CRISPR-Cas9 gene editing relies on a nuclease, a protein that cuts DNA, to make precise changes to a genome. One such nuclease is SaCas9, which originates from the bacterium Staphylococcus aureus. While less common than its SpCas9 counterpart, SaCas9 is notable for its smaller size, making it a valuable tool for specific applications in genetic research.
A component of how any Cas9 nuclease functions is its interaction with a Protospacer Adjacent Motif, or PAM. The PAM is a short, specific DNA sequence that the protein must recognize to initiate its gene-editing activity. This sequence is necessary for effectively using the SaCas9 system to target and modify DNA.
The Function of the Protospacer Adjacent Motif
The Protospacer Adjacent Motif (PAM) is a short sequence of DNA bases located immediately next to the DNA sequence targeted for editing. It serves as a binding and recognition signal for the Cas nuclease. The nuclease, guided by an RNA molecule to a location on the genome, scans the DNA for the correct PAM sequence before it binds and makes any cuts. If the correct PAM is not present, the nuclease will not engage with the target DNA.
This recognition mechanism originates from the CRISPR system’s natural role in bacteria, where it functions as an adaptive immune system to fight invading viruses. The PAM sequence is present in the viral DNA but is absent from the bacterial genome where corresponding target sequences are stored. This distinction allows the cell’s Cas nuclease to identify and destroy foreign DNA without attacking its own genetic material.
In the context of gene editing, a specific gene can only be targeted if it has the correct PAM sequence located adjacent to the desired editing site. The PAM itself is not part of the sequence that gets modified, but its presence is a prerequisite for the Cas9 protein to function.
The Specific SaCas9 PAM Sequence
The recognized Protospacer Adjacent Motif for the wild-type SaCas9 nuclease is NNGRRT. This sequence is located on the target DNA strand immediately following the region that the guide RNA binds to. The standard IUPAC nucleotide code helps break down what this sequence means:
- N can be any of the four DNA bases: Adenine (A), Guanine (G), Cytosine (C), or Thymine (T).
- G stands specifically for Guanine.
- R represents a purine, which means it can be either Adenine (A) or Guanine (G).
- T stands for Thymine.
The SaCas9 nuclease recognizes a family of related sequences, and studies have confirmed that the guanine (G) at the third position is important for efficient DNA cleavage. This requirement differs from the more commonly used SpCas9 nuclease, which recognizes the simpler NGG PAM. The longer and more complex SaCas9 PAM offers a different set of targeting parameters for researchers.
While the SaCas9 PAM may seem more restrictive, it expands the total number of editable sites when used alongside SpCas9. If a desired DNA target lacks the NGG sequence required by SpCas9, it might possess the NNGRRT sequence needed for SaCas9. This complementarity provides scientists with more options to find a suitable location for genetic modification.
Key Advantages of the SaCas9 System
A primary advantage of the SaCas9 system is the physical size of the nuclease. The SaCas9 protein is composed of approximately 1053 amino acids, making it significantly smaller than the SpCas9 protein, which has about 1368 amino acids. This size difference means its corresponding gene is more than one kilobase shorter.
This compact size is beneficial for in vivo gene therapy applications, where the editing machinery must be delivered into living organisms. The most common delivery vehicle for this purpose is the Adeno-Associated Virus (AAV), which is constrained by a packaging limit of roughly 4.7 kilobases of genetic material.
The larger gene for SpCas9 (around 4.2 kb) pushes this limit, making it challenging to package it into a single AAV with its guide RNA and regulatory elements. In contrast, the smaller SaCas9 gene (around 3.2 kb) fits comfortably within a single AAV vector with all other required components. This all-in-one system simplifies the delivery process and increases the efficiency of gene editing.
Engineered SaCas9 Variants for Expanded Targeting
While wild-type SaCas9 is effective, its targeting is limited by the need for a specific NNGRRT PAM sequence. To overcome this constraint, researchers have used protein engineering to create modified versions of SaCas9 with altered or more relaxed PAM specificities. This work expands the number of genomic sites that can be accessed for gene editing.
A notable engineered version is KKH-SaCas9. Through targeted mutations, scientists modified the part of the nuclease that recognizes the PAM. The resulting KKH-SaCas9 variant recognizes a more flexible NNNRRT sequence. This change removes the requirement for a guanine at the third position, nearly quadrupling the number of potential target sites within the human genome compared to its wild-type predecessor.
The development of such variants demonstrates how gene-editing tools can be tailored for greater utility, making previously untargetable regions of a gene accessible. Creating high-fidelity variants also reduces off-target effects, enhancing the precision and safety of these tools for therapeutic applications. This field of protein engineering remains an active area of research, with ongoing efforts to design new Cas nucleases with broader targeting capabilities.