In the rapidly evolving field of genetic research, CRISPR technology stands out as a groundbreaking tool with the potential to revolutionize medicine, agriculture, and biotechnology. One crucial aspect of CRISPR systems is the Protospacer Adjacent Motif (PAM), a short DNA sequence that is indispensable for the recognition and cutting of target DNA by CRISPR-associated (Cas) nucleases. A recent review paper published in Nature Communications delves into the complexity of PAM recognition and explores the future of engineering PAM-free nucleases, which could significantly enhance the capabilities of CRISPR systems.
CRISPR-Cas systems are a type of immune defense mechanism found in prokaryotes, such as bacteria and archaea. These systems use RNA molecules to guide Cas nucleases to specific DNA sequences, where they create double-strand breaks. The presence of a PAM is essential for the CRISPR-Cas system to distinguish between self and non-self DNA, thereby preventing autoimmunity. Without PAMs, CRISPR nucleases could mistakenly target the organism’s own DNA, leading to detrimental consequences.
The specificity and efficiency of CRISPR-Cas systems largely depend on the accurate recognition of PAM sequences. Different Cas nucleases recognize different PAM sequences, which can limit the range of targetable sites within a genome. For instance, the commonly used Streptococcus pyogenes Cas9 (SpyCas9) recognizes the PAM sequence 'NGG,' where 'N' can be any nucleotide. This limitation has spurred extensive research into engineering Cas variants and discovering new nucleases with altered or relaxed PAM requirements.
One of the pioneering efforts in PAM engineering was conducted by Kleinstiver et al., who developed SpyCas9 variants with altered PAM specificities. By combining random mutagenesis with a selection process, they produced variants that could recognize PAM sequences such as NGA, NGAG, and NGCG. Another notable example is xCas9, an evolved SpyCas9 variant capable of recognizing a broader range of PAM sequences, including NG and various NNG sequences. These advancements have expanded the targeting scope of CRISPR systems, allowing for more flexibility in genome editing applications.
In their review, the authors emphasize the challenges and benefits of creating PAM-free nucleases. On one hand, eliminating the PAM requirement would theoretically enable CRISPR nucleases to target any DNA sequence, vastly simplifying site selection and reducing off-target effects. On the other hand, the absence of PAMs could lead to unintended consequences, such as the self-targeting of guide RNA constructs, which could be disastrous, especially in therapeutic applications.
Despite these challenges, the pursuit of PAM-free nucleases continues to gain traction. Researchers are exploring various strategies, including protein engineering and ortholog mining, to achieve this goal. Ortholog mining involves searching for naturally occurring Cas nucleases with diverse PAM recognition profiles. For example, the discovery of NmeCas9, which recognizes the PAM sequence NNNNGATT, has added to the repertoire of available nucleases for genome editing.
Protein engineering, on the other hand, involves making specific modifications to the amino acid sequence of Cas nucleases to alter their PAM recognition capabilities. This approach has led to the creation of several engineered variants with relaxed PAM requirements. For instance, the SpCas9-NG variant, developed using structure-guided design, recognizes the NG PAM sequence, providing greater flexibility in target selection.
A particularly intriguing aspect of the review is the discussion of potential trade-offs associated with PAM-free nucleases. While the ability to target any sequence is highly desirable, it is also important to consider the evolutionary rationale behind PAMs. As the authors note, ""
