Written by Team Virginia
Introduction:
This blog, we are diving into the world of plants to explore the use of genome editing technologies such as CRISPR and TALEN to increase crop yield. We read an article called "Engineering crops of the future: CRISPR approaches to develop climate-resilient and disease-resistant plants" (2020) to help us understand this new synthetic biology approach to producing more food for a growing global population.
Citation:
Zaidi, S.SeA., Mahas, A., Vanderschuren, H. et al. Engineering crops of the future: CRISPR approaches to develop climate-resilient and disease-resistant plants. Genome Biol21, 289 (2020). https://doi.org/10.1186/s13059-020-02204-y
The Problem:
According to the United Nations, the global population is expected to reach nearly 9.7 billion by 2050 (1). This poses a huge problem for agriculture because as the population grows, the area of agricultural land per capita decreases. This leads to lower crop yields which are not able to satisfy the demands of a growing population. Additionally, climate change leads to warmer summers which results in grave and unforeseeable patterns of crop disease. Climate change also results in higher sea levels which decreases the amount of accessible arable land.
The Solution
Current Methods:
Genetic Modification (GM)
How does GM work?
Identification of a desirable gene
Cloning of DNA is a carrier or vector plasmid
Delivery to target plant
Generation of desired plant with desired trait
What is the problem with GM?
Regional Solution:
Make the crops climate resistant to prevent crop disease
It is possible to increase crop yield as crop yield has increased since the Green Revolution despite many abiotic factors (drought, salinity, flooding, etc.)
Mostly due to awareness, more funding, market development, infrastructure, and policy support
Functional Solution:
Use new plant breeding technologies (NPBTs) such as CRISPR and TALEN to precisely manipulate genes without introducing exogenous DNA such as antibiotic resistance gene in a plasmid vector
Advantages: shorten breeding cycle, accelerate crop research (speed breeding, next-generation genotype and phenotype platforms), genome editing, affordable, fast turnaround into food products, and fast approval by the USDA (US Department of Agriculture)
Examples
Browning-resistant mushrooms
High-amylopectin waxy corn (Zea mays)
False flax (Camelina sativa) with enhanced omega 3 oil
Introduction to CRISPR
CRISPR = clustered regularly interspaced short palindromic repeat
TALEN = transcription activator-like effector nuclease
How does CRISPR work?
Site-specific nucleases (SSNs) bind and cleave specific, precise nucleic acid sequences which introduce double stranded breaks at or near the target and then let natural DNA repair processes to insert the desired change and then rebind the DNA.
What are the three modifications possible by CRISPR/Cas9 (2)?
o Disruption o Deletion o Insertion
CRISPR Vocabulary:
1. CRISPR: short repeats of sequence used in bacteria to defend against viruses
2. Cas9: an endonuclease (enzyme that cleaves nucleotides that are not at the two ends) that cleaves viral DNA in prokaryotes at a precise location with the guidance of guide RNA
Classified into Class I (type I, III, IV) with multiple subunits and Class 2 (type II, V, VI) with a single subunit
Type II is most common in genetic engineering
3. Guide RNA: binds to Cas9 to guide it to a specific location on the DNA to be cleaved
4. dsDNA: double stranded DNA
5. PAM: sequence of DNA that is conserved downstream from the cut site
Steps of CRISPR Technology
How does CRISPR work and what exactly happens?
Steps of CRISPR (3):
Recognition
Guide RNA binds to Cas9 enzyme and targets a particular sequence in dsDNA through its complementary component crRNA.
Cleavage
Cas9 recognizes the appropriate PAM site near the cut site and then creates a double stranded break in the DNA sequence at the target site
Repair
Two mechanisms, non-homologous end joining (NHEJ) and homology-directed repair (HDR), repair the DNA and introduce a modification
NHEJ uses an enzyme to join two parts imprecisely of DNA and does not require an exogenous homologous DNA
HDR is more precise than NHEJ but requires the use of homologous DNA to insert or replace a sequence of DNA.
How does CRISPR lead to increased drop yield (4):
1. Gene disruption via NHEJ/HDR mechanism: insert or delete a sequence to disrupt gene production
2. Gene disruption via promoter modification: disrupt the promoter to block gene expression or block pathogen binding to the promoter
3. Gene deletion: multiple guide RNAs are introduced to the DNA to create double stranded breaks around a gene so that the gene of interest is deleted when the DNA is joined back together
4. Biomimicking via promoter, allele, and gene replacement: a series of CRISPR-mediated mutations that change the sequence of the DNA to a desired gene sequence from a disease-resistant variety
Careers related to increasing crop yield:
1. USDA officer
2. Synthetic Biologist/Genetic Engineer
3. Farmer
References:
1. Growing at a slower pace, world population is expected to reach 9.7 billion in 2050 and could peak at nearly 11 billion around 2100 | UN DESA | United Nations Department of Economic and Social Affairs [Internet]. [cited 2022 Oct 12]. Available from: https://www.un.org/development/desa/en/news/population/world-population-prospects-2019.html
2. CRISPR/Cas9 [Internet]. CRISPR. [cited 2022 Oct 12]. Available from: http://crisprtx.com/gene-editing/crispr-cas9
3. Asmamaw M, Zawdie B. Mechanism and Applications of CRISPR/Cas-9-Mediated Genome Editing. Biologics. 2021 Aug 21;15:353–61.
4. Zaidi SS e A, Mahas A, Vanderschuren H, Mahfouz MM. Engineering crops of the future: CRISPR approaches to develop climate-resilient and disease-resistant plants. Genome Biology. 2020 Nov 30;21(1):289.