Part:BBa_K3096001

The Cas3 Nuclease is part of the Type I CRISPR/Cas system in E.coli

This is the coding region of the Cas3 Nuclease is part of the Type I CRISPR/Cas system in E.coli.

Background

The CRISPR/Cas3 system can be used to not only cut bud degrade the DNA targeted by the CRISPR Arrays.

Usage & Biology

Similar to the type II CRISPR/Cas9 system, the type I CRISPR/Cas3 system is part of the microbial adaptive immune system, however its function and mechanism of action differs from the CRISPR/Cas9 system as it is able to cut double stranded DNA and degrade longer strands of DNA by cutting several times. The CRISPR/Cas-based defence relies on the recognition of known e.g. viral DNA sequences via the crRNA. After the primary invasion of a virus, parts of its DNA are cut and integrated as short spacers between repeating palindromic sequences into the bacterial genome at the CRISPR locus. The reinfection with the same virus leads to the whole CRISPR array’s transcription and the pre crRNA is cut into small sequences containing only one spacer and one palindrome motif. In case of an type I system this crRNA then guides the surveillance complex Cascade ABCDE to complementary sequences in the viral DNA which is unwound, generating the so-called R-loop. The Cas3 helicase-nuclease protein attaches to this R-loop and unidirectionally degrades the invader DNA in a 3′–5′ direction at the site of a protospacer adjacent motif (PAM) by inducing a sequence of cuts by a reeling motion. Hereby, the large Cas3/Cascade complex pulls the substrate DNA towards itself, introducing single strand breaks. The degradation products are approximately the length of a spacer sequence and the cuts are enriched in PAM-like NTT motives at the 3´-end, making many of these degradation products suitable for the integration into the CRISPR locus.

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]

References

1. Xia, Peng-Fei, et al. "Synthetic genetic circuits for programmable biological functionalities." Biotechnology advances (2019).
2. Terns, Michael P., and Rebecca M. Terns. "CRISPR-based adaptive immune systems." Current opinion in microbiology 14.3 (2011): 321-327.