Home › Microbiology › Substrate Generation for Endonucleases of CRISPR/Cas Systems
Microbiology JoVE (Open Access) Citable · DOI

Substrate Generation for Endonucleases of CRISPR/Cas Systems

DOI: 10.3791/4277-v
What you'll learn
  • ✓ Generate long pre-crRNA substrates using PCR amplification
  • ✓ Prepare intermediate pre-crRNA via DNA oligonucleotide annealing
  • ✓ Synthesize short Cas RNA substrates by custom oligo synthesis
  • ✓ Perform in vitro T7 transcription and Cas6 endonuclease assay
Protocol

Biopharma Insights CRISPR/Cas systems mediate adaptive immunity in Bacteria and Archaea. Many Cas proteins are proposed to act as endoribonucleases acting on crRNA precursors of varying length. Here we illustrate three different approaches to generate pre-crRNA substrates for the biochemical analysis of Cas endonuclease activity.

Difficulty
advanced
Total time
~2 days
Biosafety
BSL-1

Steps

1
Review CRISPR/Cas adaptive immunity mechanisms

Overview of CRISPR/Cas systems in Bacteria and Archaea and proposed endoribonuclease activity of Cas proteins on crRNA precursors.

▶ 01:37
2
Amplify long pre-crRNA by PCR

Generate long pre-crRNA substrates through PCR amplification for biochemical analysis.

▶ 02:29
3
Anneal DNA oligos for intermediate pre-crRNA

Generate intermediate-length pre-crRNA substrates by annealing complementary DNA oligonucleotides.

▶ 04:14
4
Synthesize short Cas RNA substrates

Obtain short Cas RNA substrates via custom RNA oligonucleotide synthesis.

▶ 06:10
5
Transcribe RNA with T7 polymerase

Perform in vitro T7 RNA polymerase transcription to produce RNA substrates.

▶ 06:41
6
Run Cas6 endonuclease assay

Set up and execute the Cas6 endonuclease cleavage assay on generated substrates.

▶ 08:04
7
Analyze representative Cas6 assay results

Interpret representative results from the Cas6 endonuclease assay.

▶ 09:08
8
Summarize substrate generation approaches

Conclusion on the three approaches for generating pre-crRNA substrates for Cas endonuclease analysis.

▶ 11:10
💬 Comments coming soon