Introduction

This DNA template consists of a Direct Repeat (DR) loop, which is a crucial component of crRNA molecules that guide LwaCas13a proteins to their targets. The DR loop is essential for anchoring the Cas13a protein to the crRNA molecule.

This part can be directly used to design crRNA templates suitable for in vitro transcription to produce functional crRNA molecules.

Biology & Usage [1]

The Direct Repeat (DR) loop is a crucial part of the CRISPR RNA (crRNA) that Cas13 proteins use to target and cleave RNA. This loop forms a conserved stem-loop structure within the crRNA, typically about 36 nucleotides long with an 8-10 nucleotide stem. The sequence of the DR loop is specific to each Cas13 ortholog and is essential for the protein to bind to the crRNA. Notably, the DR loop is typically positioned at the 5’ end of the crRNA for Cas13a proteins, while for Cas13b, it is found at the 3’ end of the molecule. If the DR loop is deleted or its structure is disrupted, Cas13's ability to bind and target RNA is significantly impaired.

Design

To generate an effective crRNA for use with the LwaCas13a protein through in vitro transcription, position the DR loop at the 3' end of the 28-bp spacer template, ensuring that the spacer sequence is complementary to the target you intend to detect.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

Biosafety

We used the Asimov's tool — Kernel — to check the sequence's safety with the Biosecurity Sequence Scanner. The results showed no flagged sequences, confirming that this part is safe to use.

Sequence Source