Part:BBa_K1773005

I-F type CRISPR-Cas Cascade complex

I-F type CRISPR-Cas Cascade complex, which has a mutated EcoRI restriction site to make it biobrick compatible. This protein complex needs to be coexpressed with a crRNA region, to make a functional Cascade ribonucleoprotein complex. Together With Cas3 (BBa_K1773003) degrades DNA.

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
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 1790
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 2307
  

Usage and Biology

Cascade (CRISPR Associated Complex for Antiviral Defence) complex is an essential component of I-type CRISPR-Cas systems. This ribonucleoprotein complex is coded by operon of 4 genes  : Csy1, Csy2, Csy3 and Cas6f, with stoichiometry of 1:1:6:1 respectively. Also it has a crRNA molecule, witch acts like a guide, that brings the whole complex to the target dsDNA sequence. Each Cascade complex has a unique Protospacer adjacent motive (PAM) recognition site. The recognisable PAM sequence of this specific I-F Cascade complex from Aggregatibacter actinomycetemcomitans recognises CC nucleotides directly upstream of the protospacer (the target sequence). In other words, a target sequence needs to have a CC pair on the non-complementary strand of DNA -2 -1 positions of the protospacer. When Cascade complex finds and recognises the DNA target, then Cas3 (BBa_K1773003) is recruited for DNA degradation.

Quick change mutagenesis

The wild type Cascade complex had EcoRI restriction site inside the coding region of Csy1 gene (Figure1).

(Figure1. Cascade plasmid map. There is only one EcoRI restriction site present in the CsyI gene. )

To make this construct biobrick compatible we conducted quick change mutagenesis and mutated the EcoRI site. We used Invitrogen GeneArt® Site-Directed Mutagenesis PLUS System kit. The coding amino acid was not changed, but EcoRI could not digest the mutated plasmid. After mutagenesis, 6 colonies were inoculated and plasmids were extracted. Next we conducted a restriction analysis with EcoRI to determine, which mutants were successful (Figure2). All mutant plasmids were mutated successfully, and a quicker moving band appeared, compared to the digested non-mutant Cascade complex (K). Three of these plasmids were sequenced, and all of them were confirmed.

After confirmation from sequencing, we performed a PCR with primers flanking the Cas3 gene, which also contained the Prefix and suffix sequences. Later the PCR product was cloned to ta pSB1C3 linearised vector.

(Figure2. Cascade restriction analysis After mutant digestion with EcoRI no DNA linearisation was present, compared to the non-mutated Cascade control (K). )

Sources

1. Gasiunas G, Sinkunas T, Siksnys V (2014) Molecular mechanisms of CRISPR-mediated microbial immunity. Cellular and molecular life sciences : CMLS 71: 449-465

2.1.Sorek R, Lawrence CM, Wiedenheft B (2013) CRISPR-mediated adaptive immune systems in bacteria and archaea. Annual review of biochemistry 82: 237-266