06 - Introducing Your Students to CRISPR with Sickle Cell Gene Editing
of four different subunits (Figure 5) . Within adult hemoglobin, there are two types of subunits: Alpha hemoglobin (HbA, depicted in red) and Beta hemoglobin (HbB, depicted in blue). Each subunit contains an iron-containing heme group responsible for oxygen binding and transport. In healthy red blood cells (RBCs), hemoglobin exists as tetramers, facilitating its vital role in oxygen transport throughout the body. A third hemoglobin subunit, fetal hemoglobin or HbF, is expressed in utero and in newborn babies (Figure 6). Fetal hemoglobin ceases production around the age of two, giving way to the increased synthesis of HbB. Diseases like beta-thalassemia and Sickle Cell affect the HbB subunit of hemoglobin. Beta- thalassemia is a disorder where the body does not produce enough of the HbB subunit, leading to lower concentrations of hemoglobin and reduced oxygen in the body. In Sickle Cell disease, amino acid substitutions compromise the HbB subunit, changing the shape of the red blood cell and causing clumping in small blood vessels. Depending on the alleles, the symptoms can vary in severity. Common symptoms include fatigue, anemia, pain and swelling, and shortness of breath. Through medical and clinical research on hemoglobinopathies, scientists observed that individuals who continue to produce HbF past infancy experience milder symptoms. This is be- cause HbF can compensate for HbB in the hemoglobin complex. Expression of HbF is controlled by BCL11A protein, which turns off production of HbF in young children. New genetic therapies like Casgevy use CRISPR to inactivate the BCL11A gene, turning on production of fetal hemo- globin. Data from the clinical trial shows that the gene therapy was effective in the majority of patients, signaling an improvement in their quality of life. First, you will design guide RNAs (gRNA) that recruit Cas9 to target the BCL11A gene, resulting in a double stranded break. Next, DNA samples will be analyzed from five CRISPR experiments. In each sample, DNA from the BCL11A gene has been amplified and combined with Cas9 and a unique gRNA. If the Cas9:gRNA complex is successfully able to cleave the BCL11A gene, it will reveal multiple bands during agarose gel electrophoresis. This will allow you to select the gRNAS that can potentially be used to inactivate the BCL11A gene. After successful gene editing, the HbF gene will turn on, expressing the fetal hemoglobin subunit and alleviating the symptoms of beta-thalassemia or sickle cell anemia.
Figure 6: Gene expression of hemoglobin before and after birth. 2
Image Attributions: 1 Zephyris at the English-language Wikipedia, CC BY-SA 3.0 <http://creativecommons.org/licenses/by-sa/3.0/>, via Wikimedia Commons 2 Postnatal_genetics.svg: original: Furfur, File:Haemoglobin-Ketten.svg, derivation/translation:Leonid 2 deriva- tive work: Leonid 2, CC BY-SA 3.0, via Wikimedia Commons
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