02 - Code Breakers: Using CRISPR to Rewrite Genetics
Introduction Unleash the power of gene editing with your students using REAL CRISPR-Cas technology to knock out colorful genes in E. coli. Identify successful knockout based on the cell color. Experi- ment by switching RNA templates and analyzing results, letting your students prove the specific - ity of CRISPR! Background Information CRISPR is a game-changing gene-editing tool that is catalyzing a wave of innovations across fields as diverse as biotechnology, medicine, agriculture, and energy. This system evolved in bacteria at the beginning of evolutionary history as a defense against viral attacks and was discovered by humans in the late 1980s. However, it would take scientists another 20 years to harness CRISPR as a precise and versatile gene editor. Today, CRISPR’s unmatched ability to rewrite the code of life has captured the imagination of re- searchers and the public alike. CRISPR technologies are used to cure diseases like cancer and sickle cell anemia, develop antibiotics, study complex genetic traits, understand evolutionary history, create drought-resistant crops, and develop new materials like bioplastics and biofuels. As CRISPR continues to advance its transformative potential will be limited only by our imagination and by the regulations we create. WHERE DOES THE NAME CRISPR COME FROM? CRISPR, short for Clustered Regularly Interspaced Short Palindromic Repeats describes uniquely patterned regions of bacterial DNA. An enzyme tightly linked to these CRISPR region was identified by scientists and called Cas for “CRISPR-associated proteins”. In bacteria and archaea, Cas enzymes cut and destroy foreign DNA, guided by the nucleotide sequences stored in CRISPR regions. Cas enzymes
are central to new DNA editing technologies but the CRISPR region itself is not. Never the less the CRISPR name has be- come synonymous with these technologies. EXPLORING THE CRISPR-CAS SYSTEM At the heart of CRISPR gene editing are two molecules: (1) an enzyme called Cas and (2) a
Figure 1: Bacterial CRISPR Region.
piece of RNA known as guide RNA (gRNA). These molecules collaborate to form a complex capable of identifying and cutting a specific DNA sequence within a genome, known as the target DNA. Following a cut, a cell’s intrinsic repair mechanisms mend the targeted DNA often with the help of additional DNA instructions, called template DNAs, that the experimenter provides. This repair process leads to changes in the DNA sequence that can induce a loss of function mutation, correct a mutation, or introduce a new trait in an organism. Cas proteins are endonucleases. These are enzymes that cut DNA by breaking the phosphodiester bonds between nucleotides. Some endonuclease cut DNA nonspecifically, but most cleave at specific nucleotide sequences called target sites. Cas enzymes fall into this latter category. They target specific sites within a DNA molecule and create a double-strand break. However, unlike most endonucle- ases (like restriction enzymes), which have a target DNA sequence built into their structure, CRISPR outsources its specificity to interchangeable molecules of guide RNA. This means that Cas enzymes can be programmed to cut any DNA region by creating guide RNA molecules that are complementary to the target’s sequence.
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