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  • What is the PAM?

  • A CRISPR White Board Lesson presented by the IGI.

  • Editing genomes with CRISPR proteins involves something that often confuses people

  • a little sequence called the PAM.

  • What is a PAM?

  • Why does it exist?

  • And why does it matter?

  • We're going to help you understand.

  • It all starts with what CRISPR systems originally evolved to do: defend bacteria against viruses.

  • Bacteria face the constant threat of infection and destruction by a special type of virus

  • - the bacteriophage.

  • If bacteriophages are able to inject their DNA genomes into the bacterial cell, replicate

  • inside, and burst out, the cell is toast!

  • In response, bacteria have evolved a protective immune system called CRISPR.

  • The CRISPR array is a short stretch of DNA in bacteria composed of alternating repeated

  • sequences and target-specific spacers.

  • These spacers contain the DNA of invading viruses collected from past infections.

  • When a virus infects a bacterium, a new spacer is added in to the growing CRISPR array.

  • This process begins when a protein complex, known as Cas1 and Cas2, identifies the invading

  • viral DNA, and cuts out a segment of a specific length.

  • This segment of DNA is known as the protospacer.

  • The protospacer is inserted into the front of the CRISPR array.

  • Now the bacteria has an embedded memory of this phage infection.

  • If the phage returns, the bacteria is armed to defend itself.

  • This defense starts with transcribing a long CRISPR RNA (crRNA) from the repeats and spacers

  • of the CRISPR array.

  • Another RNA called the trans-acting or tracrRNA comes along and links up with the crRNA through base pairing

  • A protein, known as Cas9, grabs onto the dual RNAs, and theyre trimmed to a more manageable

  • size to form a complete search complex.

  • If the sequence of the crRNA matches the sequence of the invading virus, Cas9 cuts the viral

  • DNA and destroys the phage, allowing the bacterial cell to survive.

  • *Snip! *Snip!

  • But waitthe viral DNA that is targeted by the search complex is the exact same sequence

  • as the DNA in the CRISPR array, so how exactly is Cas9 able to distinguish between itself

  • and the enemy?

  • This is where the PAM comes in.

  • The PAM, which stands for the protospacer adjacent motif, is a specific sequence of

  • nucleotides, around 2–6 base pairs, that follows the protospacer sequence in a viral genome

  • In Streptococcus pyogenes, Cas9 recognizes the PAM sequence "GG," with an additional

  • nucleotide between it and the protospacer.

  • This PAM sequence must be present for the Cas9 protein to know that it’s okay to latch

  • onto and cut this region of DNA.

  • How does this keep the bacterium from hurting itself?

  • The key is that the spacer sequences within the CRISPR array are NOT followed by a "GG."

  • The sequence of the repeat is always the same -"GTT."

  • This means that the Cas9 is unable to bind to the CRISPR array and thereby avoids cutting

  • the bacterium's own genome.

  • But how is it that there’s always a PAM sequence at a true Cas9 target?

  • In the DNA acquisition step we covered previously, we mentioned that the Cas1–Cas2 protein

  • complex is in charge of capturing new spacers from incoming viral DNA.

  • Cas9 works with Cas1 and Cas2 to find a PAM sequence and remove the protospacer next to it.

  • Only picking targets with PAMs guarantees that when the same virus infects again and

  • Cas9 is armed with a matching crRNA guide, nothing will stop it from destroying the enemy DNA.

  • The PAM sequence also serves an additional role.

  • Searching through all the DNA inside a bacterial cell can take a very long time, but the PAM

  • sequence accelerates the search process.

  • Instead of trying to unwind every bit of DNA to check for a match, Cas9 bounces around

  • the cell, searching for a tiny PAM sequence.

  • If it finds one, only then does it check to see if the crRNA matches.

  • So how is the PAM involved in Cas9 genome editing?

  • If scientists want to use Cas9 to cut human DNA or the DNA of any other organism, they

  • first look for a PAM sequence within the target genome and then design an RNA to match the

  • sequence next to it.

  • But what if there is noGGnext to what scientists want to cut?

  • Fortunately, “GGisn’t the only PAM in town.

  • Scientists can edit genes with Cas9s from different organisms and even different CRISPR proteins

  • These different proteins have all evolved to recognize distinct PAMs.

  • Scientists have even engineered the S. pyogenes Cas9 to recognize other PAMs.

  • The future of genomics is in our hands, so make sure not to forget the PAM!

What is the PAM?

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B2 US pam crispr dna sequence array bacteria

What is the PAM? - A CRISPR Whiteboard Lesson

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    zywang posted on 2020/03/09
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