SpRY Cas9: New Horizons in Genome Editing

Advancements in genome editing have led to the development of SpRY Cas9, a highly versatile variant of the widely used CRISPR-Cas9 system. This enzyme expands the range of DNA sequences that can be targeted, addressing limitations of earlier Cas9 versions and improving precision for genetic modifications. Its flexibility holds promise for research, therapeutic applications, and biotechnology.

Mechanisms Of DNA Cleavage

SpRY Cas9 functions as a programmable endonuclease, introducing double-strand breaks (DSBs) in DNA with precision. Like Streptococcus pyogenes Cas9 (SpCas9), it employs a catalytic HNH domain to cleave the complementary DNA strand and a RuvC domain to cut the non-complementary strand. However, modifications reduce its dependence on protospacer adjacent motifs (PAMs), expanding its targeting range and enabling access to previously inaccessible genomic regions.

Once SpRY Cas9 binds to its target site, the enzyme undergoes a conformational shift that positions the HNH domain for cleavage. Structural studies using cryo-electron microscopy reveal increased flexibility in this domain, allowing it to accommodate a wider array of DNA sequences. This adaptability is particularly useful for therapeutic applications requiring precise gene correction. The RuvC domain follows shortly after HNH activation, ensuring coordinated cleavage.

Following DNA cleavage, the cell’s repair mechanisms determine the edit’s outcome. Non-homologous end joining (NHEJ) often introduces small insertions or deletions (indels), disrupting gene function. Alternatively, homology-directed repair (HDR) enables precise sequence modifications when a donor template is available. The efficiency of these repair pathways varies by cell type and cycle phase. Researchers are exploring strategies to influence repair outcomes, such as small-molecule inhibitors that suppress NHEJ or engineered templates that enhance HDR efficiency.

PAM Recognition

Protospacer adjacent motif (PAM) recognition dictates where CRISPR-Cas9 can bind and initiate DNA cleavage. Traditional SpCas9 requires an NGG PAM sequence, limiting its targeting range. SpRY Cas9 has been engineered to exhibit near-PAMless activity, broadening the number of accessible genomic sites. By reducing reliance on specific nucleotide motifs, it enables modifications in regions previously difficult to edit.

Structural analyses show that SpRY Cas9 achieves this flexibility through alterations in its PAM-interacting domain, which traditionally binds NGG sequences. Mutations in this domain weaken its preference, allowing interaction with a broader spectrum of DNA motifs, including NRN and NYN sequences, where N represents any nucleotide and R/Y denote purines or pyrimidines. This expanded compatibility improves genome editing efficiency, especially in organisms or regions with limited conventional PAM sites.

While the relaxed PAM requirement increases target accessibility, it also raises concerns about unintended interactions. High-throughput sequencing techniques such as GUIDE-seq and CIRCLE-seq have been used to assess off-target activity, showing that SpRY Cas9 maintains specificity comparable to traditional SpCas9. Researchers are refining computational models to predict potential off-target sites and optimize guide RNA design for greater precision.

Guide RNA Binding

SpRY Cas9’s ability to recognize and bind guide RNA (gRNA) is central to its function. The enzyme associates with a single-guide RNA (sgRNA), forming a ribonucleoprotein complex that directs it to the target sequence. The sgRNA consists of a scaffold region stabilizing Cas9 binding and a spacer sequence complementary to the DNA target. SpRY Cas9 maintains high binding affinity across a wider set of target sites due to its relaxed PAM constraints, increasing flexibility in gRNA design.

Cryo-electron microscopy studies show that SpRY Cas9 undergoes conformational changes upon sgRNA binding, optimizing DNA recognition. These shifts enhance its ability to distinguish between on-target and near-target sequences, which is crucial for precise genetic modifications. Researchers have found that GC-rich spacers improve binding stability, while certain mismatches near the 5’ end of the spacer reduce cleavage efficiency. Computational tools now help predict optimal gRNA sequences for improved editing accuracy.

Chemical modifications to gRNA have been explored to enhance SpRY Cas9 performance. Alterations such as 2′-O-methylation and phosphorothioate linkages improve stability and reduce degradation by cellular nucleases, increasing editing efficiency in mammalian cells. These modifications are particularly valuable for therapeutic applications requiring prolonged gRNA activity. Additionally, truncated gRNAs with shorter spacer regions have been investigated as a strategy to minimize off-target effects while maintaining high specificity.