CRISPR/Cas9 USED TO ALTER PHOTOSYNTHESIS

Source: Phys.org

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Context

• A research team from the Innovative Genomics Institute at the University of California in collaboration with the Realizing Increased Photosynthetic Efficiency (RIPE) project led by the University of Illinois, has achieved a significant breakthrough.
• They used the CRISPR/Cas9 gene-editing technology to increase gene expression in rice, a staple food crop, thereby enhancing photosynthetic activity.
• This research marks the first unbiased gene-editing approach to boost gene expression for improved photosynthesis

Details

Background

• CRISPR/Cas9 Technology:
  • Traditionally used to knock out or decrease the expression of genes.
  • This study leverages CRISPR/Cas9 to increase gene expression by modifying upstream regulatory DNA.
• Previous Research:
  • Prior studies often utilized synthetic biology strategies, introducing genes from other organisms to enhance photosynthesis.
  • Inspired by a 2018 Nature Communications paper that improved water-use efficiency in a model crop by overexpressing the PsbS gene.
• Significance of Rice:
  • Rice provides at least 20% of the world's calories.
  • It possesses only one copy of each of the three key photoprotection genes, making it an ideal model for this study.

Research Methodology

• Targeting Regulatory DNA: The team focused on altering the DNA upstream of the PsbS gene, which controls gene expression levels and timing.
• CRISPR/Cas9 Editing: Utilized CRISPR/Cas9 to induce inversions, or "flipping," of the regulatory DNA to enhance gene expression.
• RNA Sequencing: Conducted RNA sequencing to compare the activity of all genes in the rice genome before and after the modifications.

Key Findings

• Increased Gene Expression: The alterations led to a substantial increase in the expression of the PsbS gene, surpassing initial expectations.
• Impact on Downstream Photosynthetic Activity: Enhanced gene expression resulted in improved downstream photosynthetic activity.
• Plasticity of Plant Genomes: Demonstrated the inherent plasticity of plant genomes, which have adapted to significant changes over millions of years of evolution and thousands of years of domestication.
• Efficiency of the Method: Approximately 1% of the modified plants exhibited the desired phenotype, indicating room for improvement in the efficiency of the gene-editing process.

Implications

• Agricultural Advancements: This proof-of-concept study shows that CRISPR/Cas9 can be used to create significant changes in crop genes, potentially leading to more efficient photosynthesis and better crop yields.
• Regulatory Considerations: By modifying existing genes rather than introducing foreign DNA, this approach may circumvent some regulatory hurdles, facilitating quicker adoption by farmers.

Photosynthesis

• Photosynthesis is a fundamental biological process by which green plants, algae, and certain bacteria convert light energy into chemical energy, storing it in the bonds of glucose and other organic molecules.
• This process is crucial for the sustenance of life on Earth as it provides the primary source of energy for nearly all organisms.

Basic Equation

• The overall simplified chemical equation for photosynthesis is: 6CO2+6H2O+light energy→C6H12O6+6O2
• This represents the transformation of carbon dioxide and water into glucose and oxygen, facilitated by light energy.

Components Involved

• Chloroplasts:
  • Structure: Found in plant cells, chloroplasts have an outer membrane, inner membrane, and internal thylakoid membranes arranged in stacks called grana.
  • Function: These organelles contain chlorophyll and other pigments that capture light energy.
• Pigments:
  • Chlorophyll: The primary pigment, chlorophyll a, absorbs light mainly in the blue-violet and red parts of the spectrum and reflects green light, giving plants their color.
  • Accessory Pigments: Chlorophyll b, carotenoids, and xanthophylls help capture additional light energy and protect the plant from photo-damage.

Stages of Photosynthesis

Photosynthesis occurs in two main stages: the light-dependent reactions and the Calvin cycle (light-independent reactions).

• Light-Dependent Reactions:
  • Location: Thylakoid membranes of the chloroplasts.
  • Process:
    • Photon Absorption: Chlorophyll absorbs light energy, exciting electrons.
    • Water Splitting (Photolysis): Water molecules are split to release oxygen, protons, and electrons.
    • Electron Transport Chain (ETC): Excited electrons travel through the ETC, creating a proton gradient that drives ATP synthesis.
    • NADP+ Reduction: Electrons reduce NADP+ to NADPH.
  • Outputs: ATP, NADPH, and O₂.
• Calvin Cycle (Light-Independent Reactions):
  • Location: Stroma of the chloroplasts.
  • Process:
    • Carbon Fixation: CO₂ is fixed into a 5-carbon molecule (RuBP) by the enzyme RuBisCO, forming 3-phosphoglycerate (3-PGA).
    • Reduction: ATP and NADPH from the light-dependent reactions reduce 3-PGA to glyceraldehyde-3-phosphate (G3P).
    • Regeneration: Some G3P molecules go to form glucose and other carbohydrates, while others regenerate RuBP, enabling the cycle to continue.
  • Outputs: G3P, which can be converted into glucose and other carbohydrates.

Factors Affecting Photosynthesis

• Light Intensity: Increased light intensity boosts the rate of photosynthesis up to a point, beyond which the rate plateaus.
• Carbon Dioxide Concentration: Higher CO₂ levels enhance photosynthesis until other factors become limiting.
• Temperature: Photosynthesis is temperature-dependent, with an optimal range for most plants. Extremely high or low temperatures can inhibit the process.
• Water Availability: Water is a raw material for photosynthesis, and scarcity can limit the process.

CRISPR-Cas9

• CRISPR-Cas9, often simply referred to as CRISPR, is a groundbreaking technology for editing genomes, allowing researchers to alter DNA sequences and modify gene function with high precision.
• This tool has revolutionized genetics, molecular biology, and biotechnology, offering potential applications in medicine, agriculture, and beyond.

What is CRISPR-Cas9?

• CRISPR(Clustered Regularly Interspaced Short Palindromic Repeats) and Cas9 (CRISPR-associated protein 9) together form a system that bacteria naturally use as a defense mechanism against viruses.
• Scientists have adapted this system for gene editing.

Components of CRISPR-Cas9

• Guide RNA (gRNA):
  • Structure: A synthetic RNA molecule engineered to contain a short sequence (~20 bases) that is complementary to the target DNA sequence.
  • Function: Directs the Cas9 protein to the specific location in the genome where the cut is to be made.
• Cas9 Protein:
  • Function: An endonuclease enzyme that cuts DNA at a site specified by the gRNA.

Mechanism of Action

• Target Recognition: The gRNA binds to its complementary DNA sequence in the genome.
• DNA Cleavage: The Cas9 protein, guided by the gRNA, introduces a double-strand break (DSB) in the DNA at the target location.
• DNA Repair:
  • The cell's natural repair mechanisms are then employed:
    • Non-Homologous End Joining (NHEJ): This repair process can introduce small insertions or deletions, leading to gene disruption.
    • Homology-Directed Repair (HDR): This process can be used to introduce precise genetic changes if a repair template is provided.

Applications of CRISPR-Cas9

• Medicine:
  • Gene Therapy: Correcting genetic defects in patients with inherited diseases (e.g., sickle cell anemia, cystic fibrosis).
  • Cancer Research: Editing genes in cancer cells to understand their function and develop new treatments.
  • Infectious Diseases: Developing treatments for viral infections like HIV by targeting and disabling viral DNA within human cells.
• Agriculture:
  • Crop Improvement: Creating genetically modified crops with desirable traits such as drought resistance, pest resistance, and enhanced nutritional content.
  • Livestock: Developing disease-resistant livestock and improving traits such as growth rate and meat quality.
• Fundamental Research:
  • Functional Genomics: Studying gene function by creating knockout and knock-in models in various organisms.
  • Developmental Biology: Understanding the roles of specific genes in development by observing the effects of their manipulation.

Sources:

Phys.org