06 - Introducing Your Students to CRISPR with Sickle Cell Gene Editing
Background Information The gene editing tool CRISPR-Cas9 was developed by bacteria at the beginning of evolutionary history as a defense against viral attacks. It was created by nature, not human beings, but we discovered it in the late 1980s. We figured out how it worked in the early years of this century, and have now made it into a valuable part of our efforts to improve human health, make our food supply hardier and more resistant to disease, and advance any arm of science that involves living cells, such as biofuels and waste management. The CRISPR-Cas System in Action
In 1987 Yoshizumi Ishino and colleagues at Osaka University in Japan were researching a new microbial gene when they discovered an area within it that contained five identical segments of DNA made up of the same 29 base pairs. The segments were separated
Figure 1: Bacterial CRISPR Region.
from each other by 32-base pair blocks of DNA called spacers, and each spacer had a unique configuration (Figure 1). This section of DNA didn’t resemble anything microbiologists had seen before and its biological significance was unknown. Eventually these strange segments and spac - ers would be known as Clustered Regularly Interspaced Short Palindromic Repeats – or CRISPR. Scientists also discovered that a group of genes coding for enzymes they called Cas (CRISPR- associated enzymes) were always next to CRISPR sequences. In 2005, three labs noticed that the spacer sequences resembled viral DNA and everything fell into place. When a virus invades a bacterium, the bacterium identifies the virus as foreign and collects some of its DNA so it can be recognized the next time it shows up. The bacterium puts the viral DNA into a spacer in the CRISPR section of its own DNA. As the spacers fill up with viral DNA, they become a database of viral enemies. To set up an ongoing defense system, the bacterium takes each piece of viral DNA out of storage in the spacers and transcribes it into a strand of RNA, then a Cas enzyme binds to one of these loaded RNA strands. Together, the viral-loaded RNA and the Cas enzyme drift through the cell. If they encounter foreign DNA that matches the spacer sequence, the RNA will base-pair so the Cas enzyme can chop the invader’s DNA into pieces and prevent it from replicating. This system made other bacterial defenses, such as restriction enzymes, look very primitive. When they used CRISPR-Cas, bacteria could find any short sequence of DNA and attack it with precision. CRISPR-Cas9 History Because DNA sequencing technology was in its infancy in 1987, the Japanese scientists didn’t know if the mysterious structure they had discovered only occurred in E. coli ; but by the late 1990s technology had advanced and microbiologists could sequence most of the microbial DNA in seawater and soil samples.
Thanks in part to the newly available DNA sequencing data, a study led by Ruud Hansen found that the Cas enzymes could snip DNA but didn’t know why. At the same time, Alex- ander Bolotin’s team at the French National Institute for Agricultural Research found that the spacers all share a common sequence they called the protospacer adjacent motif (PAM). The PAM enables Cas enzymes to recognize their target. Different Cas enzymes recognize different PAM sequences; the most
Figure 2: Target DNA and PAM site.
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