Session 5
Authors: Shani Eilon , Zainal Adawiah , Stephens Timothy , Khan Adil , Gagliardi Pedro Garcia 
Published: July 2023

Multi-Knock – a multi-targeted genome-scale CRISPR toolbox to overcome functional redundancy in plants

Plant genomes are characterized by large and complex gene families that often result in similar and partially overlapping functions. This genetic redundancy severely hampers current efforts to uncover novel phenotypes, delaying basic genetic research and breeding programs. To address the problem of masked phenotypic variation due to functional redundancy, we developed and validated Multi-Knock, a novel genetic approach in plants that combines forward-genetics with dynamically targeted genome-scale CRISPR/Cas9 tools. A total of 59,129 multi-targeted sgRNAs, divided into 10 functional sub-libraries targeting 16,152 genes in Arabidopsis, were designed, synthesized, and cloned into a genome-editing intronized Cas9 vector. From this collection, 5,635 sgRNAs targeting 1,123 of the 1,327 transporters (TRP) in Arabidopsis were cloned into four different Cas9 vectors generating independent CRISPR libraries, wherein each sgRNA was designed to target closely related homologues within sub-clades in transporter families. A proof of concept forward-genetic screen using over 3,500 CRISPR lines targeting the plant transportome recovered multiple known phenotypes in Arabidopsis, demonstrating the validity of the approach. Moreover, our screen allowed us to uncover novel transporters whose function has been hidden due to genetic redundancy. Specifically, we identified a homologous subfamily of three previously unstudied genes with partially overlapping function, PUP7, PUP21, and PUP8. We discovered that while all three proteins biochemically function as cytokinin transporters, PUP8 localizes to the plasma membrane, while PUP7 and PUP21 are localized to the tonoplast. We show that these proteins regulate meristem size, phyllotaxis, and plant growth, revealing complex redundant activity within this sub-family and providing a demonstration of the power of the Multi-Knock approach to discover new biological functions.

Gene co-expression network analysis identifies molecular regulators for improving stress tolerance traits in papaya and rice

Discovering molecular mechanisms that govern traits that boost crop productivity under environmental stress, including pathogen attack is an urgent need. Gene co-expression network (GCN) analysis has been widely used to reveal hub genes and key pathways from gene expression datasets. To understand the molecular mechanism of disease progression in papaya dieback disease (PDD) and prioritise hub genes for increasing antioxidant content in pigmented rice, we performed GCN analysis from RNA-seq datasets. A modified Markov Cluster (MCL) algorithm was used to identify co-expression modules and hub genes within these modules in the rice and papaya. The papaya GCN consisted of 53 modules that defined the papaya-pathogen (Erwinia mallotivora) interaction network. The modules revealed not only classical disease resistance-related pathways, such as pattern recognition receptors (PRR) and pathogenesis-related (PR) genes but also new molecular regulators that could be prioritised for further validation and papaya breeding programme. The pigmented rice GCN identified three established functional modules related to flavonoid and terpenoid biosynthesis as well as transcriptional regulators that modulate these pathways in the rice seed. The rice and papaya breeding programmes in Malaysia will use the information generated here as a foundation for marker-assisted breeding or gene editing, for improving crop productivity.

Exploring the origin and evolution of primary plastids using Paulinella as a model system

Plastid primary endosymbiosis has occurred twice, once  1.5 Bya in the Archaeplastida ancestor and once  120 Mya in the Paulinella (Rhizaria) lineage. Both events precipitated massive evolutionary changes, including the recruitment and activation of genes that are horizontally acquired, redeployment of existing genes and pathways in novel contexts, and the evolution of mechanisms for controlling the new endosymbiont by the host. Whereas significant steps have been made into understanding organelle evolution using the Archaeplastida model, its ancient provenance limits our ability to investigate the factors governing the early stages of endosymbiosis. Our work using multi-omics analysis of the recently evolved photosynthetic Paulinella lineage, has provided novel insights into the evolutionary strategies that facilitated the transition of a eukaryotic organisms from a heterotrophic to a photosynthetic lifestyle. These insights include the role of retrotransposition in the integration of endosymbiont-derived genes and the evolution of control of endosymbiont pathways by the host, results which have led to testable hypotheses about the forces that shape the genome evolution of both the host and endosymbiont after primary endosymbiosis. These results not only open new avenues for studying Archaeplastida evolution, but, in ongoing work, allow the exploration and optimization of photosynthesis in traditional systems.

CRISPRi-based circuits for genetic computation in plants

Synthetic gene circuits can enable new cellular behaviours by integrating multiple input signals into customisable genetic programs. However, gene circuit development in plants has been limited by a lack of orthogonal and modular parts required for their construction. Here, we present the first implementation of reversible CRISPRi-based gene circuits in plants. First, we created a toolkit of engineered repressible promoters of different strengths and used them as integrators for the construction of NOT and NOR gates in Arabidopsis thaliana. Next, we determined the optimal processing system to express sgRNAs from RNA Pol II promoters to introduce NOR gate programmability for interfacing with host regulatory sequences. Finally, we layered multiple NOR gates together to create OR, NIMPLY, and AND logic functions and tested NOR gate activity in Physcomitrium patens to demonstrate cross-species utility. Our CRISPRi circuits are orthogonal, compact, reversible, programmable, and modular, providing a new platform for sophisticated spatio-temporal control of gene expression in plants.

Cracking the vascular origin of potato tuber formation through a single cell RNA-Seq approach

Potatoes represent the most important non-cereal source of calories for human consumption reaching a production of 376 million metric tons in 2021. Even though many of the tuber inducing pathways have been identified, the process by which stolons, a specialized underground stem, swells to become the tuber remains unknown. To understand the cellular processes behind this we first performed transverse sections of potato stolons in different stages of tuberization and looked for patterns of cellular proliferation. To identify the gene regulatory network behind this, we performed single cell RNA-seq of induced and un-induced in vitro tubers. This was the first time something like this was attempted in a storage organ. We validated the identity of our cluster analysis through in-situ assays of in-vitro and stolon derived tubers. Our results show a new kind of cellular identity, enriched in tuber identity and starch accumulation genes.