Is Cas9 an endonuclease

Cas9

Synonyms: CRISPR-associated protein 9, Cas5 (obsolete)
English: Cas9, CRISPR-associated protein 9

1 definition

Cas9 is a bacterial endonuclease that cleaves strands of DNA with a double-strand break. The protein is used by bacteria to defend themselves against bacteriophages.

2 structure

Cas9 is made up of two lobes between which the guide RNA (gRNA) is threaded:

  • REC lobe ("recognition lobe"): It forms the globular and larger part of the enzyme. It comprises a bridging helix and the two domains REC1 and REC2. Together they act as a DNA binding site that fixes the enzyme in the desired area.
  • NUC lobe ("nuclease lobe"): It mediates a double endonuclease function and is composed of 3 domains:
    • PAM-interacting domain: It recognizes the Protospacer Adjacent Motif (PAM triplet) of the foreign DNA and thereby initiates the enzyme action.
    • HNH domain. It cleaves the DNA target strand that is complementary to the gRNA.
    • RuvC domain: It cuts the opposite strand, i.e. strand complementary to the target strand. Both nuclease activities together introduce the "smooth" double-strand break.

The smallest Cas9 gene to date was isolated from the bacterium Staphylococcus aureus and comprises 3,159 bp.

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Cas9

3 classification

The Cas protein family can be divided into three types:

  • Type I, II and IIIa bind and cut double-stranded DNA
  • Type IIIb binds and cuts single-stranded RNA

Cas9 is the lead molecule of type II.

4 function

The Cas9 forms a natural defense system for bacteria, which is used, for example, in the event of an attack by a bacteriophage. The virus-like foreign DNA is destroyed and the phage can no longer multiply.

Since the DNA of the bacterium itself does not have any PAM triplets, it is protected from self-destruction. However, some bacteriophages have adapted to this defense mechanism and produce anti-CRISPR proteins such as AcrF1 or AcrF2 to circumvent the process.

5 biotechnology

In biotechnology, Cas9 forms the enzymatic part of the CRISPR / Cas system. In order for the Cas9 to be active, it needs

  • a PAM triplet
  • a specific gRNA, in the immediate vicinity of which the DNA sequence is cut.

The bacterial gRNA always consists of a dimeric RNA complex that is created by the aggregation of crRNA and tracrRNA.

In the context of genetic engineering, an artificial variation can also be produced, which is then referred to as sgRNA (single guided RNA). The sgRNA combines both functions of crRNA and tracrRNA in just one strand and thereby imitates the natural effect of the secondary structure Synthesis of the RNA complex is omitted, the sgRNA acts faster, but also represents a significantly more complex and above all more cost-intensive method of genome editing.

6 literature

  • Yadav SK, Kumar V, Singh SP. Recent Trends and Techniques in Plant Metabolic Engineering, Springer, 2018

7 Image source

  • saCas9 CC by Frankensteinper Model ID 3DPX-011815