| Products/Services Used | Details | Operation |
|---|---|---|
| Codon Optimization | The TranC protein sequences, which were codon-optimized for expression in E. coli, as well as their corresponding guide RNA sequences, were commercially synthesized by Nanjing GenScript Biotech Co., Ltd. A codon-optimized LaTranC variant, featuring N-terminal and C-terminal nuclear localization signal (NLS) sequences tailored for human, was commercially synthesized by Nanjing GenScript. The sgRNA scaffold sequence was commercially synthesized by Nanjing GenScript and subsequently cloned into the human U6-driven sgRNA expression vector142 via Gibson assembly, yielding phU6-LaTranC-sgRNA. A total of 353 single-residue sites meeting these criteria were selected for mutagenesis (Table S2), and a pooled plasmid library of LaTranC singlearginine mutants at these positions was synthesized commercially (Nanjing GenScript). Guide RNAs—including TranC CRISPR RNAs and TnpB-derived reRNAs—were synthesized by Nanjing GenScript. | Get A Quote |
| PCR Cloning and Subcloning | Get A Quote |
Transposon-encoded TnpB nucleases gave rise to type V CRISPR-Cas12 effectors through multiple independent domestication events. These systems use different RNA molecules as guides for DNA targeting: transposon-derived right-end RNAs (reRNAs or omega RNAs) for TnpB and CRISPR RNAs for type V CRISPR-Cas systems. However, the molecular mechanisms bridging transposon activity and CRISPR immunity remain unclear. We identify TranCs (transposon-CRISPR intermediates) derived from distinct IS605- or IS607-TnpB lineages. TranCs utilize both CRISPR RNAs and reRNAs to direct DNA cleavage. The cryoelectron microscopy (cryo-EM) structure of LaTranC from Lawsonibacter sp. closely resembles that of the ISDra2 TnpB complex; how... More
