06 - Introducing Your Students to CRISPR with Sickle Cell Gene Editing
system contained two RNAs (tracrRNA and CRISPR RNA) and only one protein – Cas9. This system was so much simpler than other CRISPR systems that the team thought it could be har- nessed as a powerful genetic engineering tool. Charpentier predicted that the two RNAs worked together to guide Cas9 to specific viral DNA sequences, and she was right. Charpentier presented her findings in 2010 at a CRISPR meeting in Wageningen, The Nether - lands, and it was the highlight of the conference. In 2011, she and Doudna met at an American Society for Microbiology meeting in Puerto Rico and agreed to collaborate on the problem of how Cas9 cleaved DNA and how it could be adapted to make targeted cuts in a genome. They solved this problem and their results have been used successfully around the world. At the same time, Feng Zhang, an MIT researcher exploring the genetics of complex psychiat - ric and neurological diseases, was looking at ways to edit eukaryotic human and mammalian cells. In 2010, he published a report on how to do so using a previously developed gene editing system called TALEN. He published a second paper in 2012 outlining how he and his team had used CRISPR-Cas9 to edit the genome of mammalian cells, and in 2015 announced the creation of a simpler and more precise tool called CRISPR-Cpf1. In 2016, CRISPR-CasC2c2, that targets RNA rather than DNA, was unveiled. Putting CRISPR-Cas to Work The ability of CRISPR-Cas to specifically target and cut DNA, combined with modern DNA sequencing, has opened new avenues in genetic engineering, molecular biology, and synthetic biology. Researchers can determine the sequence of a segment of a gene, design a CRISPR guide RNA (gRNA) to specifically cut the DNA, and combine everything within a cell to efficiently change the DNA. The gRNA combines the tracrRNA and CRISPR RNA into a single DNA molecule, simplifying delivery into a cell. One of the most common uses of CRISPR technology is to digest a gene to disrupt its function. Once cut, DNA repair mechanisms will try to mend the double stranded break, often resulting in small insertions, deletions, or other mutations that disrupt gene function. In addition to using CRISPR-Cas systems to disrupt mutated genes, scientists can use CRISPR to replace them with genes Figure 4 - Repairing DNA Using HDR
that function the way they are supposed to (Figure 4). First, the DNA is cut using CRISPR-Cas to create a double stranded break. Next, the cells are given a template DNA strand, containing the correct sequence, which can be incorporated into the cut DNA using homology directed repair (HDR). With HDR, the natural cel- lular machinery will incorporate the template DNA into the ge- nome at the site of the CRISPR di- gest. By controlling the template DNA strand, researchers can repair mutated genes or even insert entirely new genes into an organism. CRISPR-Cas systems allow researchers to easily place the new genes precisely where they want them, unlike some of the older methods of gene therapy where the new genes are randomly inserted into the plant or animal genomes. Scientists are already using CRISPR to insert new genes into healthy genomes that will make plants, in particular, more
CRISPR-Cas targeting DNA
Corrected DNA sequence
Homologous DNA sequences
Homology Directed Repair (HDR)
Repaired DNA
Figure 4: Repairing DNA Using HDR
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