Part:BBa_K1218011

Cas9

CRISPR-Cas is a bacterial immune system that remembers and targets foreign viral DNA by storing DNA sequences, or spacers, between clustered regularly interspaced short palindromic repeats (CRISPRs). RNA transcripts of the spacers are then used to sense homologous DNA, which is cleaved by CRISPR-associated (Cas) proteins.

This part codes for the tracrRNA, Cas9 protein, and minimal CRISPR array of a type II CRISPR-Cas system. The CRISPR array includes two CRISPR repeats separated by a spacer with two BsaI sites. Digestion with BsaI allows for insertion of a new spacer, thus changing the sequence targeted by Cas9.

Part improvement

Cambridge-JIC 2016 has submitted a new part (BBa_K2148013) consisting of cas9 codon-optimized for Chlamydomonas reinhardtii chloroplast chassis. This has been achieved through software developed at Saul Purton's lab at UCL. All illegal sites have been removed whilst maintaining the codon information and genetic A/T bias of the system.

The cas9 submitted additionally has a fusion tag to link reporter genes such as fluorescent markers.

For more information on the contruction of this part please refer to the design page.

The aim behind this part improvement is to use CRISPR/CAS9 technology to accelerate homoplasmy in chloroplast transformation and overcome this important bottleneck in plastid engineering.

Usage and Biology

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 1642
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 3921
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 4863
  Illegal BsaI.rc site found at 4840