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What is the PAM?
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A CRISPR White Board Lesson presented by the IGI.
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Editing genomes with CRISPR proteins involves something that often confuses people
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a little sequence called the PAM.
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What is a PAM?
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Why does it exist?
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And why does it matter?
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We're going to help you understand.
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It all starts with what CRISPR systems originally evolved to do: defend bacteria against viruses.
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Bacteria face the constant threat of infection and destruction by a special type of virus
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- the bacteriophage.
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If bacteriophages are able to inject their DNA genomes into the bacterial cell, replicate
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inside, and burst out, the cell is toast!
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In response, bacteria have evolved a protective immune system called CRISPR.
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The CRISPR array is a short stretch of DNA in bacteria composed of alternating repeated
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sequences and target-specific spacers.
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These spacers contain the DNA of invading viruses collected from past infections.
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When a virus infects a bacterium, a new spacer is added in to the growing CRISPR array.
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This process begins when a protein complex, known as Cas1 and Cas2, identifies the invading
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viral DNA, and cuts out a segment of a specific length.
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This segment of DNA is known as the protospacer.
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The protospacer is inserted into the front of the CRISPR array.
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Now the bacteria has an embedded memory of this phage infection.
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If the phage returns, the bacteria is armed to defend itself.
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This defense starts with transcribing a long CRISPR RNA (crRNA) from the repeats and spacers
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of the CRISPR array.
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Another RNA called the trans-acting or tracrRNA comes along and links up with the crRNA through base pairing
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A protein, known as Cas9, grabs onto the dual RNAs, and they’re trimmed to a more manageable
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size to form a complete search complex.
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If the sequence of the crRNA matches the sequence of the invading virus, Cas9 cuts the viral
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DNA and destroys the phage, allowing the bacterial cell to survive.
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*Snip! *Snip!
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But wait… the viral DNA that is targeted by the search complex is the exact same sequence
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as the DNA in the CRISPR array, so how exactly is Cas9 able to distinguish between itself
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and the enemy?
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This is where the PAM comes in.
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The PAM, which stands for the protospacer adjacent motif, is a specific sequence of
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nucleotides, around 2–6 base pairs, that follows the protospacer sequence in a viral genome
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In Streptococcus pyogenes, Cas9 recognizes the PAM sequence "GG," with an additional
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nucleotide between it and the protospacer.
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This PAM sequence must be present for the Cas9 protein to know that it’s okay to latch
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onto and cut this region of DNA.
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How does this keep the bacterium from hurting itself?
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The key is that the spacer sequences within the CRISPR array are NOT followed by a "GG."
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The sequence of the repeat is always the same -"GTT."
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This means that the Cas9 is unable to bind to the CRISPR array and thereby avoids cutting
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the bacterium's own genome.
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But how is it that there’s always a PAM sequence at a true Cas9 target?
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In the DNA acquisition step we covered previously, we mentioned that the Cas1–Cas2 protein
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complex is in charge of capturing new spacers from incoming viral DNA.
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Cas9 works with Cas1 and Cas2 to find a PAM sequence and remove the protospacer next to it.
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Only picking targets with PAMs guarantees that when the same virus infects again and
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Cas9 is armed with a matching crRNA guide, nothing will stop it from destroying the enemy DNA.
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The PAM sequence also serves an additional role.
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Searching through all the DNA inside a bacterial cell can take a very long time, but the PAM
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sequence accelerates the search process.
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Instead of trying to unwind every bit of DNA to check for a match, Cas9 bounces around
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the cell, searching for a tiny PAM sequence.
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If it finds one, only then does it check to see if the crRNA matches.
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So how is the PAM involved in Cas9 genome editing?
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If scientists want to use Cas9 to cut human DNA or the DNA of any other organism, they
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first look for a PAM sequence within the target genome and then design an RNA to match the
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sequence next to it.
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But what if there is no “GG” next to what scientists want to cut?
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Fortunately, “GG” isn’t the only PAM in town.
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Scientists can edit genes with Cas9s from different organisms and even different CRISPR proteins
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These different proteins have all evolved to recognize distinct PAMs.
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Scientists have even engineered the S. pyogenes Cas9 to recognize other PAMs.
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The future of genomics is in our hands, so make sure not to forget the PAM!