SMART Templates Break CRISPR’s PAM Barrier With 10× Editing Reach

CRISPR/Cas9 gene editing has revolutionized molecular biology but remains constrained by the need for a nearby PAM site. A team from The Scripps Research Institute has developed SMART (silently mutate and repair template), a strategy that dramatically expands the editable range of CRISPR targets by redesigning the repair template – without changing the enzyme. Their work, published in Nature Communications, demonstrates in vivo protein labeling in the retina and opens new doors for therapeutic and research applications.

Why CRISPR needed a smarter template

Although CRISPR/Cas9 is powerful and widely used, one of its fundamental limitations is the requirement for a nearby PAM (protospacer adjacent motif) sequence. This restricts where edits can be made, particularly for precise modifications like inserting tags or correcting point mutations. Engineering new PAM-flexible Cas variants is possible, but often labor-intensive, costly and not always efficient. Our goal was to create a solution that works with standard Cas9 proteins by leveraging a smarter design of the repair template itself.

Engineering around PAM

We developed the SMART strategy, which introduces silent mutations in the gap region of the DNA repair template – the space between the cut site and the modification site. This prevents unintended homology-based repair in this region and ensures the entire template is used efficiently (Figure 1). We also extended this design into a second-generation version (SMART-RC), where the edit is made directly at the cut site, enabling even more flexible applications. We tested both strategies in the mouse retina using in vivo CRISPR electroporation.

Figure 1: Comparison of traditional and SMART templates. Credit: Chuanping Zhao, Martemyanov Lab, The Scripps Research Institute.

The key findings of this study were:

• SMART expands the usable guide RNA (gRNA) selection window up to 10-fold.
• Over 99% of genome targets become editable with efficient gRNAs.
• SMART enables high-efficiency labeling of endogenous proteins in vivo.
• SMART-RC further improves edit precision by aligning the cut and insert sites.
• The technique could be successfully applied in diverse retinal cell types including photoreceptors.

What SMART means for the future of genome editing

Our results demonstrate that the limitations imposed by PAM proximity can be overcome through intelligent repair template design. By decoupling PAM position from the edit site, SMART allows researchers to make precise edits at virtually any desired location without altering the enzyme itself. This greatly increases the versatility of CRISPR/Cas9 while keeping the system simple and accessible. It also means that legacy gRNA libraries and standard Cas9 tools can be used more effectively across a broader range of genomic contexts.

SMART could transform how researchers approach gene tagging, disease modeling and therapeutic editing. It has immediate applications in neuroscience and developmental biology and longer-term potential in gene therapy – especially for correcting pathogenic mutations where PAM options are limited.

There are, however, some limitations to note. While SMART works with standard Cas9, it still ultimately depends on PAMs for Cas9 recognition and binding. The editing efficiency may vary depending on cell type, delivery method and DNA repair environment and in vivo electroporation may not translate directly to clinical contexts without further optimization. Future work is needed to validate SMART’s performance across other tissues and organisms, and in combination with other editing systems like base or prime editors.

What comes next

We are now exploring how SMART can be adapted for other genome editing platforms and expanded into non-dividing cell types such as adult neurons. Further research will focus on applying this strategy to clinically relevant disease genes and integrating SMART with other advanced delivery systems. Long-term, SMART could become a standard toolkit component for precise, safe and efficient genome editing in therapeutic settings.

Reference: Zhao C, Cao Y, Ibrahim N et al. Efficient in vivo labeling of endogenous proteins with SMART delineates retina cellular and synaptic organization. Nat Commun 16, 3768 (2025). doi:10.1038/s41467-025-58945-6

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