AI-Designed Synthetic CRISPR with Reverse Protein Folding Achieves Enhanced Genome Editing Efficiency by Modifying 30% of the Sequence
Background
The limitations of gene editing tools that have evolved over hundreds of millions of years in nature. The CRISPR-Cas9 system has become a representative gene editing tool that precisely cleaves and corrects specific DNA within cells in gene therapy and agricultural biotechnology. However, directly utilizing naturally occurring Cas proteins as therapeutic agents or research tools has practical limitations. This is because their large molecular size makes it difficult to load them into adeno-associated viruses (AAV), which are used for in vivo delivery, or they may cause off-target effects.
Researchers have attempted to improve the performance of naturally occurring gene editing tools by making minor modifications. Existing protein engineering techniques have been limited to sequence modifications that replace only 1-2% of amino acids in the native sequence. Excessive sequence changes could disrupt the three-dimensional structure of the protein, potentially leading to a complete loss of its catalytic activity. As a result, CRISPR technology has faced the limitation of being improved only within the range of natural gene resources.
Key Findings
SynTnpB, a protein redesigned based on artificial intelligence. A research team led by Professor Jennifer Doudna at the University of California, Berkeley, overcame this limitation by introducing generative artificial intelligence (AI) technology. The team utilized an inverse protein-folding AI model that takes a desired three-dimensional protein structure and function as input and calculates the optimal amino acid sequence in reverse.
The target of this study was TnpB, an evolutionary precursor of the Cas12 protein and a small RNA-guided cleavage enzyme. The AI model designed a large number of synthetic variants (SynTnpBs) with completely new sequences based on the three-dimensional structure of natural TnpB. Surprisingly, the amino acid sequence of the synthetic enzymes designed by AI differed by up to 30% compared to natural TnpB.
The research team applied the generated synthetic enzymes to bacteria, plants, and human cell lines to verify their actual gene editing ability. The results showed that SynTnpB exhibited higher cleavage efficiency than natural TnpB, and it also demonstrated excellent precision in accurately recognizing the target DNA sequence. This not only surpassed the 1-2% modification limit of existing protein engineering but also demonstrated that artificially redesigned proteins with large-scale sequence modifications can exhibit much better function in vivo.
Significance and Prospects
A new milestone in biotechnology and therapeutic development. This study shows that the focus of gene editing technology has shifted from the discovery of natural products to AI-driven custom design. The small and highly precise synthetic TnpB is easy to load into AAV viral vectors. This directly addresses the dose limitation problem that occurs when delivering therapeutic agents for intractable genetic diseases into the body.
However, there are still challenges to overcome before it can be commercialized as an actual therapeutic agent. The immunogenicity of the non-natural amino acid sequences generated by AI when administered to the human immune system needs to be carefully evaluated. In addition, follow-up clinical studies are essential to ensure long-term stability in various tissues and biological environments. The research team plans to improve the AI model in the future to develop next-generation gene editing tools that minimize immune response and further enhance editing precision.
Nature, Published online: 17 July 2026; doi:10.1038/d41586-026-02272-3Synthetic CRISPR proteins edit the genome more efficiently than their natural counterparts. Plus, a rocky exoplanet with an atmosphere and the mathematical wonderland of footballs.
The small gene editing tool SynTnpB, designed by AI, provides concrete solutions in the fields of biopharmaceutical therapeutic development and the creation of gene-edited crops. The existing Cas9 enzyme consists of more than 4,000 amino acids, making it difficult to load into AAV for gene therapy. In contrast, SynTnpB has a significantly lower molecular weight, allowing it to load the gene editing tool, target guide RNA, and therapeutic gene fragment into a single viral vector.
This characteristic greatly improves the therapeutic efficacy of intractable diseases that require in vivo gene delivery, such as retinitis pigmentosa and muscular dystrophy. In the field of plant biotechnology, it can also significantly shorten the breeding period by minimizing off-target mutations when inducing desired agricultural traits. Custom protein design technology that overcomes the limitations of natural proteins is expected to become a core platform in the next generation of biotechnology.