RNA plays an important role in gene regulation. Although RNA overexpression in cells has made some achievements, in vitro transcription is still a common research method, which is popular due to convenient operations and wide uses. In vitro transcription is to use DNA as a template to simulate the intracellular transcription process to generate RNA in vitro cell-free system. That can control the genes of transcription, the process of transcription and the use of post transcriptional RNA.
In vitro transcription of RNA is mainly applied to labeled probes synthesis and lots of unlabeled RNA generation. In addition, the capped RNA synthesized in the transcription reaction can be used for hybridization analysis, microinjection, biochemical and genetic researches, etc. Synbio Technologies has developed nucleotides, polymerases, modified enzymes and kits for highly-efficient transcription of capped RNA, large-scale transcription and transcription reaction based on short DNA template.
CRISPR-Cas system offers versatile genome engineering technologies. While the ongoing discoveries of new Cas nucleases and anti-CRISPR proteins far exceeds the rate of proficient implementation. Here, researchers used E. coli cell-free transcription-translation (TXTL) systems to significantly improve the speed and scalability of CRISPR characterization and validation.
TXTL can express active CRISPR mechanism from added plasmids and linear DNA. Researchers used TXTL to measure the quantitative dynamics of DNA cleavage and gene repression for single- and multi-effector CRISPR nucleases, predict gene repression strength, determine specificities of 24 anti-CRISPR proteins, and develop a fast and scalable screen method for protospacer-adjacent motifs that was successfully applied to five uncharacterized Cpf1 nucleases. This example illustrated how TXTL can facilitate the characterization and application of CRISPR technologies in different uses.
Applications of in vitro transcription
- sgRNA synthesis for gene editing
- RNA probe synthesis for hybridization
- dsRNA / siRNA synthesis for RNA interference
- RNA virus genome synthesis for virus research
- mRNA synthesis for in vitro transcription
- Synthesis of mRNA / cRNA for electrophysiological research
- Synthesis of various types of RNA for noncoding RNA research
CRISPR sgRNA Design and Construction
DNA Plasmid Preparation
 RyanMarshall, Colin S.Maxwell, Scott P.Collins, etc. Rapid and Scalable Characterization of CRISPR Technologies Using an E. coli Cell-Free Transcription-Translation System. Molecular Cell, 2018. 69: 146-157. https://doi.org/10.1016/j.molcel.2017.12.007