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Functionalizing Cell-Free Systems with CRISPR-Associated Proteins: Application to RNA-Based Circuit Engineering

ACS Synth Biol. 2021-08; 
François-Xavier Lehr, Alina Kuzembayeva, Megan E Bailey, Werner Kleindienst, Johannes Kabisch, Heinz Koeppl
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DNA Sequencing … The sRNA operons and the tandem repeat of MGAs were ordered from Genscript. Sanger sequencing verified all constructed plasmids. Plasmids were prepared using a NucleoBond Extra Midi Plus (Macherey-Nagel) and followed by isopropanol precipitation and eluted with … Get A Quote

摘要

Cell-free systems have become a compelling choice for the prototyping of synthetic circuits. Many robust protocols for preparing cell-free systems are now available along with toolboxes designed for a variety of applications. Thus far, the production of cell-free extracts has often been decoupled from the production of functionalized proteins. Here, we leveraged a recent protocol for producing an -based cell-free expression system with two CRISPR-associated proteins, Csy4 and dCas9, expressed prior to harvest. We found that pre-expression did not affect the resulting extract performance, and the final concentrations of the endonucleases matched the level required for synthetic circuit prototyping. We demonstrat... More

关键词

CRISPR-associated systems, RNA-based circuits, cell-free extract, cell-free synthetic biology, malachite green aptamer, prototyping