gRNA and Cas9 Coexpression
CRISPR-Based Gene Activation
In Vitro Transcription Vector (for Small RNA)
Our in vitro transcription vectors are simple and efficient systems for RNA synthesis, applicable for a variety of research purposes. The small RNA version of the in vitro transcription vector system is ideally suited for producing small RNAs for biochemical studies, injection into cells or embryos for shRNA knockdown or CRISPR/gRNA-mediated genome editing, and other applications requiring short RNAs of defined sequence.
This system utilizes a T7 promoter upstream of your sequence of interest, facilitating highly efficient production of small RNAs by T7 bacteriophage RNA polymerase (T7 RNAP) under appropriate reaction conditions and in the presence of nucleotide triphosphates. We recommend that you follow established in vitro transcription protocols available in published literature.
T7 RNAP has certain base requirements for efficient transcription initiation which have been already incorporated into the vector design. The first two nucleotides of the RNA transcript will be GG, corresponding to the 3’-end of the T7 promoter sequence, followed by your transcript sequence of interest.
Our in vitro transcription vector for small RNA synthesis is engineered for run-off transcription. This means that the T7 RNAP proceeds to the end of the DNA template during transcription and does not terminate at any specific site within the plasmid. For this reason, the circular plasmid template should be linearized by restriction digestion with SapI prior to in vitro transcription. SapI will cut the plasmid at a unique site immediately downstream of your sequence of interest. Care should be taken that the sequence to be transcribed does not contain any SapI restriction site, as this would result in truncated RNA transcripts. Contaminants from the digestion reaction may inhibit the subsequent T7 RNAP transcription reaction, so purification via column or phenol:chloroform extraction following digestion is recommended. However, it is generally not necessary to purify the promoter and insert fragment away from other fragments, because only the fragment containing the T7 promoter sequence will serve as a template for transcription.
In vivo, many types of RNAs undergo modification of their 5’ end, known as 5’ capping. For many experimental purposes, uncapped, in vitro transcribed small RNAs frequently yield good results. You should consider whether 5’ capping of the generated transcripts is a requirement for your experimental design. If desired, capping of your transcripts can be carried out either co-transcriptionally, using cap analogs, or post-transcriptionally, using capping enzymes.
For further information about this vector system, please refer to the papers below.
|Nucleic Acids Res. 7:1931 (1979)||Cloning and characterization of the T7 promoter|
|Methods Mol Biol. 703:29 (2011)||Protocols for the synthesis of RNA by in vitro transcription|
|Methods Mol Biol. 941:59 (2012)||Preparation of short RNA by in vitro transcription|
|Protein Expr Purif. 9:142 (1997)||Protocol for expression and purification of T7 RNAP for in vitro transcription|
Our in vitro transcription vectors for small RNA synthesis are designed to serve as highly effective templates for T7 RNAP-mediated in vitro transcription. This vector system is optimized for high copy number replication in E. coli, efficient restriction digestion, and abundant small RNA production.
High efficiency: T7 RNAP is a robust and highly efficient enzyme. T7 RNAP-dependent in vitro transcription reactions can produce large amounts of functional RNA.
Technical simplicity: In vitro transcription using a plasmid template and T7 RNAP is technically straightforward, and far easier than RNA expression in cells, which requires transfection and cell culture.
Specified orientation: This vector system contains a single promoter for in vitro transcription. The orientation of the sequence of interest relative to the T7 promoter will determine the sequence transcribed. If both sense and anti-sense transcripts are desired, two vectors will be needed.
Run-off transcription: Efficient in vitro transcription using this vector system requires linearization of the plasmid by restriction digestion prior to the transcription reaction.
T7 promoter: A promoter for the RNA polymerase from T7 bacteriophage. Drives high-level transcription of the downstream sequence of interest.
Transcribed sequence: Your chosen DNA sequence of interest to be transcribed into RNA is placed here.
SapI: Unique restriction endonuclease site that can be used to linearize the plasmid prior to in vitro transcription.
pUC ori: pUC origin of replication. Plasmids carrying this origin exist in high copy numbers in E. coli.
Ampicillin: Ampicillin resistance gene. It allows the plasmid to be maintained by ampicillin selection in E. coli.