{"id":1461,"date":"2025-05-22T19:06:00","date_gmt":"2025-05-22T23:06:00","guid":{"rendered":"https:\/\/pressbooks.bccampus.ca\/kathleef\/?post_type=chapter&p=1461"},"modified":"2025-06-16T16:18:43","modified_gmt":"2025-06-16T20:18:43","slug":"bioinformatics-assignment-2","status":"publish","type":"chapter","link":"https:\/\/pressbooks.bccampus.ca\/kathleef\/chapter\/bioinformatics-assignment-2\/","title":{"raw":"Bioinformatics assignment 2","rendered":"Bioinformatics assignment 2"},"content":{"raw":"
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Contents:<\/b><\/h1>\r\nWhy do we want to do this assignment?<\/a>\r\n

CRISPR-Cas in Bacteria and Archaea: A Natural Immune System<\/a><\/p>\r\n

How CRISPR-Cas was modified for gene editing<\/a><\/p>\r\n

What is the PAM sequence and why is it important?<\/a><\/p>\r\n

Key Takeaways<\/a><\/p>\r\nWhat do we want to do?<\/a>\r\n\r\nWhat steps are involved?<\/a>\r\n\r\nWhat is happening in each step?<\/a>\r\n

1. Find your assigned gene\u2019s genomic DNA (gDNA) sequence<\/a><\/p>\r\n

1.1. Access the Phytozome database<\/a><\/p>\r\n

1.2. Search for your gene<\/a><\/p>\r\n

1.3. Locate and download the genomic DNA sequence<\/a><\/p>\r\n

Common Mistakes & Troubleshooting<\/a><\/p>\r\n

2. Choose an appropriate oligo sequence amongst the available options<\/a><\/p>\r\n

2.1. Access a CRISPR design tool<\/a><\/p>\r\n

2.2. Input selected exon sequence<\/a><\/p>\r\n

2.3. Adjust the settings before submitting<\/a><\/p>\r\n

Why do we add these overhangs to CRISPR oligos?<\/a><\/p>\r\n

2.4. Interpret the results<\/a><\/p>\r\n

2.5. Select a target that meets these criteria<\/a><\/p>\r\n

2.6. Confirm location<\/a><\/p>\r\n

What is efficacy?<\/a><\/p>\r\n

Common Mistakes & Troubleshooting<\/a><\/p>\r\nReferences<\/a>\r\n\r\n<\/div>\r\n

<\/a>Why do we want to do this assignment?<\/strong><\/h2>\r\n

Global food demand is increasing rapidly, with projections indicating that farmers must produce 70% more food by 2050 to feed the growing world population (Alston et al.<\/em> 2009; The State of Food and Agriculture 2020<\/em>; Ray et al.<\/em> 2023). However, traditional farming methods are reaching their limits, and climate change is exacerbating conditions by reducing the efficiency of photosynthesis in crops like rice (Figure 1) (Alston et al.<\/em> 2009; Nahar et al.<\/em> 2018).<\/p>\r\n\r\n\r\n[caption id=\"attachment_1390\" align=\"alignnone\" width=\"1024\"]\"\" Figure 1. Yield increases are now smaller than before (Alston et al.<\/em> 2009).[\/caption]\r\n

One key way climate change threatens food production is by raising temperatures, which in turn reduces the efficiency of photosynthesis, especially in C3 plants like rice (Yamori et al.<\/em> 2014). Under heat stress, C3 plants experience increased rates of photorespiration, a wasteful process that competes with normal photosynthesis (Peterhansel et al.<\/em> 2010).<\/p>\r\n

Photosynthesis relies on the enzyme RuBisCO, R<\/strong>ibu<\/strong>lose bis<\/strong>phosphate c<\/strong>arboxylase\/o<\/strong>xygenase, which ideally uses CO\u2082 as a substrate to fix carbon and build sugars. However, RuBisCO can also mistakenly use O\u2082 instead of CO\u2082, triggering photorespiration, which consumes energy without producing sugars, thereby reducing overall photosynthetic efficiency (Peterhansel et al.<\/em> 2010).<\/p>\r\n

This issue escalates with rising temperatures: CO\u2082 solubility drops more rapidly than O\u2082 solubility in water, resulting in a reduced CO\u2082\/O\u2082 ratio within the plant (Figure 2). Consequently, RuBisCO is increasingly inclined to bind to O\u2082 rather than CO\u2082, which increases photorespiration and reduces sugar yields (Ku and Edwards 1977). Therefore, C3 crop productivity will decline in a warming climate, directly contributing to future food security.<\/p>\r\n\r\n\r\n[caption id=\"attachment_1392\" align=\"alignnone\" width=\"868\"]\"\" Figure 2. The solubility of atmospheric O\u2082 and CO\u2082, along with the O\u2082\/ CO\u2082 solubility ratio in pure water as a function of temperature (Ku and Edwards 1977).[\/caption]\r\n

In contrast, C4 plants have evolved specialized anatomy and biochemistry to concentrate CO\u2082 inside their cells. By capturing CO\u2082 in mesophyll cells and transporting it to bundle sheath cells, where O\u2082 concentration is minimized, C4 plants significantly avoid photorespiration (Figure 3) (Yamori et al.<\/em> 2014). This anatomical advancement allows them to sustain higher photosynthetic efficiency, especially in high-temperature environments.<\/p>\r\n\r\n\r\n[caption id=\"attachment_1394\" align=\"alignnone\" width=\"659\"]\"\" Figure 3. Photosynthetic reactions in C3 and C4 plants (Yamori et al. 2014).[\/caption]\r\n

Our long-term goal is to engineer C3 plants, such as rice, to perform C4-like photosynthesis by integrating traits such as higher vein density and carbon-concentrating anatomy. To accomplish this, we first identify and understand the genes that control vein development and other structural characteristics essential for C4 function.<\/p>\r\n

In this assignment, we are targeting genes in the C4 plant Setaria italica<\/em> (foxtail millet), which serves as a model system that is simple enough to modify genetically. Utilizing CRISPR, we plan to knock out genes related to auxin signaling and vein formation. By examining the resulting phenotypes, we aim to identify crucial genes for developing C4 traits, knowledge that could ultimately aid in engineering higher-yield and climate-resilient rice.<\/p>\r\n

Before getting into more details, we have to get familiar with the CRISPR-Cas system.<\/p>\r\n\r\n

<\/a>CRISPR-Cas in Bacteria and Archaea: A Natural Immune System<\/b><\/h5>\r\n

CRISPR (C<\/strong>lustered R<\/strong>egularly I<\/strong>nterspaced S<\/strong>hort P<\/strong>alindromic R<\/strong>epeats) systems originated in bacteria and archaea and serve as a genetic immune mechanism against viruses and foreign DNA. When a virus invades a bacterial cell, the bacterial immune system captures small segments of the viral DNA, which are then integrated into the cell's genome at a designated locus known as the CRISPR locus.<\/p>\r\n

These integrated sequences, referred to as \u201cspacers,\u201d function as a genetic memory of previous infections. If the same virus attacks again, the bacteria transcribe these spacers into short RNA molecules known as CRISPR RNAs (crRNAs), each containing a sequence that matches part of a known invader.<\/p>\r\nThe crRNA interacts with a small RNA known as tracrRNA (trans-activating CRISPR RNA). This tracrRNA binds to crRNA, assisting it in directing the Cas9 protein, an endonuclease, towards the target DNA. Once the corresponding sequence is located, Cas9 cleaves both strands of the viral DNA, thereby destroying it and protecting the cell.\r\n\r\n[caption id=\"attachment_1395\" align=\"alignnone\" width=\"1024\"]\"\" Figure 4. CRISPR-Cas system (credit: Sebasti\u00e1n Felipe Gonz\u00e1lez Moraga (creator) and Nima Vaezzadeh; Biorender).[\/caption]\r\n

<\/a>How CRISPR-Cas was modified for gene editing<\/b><\/h5>\r\n

To modify the system for genome editing, scientists simplified the bacterial CRISPR-Cas machinery by combining the crRNA and tracrRNA into a single RNA molecule called the single-guide RNA (sgRNA or gRNA). This modified sgRNA contains a 20-nucleotide \u201cspacer\u201d sequence corresponding to the target gene and a scaffold region binding to Cas9. When introduced into cells with the Cas9 protein, the sgRNA guides Cas9 to a specific location in the genome, producing a double-stranded break in the DNA. Following the cut, the cell attempts to repair it through one of two pathways:<\/p>\r\n\r\n