An Undergraduate Research Project Utilizing CRISPR-Cas9 Gene Editing Technology to Study Gene Function in .

The CRISPR-Cas9 system functions in microbial viral pathogen recognition pathways by identifying and targeting foreign DNA for degradation. Recently, biotechnological advances have allowed scientists to use CRISPR-Cas9-based elements as a molecular tool to selectively modify DNA in a wide variety of other living systems. Given the emerging need to bring engaging CRISPR-Cas9 laboratory experiences to an undergraduate audience, we incorporated a CRISPR-based research project into our class laboratories, emphasizing its use in plants.

Mutations in GERANYLGERANYL DIPHOSPHATE SYNTHASE 1 affect chloroplast development in Arabidopsis thaliana (Brassicaceae).

Premise Of The Study: Within plastids, geranylgeranyl diphosphate synthase is a key enzyme in the isoprenoid biosynthetic pathway that catalyzes the formation of geranylgeranyl diphosphate, a precursor molecule to several biochemical pathways including those that lead into the biosynthesis of carotenoids and abscisic acid, prenyllipids such as the chlorophylls, and diterpenes such as gibberellic acid. •

Methods: We have identified mutants in the GERANYLGERANYL DIPHOSPHATE SYNTHASE 1 (GGPS1) gene, which encodes the major plastid-localized enzyme geranylgeranyl diphosphate synthase in Arabidopsis thaliana. •

Key Results: Two T-DNA insertion mutant alleles (ggps1-2 and ggps1-3) were found to result in seedling-lethal albino and embryo-lethal phenotypes, respectively, indicating that GGPS1 is an essential gene.

Functional diversification of thylakoidal processing peptidases in Arabidopsis thaliana.

Thylakoidal processing peptidase (TPP) is responsible for removing amino-terminal thylakoid-transfer signals from several proteins in the thylakoid lumen. Three TPP isoforms are encoded by the nuclear genome of Arabidopsis thaliana. Previous studies showed that one of them termed plastidic type I signal peptidase 1 (Plsp1) was necessary for processing three thylakoidal proteins and one protein in the chloroplast envelope in vivo.

Keep the balloon deflated: the significance of protein maturation for thylakoid flattening.

Thylakoidal processing peptidase (TPP) catalyzes the removal of signal peptide which leads to maturation of a subset of proteins including photosynthetic electron transport components in thylakoids. The biochemical properties of TPP were highly defined during the 1980's and 1990's, but the physiological significance of the TPP activity had remained undefined. Completion of genome sequencing revealed the presence of three TPP isoforms in the model plant Arabidopsis thaliana.

A mutation in Arabidopsis seedling plastid development1 affects plastid differentiation in embryo-derived tissues during seedling growth.

Oilseed plants like Arabidopsis (Arabidopsis thaliana) develop green photosynthetically active embryos. Upon seed maturation, the embryonic chloroplasts degenerate into a highly reduced plastid type called the eoplast. Upon germination, eoplasts redifferentiate into chloroplasts and other plastid types.

The significance of protein maturation by plastidic type I signal peptidase 1 for thylakoid development in Arabidopsis chloroplasts.

Thylakoids are the chloroplast internal membrane systems that house light-harvesting and electron transport reactions. Despite the important functions and well-studied constituents of thylakoids, the molecular mechanism of their development remains largely elusive. A recent genetic study has demonstrated that plastidic type I signal peptidase 1 (Plsp1) is vital for proper thylakoid development in Arabidopsis (Arabidopsis thaliana) chloroplasts.

Mutations in a plastid-localized elongation factor G alter early stages of plastid development in Arabidopsis thaliana.

Background: Proper development of plastids in embryo and seedling tissues is critical for plant development. During germination, plastids develop to perform many critical functions that are necessary to establish the seedling for further growth. A growing body of work has demonstrated that components of the plastid transcription and translation machinery must be present and functional to establish the organelle upon germination.