{"id":196,"date":"2020-05-29T12:00:00","date_gmt":"2020-05-29T16:00:00","guid":{"rendered":"http:\/\/anyonecanscience.com\/?p=196"},"modified":"2020-05-28T21:41:31","modified_gmt":"2020-05-29T01:41:31","slug":"crispr-and-genetic-engineering","status":"publish","type":"post","link":"https:\/\/anyonecanscience.com\/index.php\/2020\/05\/29\/crispr-and-genetic-engineering\/","title":{"rendered":"Molecular Manipulation: CRISPR and Genetic Engineering"},"content":{"rendered":"\n

\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0If you\u2019ve ever seen the movie\u00a0Gattaca,\u00a0<\/em>then you probably know something about the concept of genetic engineering. However fictionalized that movie may have seemed (my favorite part was a full genome sequence printed on one small tube of rolled up paper), it did bring up a lot of interesting ethical questions that are suddenly becoming more and more relevant. Widespread genetic engineering isn’t yet a reality, but it certainly could be within our lifetime. In the last decade, scientists have discovered a way to hijack the CRISPR-Cas9 system in bacteria to make efficient and targeted genetic editing possible. However, as we get closer and closer to widespread implementation of this technology, there is still a looming gap in the legal and ethical framework surrounding genetic engineering. Is nuanced and unbiased regulation of gene editing even possible?<\/p>\n\n\n\n

\"CRISPR-Cas9
CRISPR-Cas9 recognizes a target<\/figcaption><\/figure>\n\n\n\n

            Last week, I went over the mechanism of CRISPR<\/a> and how it gives bacteria adaptive immunity to viruses. I gave a more generalized overview of how CRISPR operates, but actually there are three distinct classes<\/a> of CRISPR that act in diverse ways to enact this immunity. In terms of genetic engineering, we are most concerned with Class II, known as CRISPR-Cas9. Each CRISPR class utilizes specific Cas proteins to perform the three main steps of bacterial immunity<\/a>: adaptation, expression, and interference. The first step, adaptation, requires Cas1 and Cas2 to form a protein complex. This complex is responsible for chopping up viral DNA and integrating it as a spacer in the CRISPR DNA. This step is essential for establishing the molecular memory of a particular virus, and it is highly conserved across all three classes of CRISPR.<\/p>\n\n\n\n

            The second step, expression, is where the CRISPR DNA is transcribed into RNA and processed into CRISPR RNA (crRNA) by specific Cas proteins and other factors. The Cas proteins that turn this precursor crRNA (pre-crRNA) into mature crRNA differ between the three CRISPR systems. In Classes I and III, it is mostly Cas5 and Cas6 that perform this processing. But in CRISPR Class II, the pre-crRNA hybridizes with trans-activating CRISPR RNA (tracrRNA) transcribed from DNA upstream of the CRISPR region. This RNA duplex is trimmed by a RNase enzyme (a protein that can cut up RNA), and it quickly recruits the Cas9 protein, which performs the interference step.<\/p>\n\n\n\n

            The final step, interference, is perhaps the most important step in bacterial immunity. It is also the step that scientists hijack for genetic engineering. In Class II, the Cas9 protein carries the crRNA:tracrRNA duplex. The spacer in the crRNA directs Cas9 to the target viral DNA, where Cas9 uses molecular scissors to induce a double-strand break that neutralizes the DNA. <\/p>\n\n\n\n

\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0Proper targeting of the viral DNA is reliant on a specific sequence in the target DNA called\u00a0protospacer adjacent motif<\/a>\u00a0(PAM), a short sequence of DNA next to the spacer\u2019s target DNA that helps Cas9 distinguish target viral DNA from native bacterial DNA. The spacer is enough to recognize a specific target sequence, but that target sequence also exists in the bacteria\u2019s own CRISPR DNA. To avoid destroying the bacteria\u2019s immunity, Cas9 is programmed to only cut target DNA that contains the PAM sequence just downstream. But why does the viral DNA always contain a PAM next to the spacer target? Because, during the adaptation phase, the Cas1\/Cas2 complex specifically selects a viral DNA \u201cprotospacer\u201d that is next to a PAM sequence. The complex chops up the DNA around the protospacer and integrates it into the CRISPR DNA.<\/p>\n\n\n\n

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