormone gibberellin, whose levels are very sensitive to environmental changes. Here we describe an alternative pathway to destabilize these proteins. We show that DELLAs are substrate of COP1, an E3 ubiquitin ligase that increases its nuclear activity to promote growth in response to shade or warmth. Our results also show that the destabilization of DELLAs by COP1 precedes the action of gibberellins, suggesting the existence of a sequential mechanism to control the stability of these proteins.

plant biology↗

bioRxiv2019

Identification of transgene-free CRISPR edited plants of rice and tomato by monitoring DsRED fluorescence in dry seeds

CRISPR/Cas technology for specific and precise modification of genomes has transformed molecular biology. However, quick elimination of the transgene remains a challenge in plant biotechnology after genome edition, especially for crops due to their long life cycle and multiploidy, not only to avoid transgene position effects and to minimize the probability of off-target mutation appearance, but also to deliver end users with edited plants free of the recombinant gene editing machinery. Counter selection based on resistance marker genes are inconvenient in the case of CRISPR/Cas applications because plants lacking the transgene cannot survive the selection, and thus two more generations must be screened to evaluate the presence of the transgene. In the case of some crops, generations can last between 6 months and a few years, and the workload may be a limiting factor because transgene detection by PCR requires germination of seeds, and selected plants must be grown until a new generation can be harvested. The expression of fluorescent proteins as selective marker has been successfully used in Arabidopsis thaliana (Stuitje et al., 2003) as a fast method for transgene presence detection prior to seed germination. Despite this clear advantage, it has not been tested in other species yet, because of the special requirements of in vitro transformation protocols. To overcome the above mentioned inconvenient, we have adapted fluorescence-dependent monitoring of transgene to genome editing approaches in tomato and rice with the goal of obtaining transgene-free homozygous edited crop plants in two generations.

plant biology↗