Editing genomes with CRISPR requires a short DNA sequence called a “PAM” right next to the sequence targeted for editing. The Casprotein, which functions as the DNA “scissors,” initially binds at the PAM as a first step of editing DNA. But this creates a challenge — some important genes that we might wish to edit don’t have a good PAM sequence nearby.
Imagine the perfect Cas enzyme — a single enzyme that can edit any genomic sequence, without relying on specific PAM requirements. This has been a dream of genome editors for some years, but attempts to create “PAMless” or “PAM-relaxed” Cas enzymes with structure-guided engineering has so far yielded Cas enzymes with low editing efficiency and high off-target effects.
In a new paper from the Doudna lab out today in Molecular Cell, Doudna lab post-doctoral researcher and co-first author Honglue Shi uncovers the molecular basis for this surprising stumbling block. Using the PAM-relaxed Cas9 variant SpRY Cas9, Shi and the team found two key problems: First, removing the requirement for PAM binding unexpectedly made the Cas9 enzyme bind strongly and at random areas all across the genome. This strong and widespread binding slowed the enzyme down, making it harder for it to find the actual target DNA sequence the researchers wanted to edit. Second, even when it eventually reached the target sequence, this overly strong binding caused the Cas9 to get stuck at an early step, preventing it from efficiently opening and cutting the DNA as intended.
Co-first author Noor Al-Sayyad at Stanford
Co-first authors Kevin Wasko (left) and Honglue Shi in front of their gel imager, confirming that the SpRY Cas9 variant struggles to effectively open DNA
Shi’s biochemical work on the project was supported by co-first authors Kevin Wasko, a Doudna lab graduate student, and Noor Al-Sayyad, a graduate student in Zev Bryant’s lab at Stanford. Wasko did experiments with human cells to look at editing efficiency and Noor used an innovative microscopy technique called rotor bead tracking to look at DNA unwinding.
“What we’ve done here is uncover the secret sauce that makes typical Cas9 — the original Nobel molecule — so efficient and precise.” says Shi. “Its success comes from a delicate balance: Cas9 gently but precisely grabs onto its PAM site, then quickly unzips the DNA strands to reach its target. This tells us that the path forward isn’t a one-size-fits-all PAMless Cas enzyme, but rather a carefully curated ‘PAM catalog’, where researchers select the best PAM variant for their particular editing task. This ensures high efficiency and accuracy, crucial for future genetic therapies.”
“Without NIH funding, we would have a huge hurdle to doing this research,” says Shi. Researchers on the project were also funded by other NIH programs and the NSF Graduate Research Fellowship Program.
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Co-first author Noor Al-Sayyad at Stanford
Co-first authors Kevin Wasko (left) and Honglue Shi in front of their gel imager, confirming that the SpRY Cas9 variant struggles to effectively open DNA
