CRISPR-associated transposons (CASTs) use nuclease-deficient CRISPR-Cas systems for RNA-guided gene insertion, enhancing the gene-editing toolkit by enabling precise DNA integration.
Background
CRISPR-associated transposons (CASTs) represent a significant advancement in gene-editing technology, originating from the fusion of CRISPR-Cas systems with transposons. These systems have evolved to enable precise, RNA-guided DNA transposition, allowing specific insertion of genetic material without the need for double-strand breaks. The traditional gene-editing methods often use DNA double-strand breaks and rely on cellular repair mechanisms that can be error-prone.
Despite the potential of CASTs, the system faces several challenges that hinder their widespread adoption. One major issue is the rarity of known CASTs in genomic databases, which limits the diversity and understanding of these systems. Known CASTs are primarily derived from Tn7-like transposons and are relatively scarce, with only a few sub-families identified to date. Additionally, the mechanisms by which CASTs select their insertion sites and the diversity of self-targeting strategies are not understood. Current CAST systems also lack the Cas1-Cas2 adaptation machinery, raising questions about how they acquire new spacers for targeting mobile genetic elements. These gaps in knowledge and the limited availability of diverse CAST systems limit the development and optimization of CAST-based gene-editing tools.
Technology description
Through a bioinformatics pipeline applied to metagenomics databases, UT researchers have identified new CAST architectures for Type I-B, I-F, and V systems, including unique Cascade effector arrangements, new self-targeting mechanisms, and minimal V-K systems. Additionally, new families of CASTs that co-opt Type I-C and Type IV CRISPR-Cas systems were discovered. The search also revealed non-Tn7 CASTs that potentially use Cas12a for horizontal gene transfer.
The differentiation of CAST technology lies in its ability to merge the programmable DNA targeting capability of CRISPR-Cas systems with the mobility of transposons. This integration allows for precise, RNA-guided DNA insertion without the need for mutagenic and error-prone double-strand breaks, typically required in traditional CRISPR-Cas9 editing.
The discovery of various CAST architectures expands the gene-editing toolkit, offering more flexibility and specificity in genetic modifications. For instance, Type I-F CASTs show diversity in Cas genes, including TniQ-Cas8/5 fusions and split Cas7s, allowing for different targeting and insertion strategies. The ability of CASTs to utilize CRISPR arrays from other CRISPR-Cas systems for horizontal gene transfer further enhances their versatility. This unique combination of features makes CASTs a powerful tool for genetic engineering, particularly valuable for applications in biotechnology, medicine, and agriculture, where precise genetic alterations are crucial
Benefits
- Enhances understanding of CRISPR-transposon co-evolution
- Expands the gene-editing toolkit
- Discovers new CAST architectures for Type I-B, I-F, and V systems
- Identifies new families of CASTs that co-opt Type I-C and Type IV CRISPR-Cas systems
- Reveals non-Tn7 CASTs potentially using Cas12a for horizontal gene transfer
- Increases diversity of reported CAST systems
- Provides insights into CRISPR-associated transposons (CASTs) and their mechanisms
- Offers potential for novel gene-editing applications
Commercial applications
- Gene therapy
- Biotechnology research
- Genetic engineering
- Pharmaceutical development
About the inventor
Dr. Ilya J. Finkelstein and his team have evaluated the diversity of reported CRISPR-associated transposons (CASTs) systems identified in genomic databases and have discovered new CASTs architectures. Dr. Finkelstein is an Associate Professor of Molecular Biosciences at UT Austin and oversees a laboratory focused on the investigation of molecular mechanisms of genome maintenance, CRISPR biology, and epigenetic inheritance. Dr. Finkelstein has authored/co-authored numerous journal publications within his field and has been the recipient of a variety of prestigious awards. His research has broad applications in molecular biology/genetics, epigenetics, and synthetic biology, and since 2019 his lab has focused on pandemic countermeasures to better understand viral variants and the design of more effective vaccines.
