字幕表 動画を再生する 英語字幕をプリント 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 they’re 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 wait… the 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 no “GG” next to what scientists want to cut? Fortunately, “GG” isn’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!