06 - Introducing Your Students to CRISPR with Sickle Cell Gene Editing
commonly-used Cas9 from Streptococcus pyogenes recognizes the PAM sequence 5’-NGG-3’, where “N” can be any nucleotide base (Figure 2). The discovery that CRISPR spacers were related to viral DNA sequences occurred by three different groups of scientists. Eu - gene Koonin, an evolutionary biologist at the National Center for Biotechnology Information in Bethesda, Maryland, developed a theory that bacteria were using CRISPR to fight off viruses. Koo -
If you’ve eaten yogurt or cheese, chances are you’ve eaten CRISPR-ized cells. – Rodolphe Barrangou
nin’s theory was tested by Roldolphe Barrangou and Philippe Horvath, then microbiologists at the yogurt company Danisco in France. The company used bacteria to convert milk into yogurt, and entire cultures could be wiped out by bacteria-killing viruses. Barrangou and his team in- fected one of their yogurt bacteria – Streptococcus thermophilus – with two strains of viruses and
cultured the resistant bacteria that survived the assault. Upon examination, they found DNA from the viruses they had used inside CRISPR spacers. Some of the other contributors to CRISPR-Cas be- tween 2002 and 2013 include: John van der Oost of the University of Wageningen in The Netherlands (the discovery of small CRISPR RNAs), Luciano Marraffini and Erik Sontheimer at Northwestern University in the USA (CRISPR targets DNA, not RNA), Sylvain Moineau at the University of Laval in Canada (CRISPR-Cas9 can produce double-strand- ed breaks in target DNA), and Virginijus Siksnys at Vilnius University in Lithuania (CRISPR systems are self-contained units that can be cloned, and Cas9 can be reprogrammed to a site of choice by chang- ing the sequence of the CRISPR rRNA). The next step in the CRISPR story was carried out by three different scientists at almost the same time: Jennifer Doudna at the University of Califor- nia in Berkeley who worked on microbial CRISPR- Cas systems; Emmanuelle Charpentier, then at the University of Vienna in Austria, who also worked on microbial CRISPR-Cas systems; and Feng Zhang at the Broad Institute of MIT who pioneered CRISPR systems in mammalian and human cells. All three of these scientists created mechanisms that made CRISPR a real research tool and not just an interesting phenomenon.
Cas9
gRNA
Binding to target
Double-stranded cut
Figure 3: CRISPR targeting and digestion of DNA.
Jennifer Doudna was an RNA expert who was trying to discover all the things that RNA can do besides being a protein tem- plate. She had already found that it could be used as a sensor and could control the activity of genes when Blake Wiedenheft joined her laboratory. Wiedenheft wanted to study Cas en- zymes to understand how they worked, and Doudna sponsored his research because she thought the chemistry would be interesting, not because she thought CRISPR had any practical applications.
You’re not trying to get to a particular goal except understanding. – Jennifer Doudna
What they discovered was that Cas enzymes could cut DNA and were programmable. Using the CRISPR-Cas9 system from Streptococcus pyogenes , which causes strep throat, Doudna and her colleagues figured out how to hand the Cas9 enzyme an RNA molecule that matched a sequence of DNA they wanted to cut from the genome, then guide it to the target site (Figure 3). Meanwhile, Charpentier and her colleagues were mapping all the RNAs in Streptococcus pyogenes and finding a large number of new small RNA molecules they called trans-activating CRISPR RNA (tracrRNA) that lived close to the S. pyogenes CRISPR system. They also discovered that, unlike other CRISPR systems that contained one RNA strand and many proteins, S. pyogenes ’ CRISPR
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