DNA transposons have emerged as promising tools in both gene therapy and functional genomics. In particular, the Sleeping Beauty (SB) DNA transposon has advanced into clinical trials due to its ability to stably integrate DNA sequences of choice into eukaryotic genomes. The efficiency of the DNA transposon system depends on the interaction between the transposon DNA and the transposase enzyme that facilitates gene transfer. In this study, we assess the DNA-binding capabilities of variants of the SB transposase and demonstrate that the structural stability of the primary DNA-recognition subdomain, PAI, affects SB DNA-binding affinity and transposition activity. This fundamental understanding of the structure–function relationship of the SB transposase will assist the design of improved transposases for gene therapy applications.
Members of the
- PAR ID:
- 10565358
- Publisher / Repository:
- Nature Publishing Group
- Date Published:
- Journal Name:
- Nature Communications
- Volume:
- 16
- Issue:
- 1
- ISSN:
- 2041-1723
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Abstract CRISPR-associated transposases (CASTs) direct DNA integration downstream of target sites using the RNA-guided DNA binding activity of nuclease-deficient CRISPR-Cas systems. Transposition relies on several key protein-protein and protein-DNA interactions, but little is known about the explicit sequence requirements governing efficient transposon DNA integration activity. Here, we exploit pooled library screening and high-throughput sequencing to reveal novel sequence determinants during transposition by the Type I-F Vibrio cholerae CAST system (VchCAST). On the donor DNA, large transposon end libraries revealed binding site nucleotide preferences for the TnsB transposase, as well as an additional conserved region that encoded a consensus binding site for integration host factor (IHF). Remarkably, we found that VchCAST requires IHF for efficient transposition, thus revealing a novel cellular factor involved in CRISPR-associated transpososome assembly. On the target DNA, we uncovered preferred sequence motifs at the integration site that explained previously observed heterogeneity with single-base pair resolution. Finally, we exploited our library data to design modified transposon variants that enable in-frame protein tagging. Collectively, our results provide new clues about the assembly and architecture of the paired-end complex formed between TnsB and the transposon DNA, and inform the design of custom payload sequences for genome engineering applications with CAST systems.
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