With the recent award of the Nobel Prize in chemistry to Emmanuelle Charpentier and Jennifer Doudna, the terms CRISPR and Cas9 are currently drawing much attention throughout news and social media outlets. Meanwhile, scientists at the Würzburg-based Helmholtz Institute for RNA-based Infection Research (HIRI), a site of the Helmholtz Centre for Infection Research (HZI), have identified novel DNA motifs called PAMs (Protospacer Adjacent Motifs) for Cas9 that can be applied to sharpen the precision of CRISPR technologies. The research team led by Professor Chase Beisel, head of HIRI’s RNA Synthetic Biology group, in collaboration with scientists from North Carolina State University and University of North Texas, published their findings earlier on this year in the journal Science Advances.
While many different Cas nucleases exist, the most commonly used has been the Cas9 nuclease from a bacterium called Streptococcus pyogenes. “Like other Cas nucleases, Cas9 can only target DNA sequences flanked by a specific PAM,” explains Beisel. The acronym PAM, short for protospacer adjacent motif, refers to a specific nucleotide sequence whose presence and location dictates the genomic regions that can be targeted for editing by CRISPR. These motifs are nuclease specific, which means that while many different Cas nucleases exist, each one only responds to a specific PAM. However, Cas nucleases can have off-target effects, where similar sequences elsewhere in the genome are inadvertently targeted, resulting in reduced precision and unintended edits.
Despite being extensively studied, until now it had remained unclear whether additional sequences could be recognized as a PAM by Cas9. The team found that this widely used Cas9 could recognize additional sequences as PAMs. The unexpected findings “potentially impact every application in which this Cas9 is used” states Prof Beisel. As Cas9 is currently being used to study human diseases and for human gene therapy in ongoing clinical trials, a better idea of which DNA sequences can and cannot be targeted would therefore impact all of these areas. In the future, such ‘non-canonical’ PAMs could potentially be included into off-target prediction algorithms, not only for Cas9 but for other nucleases as well, increasing the precision of CRISPR technologies altogether.
Moreover, their work introduces the first PAM screen that links recognition of a PAM to E. coli growth on sugar, simplifying how PAM preferences can be identified. This unique high-throughput screen is called PAM-SEARCH. The researchers confirmed that the novel PAM sequences could be recognized under a wide array of assays, in both bacterial and human cells, such as base editors that are being developed to fix disease-causing mutations. The study also observed the same results in “high-fidelity” versions of Cas9 that are known to reject any off-target sites. Further molecular dynamics simulations conducted by the researchers shed light on how Cas9 recognizes this unique PAM sequence at a molecular level.
CRISPR technologies: What are genetic scissors and why do they matter?
“CRISPR technologies are revolutionary tools for genome editing that possess a myriad of applications in various fields, including medicine, agriculture, biotechnology and scientific research,” explains Prof Beisel. Often referred to as genetic scissors, CRISPR/Cas involves two components that allows scientists to target and edit specific segments of DNA from animals, plants and microorganisms. The system requires a ‘guide’ RNA that allows scientists to target a specific DNA region while Cas, a nuclease guided by the RNA, cuts the DNA like scissors. Once the DNA is cut, it is then easy to rewrite the code, omitting or including specific sequences.
D. Collias, R. T. Leenay, R. A. Slotkowski, Z. Zuo, S. P. Collins, B. A. McGirr, J. Liu and C. L. Beisel: A positive, growth-based PAM screen identifies noncanonical motifs recognized by the S. pyogenes Cas9. Science Advances (2020), DOI: 10.1126/sciadv.abb4054