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.

Amazonas_Brazil team improvement

Amazonas_Brazil team has designed news BioBricks parts (BBa_K2457001 AND BBa_K2457002) aiming to implement bioengineering principles into CRISPR locus. Our proposal was to rationally design parts to be interchangeable and standardized, providing a BioBrick toolbox to attend multiple SynBio purposes. For that matter, Amazonas_Brazil team has:

• Engineered abstracted CRISPR locus, building two standard BioBricks parts: the BBa_K2457001 - composed of Cas9 coding sequence + BBa_B0015 - and BBa_K2457002 - composed of an optimized sgRNA device.

• Built BBa_K2457001 without regulatory modules to provide an approach to easily switch the promoter and RBS from Cas9 coding sequence through 3A Assembly.

• Designed abstracted parts with ALL RFCs assemblies compatibility, paving the way to many engineering possibilities.

• Constructed BBa_K2457002 composed of both crRNA protospacer sequence and tracrRNA structure with an optimized design.

• Modified the Shine-Dalgarno (AGGAGG) from the RBS at the end of Cas9 coding sequence to avoid ribosome recruitment on the wrong place of our synthetic BioBrick. It was necessary as the Cas9 is located in an operon complex in native CRISPR locus.

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.

Egypt-AFCM Team Improvement

[http://2017.igem.org/Team:AFCM-Egypt# Egypt-AFCM Team] aimed to use cas9 for non-coding RNAs editing represented in regulation of circular RNA based circuit to knock-in hsa_circ_0000064 at BBa_K2217001 into Hepatocellular carcinoma cells. To improve characterization of BBa_K1218011, We designed a CRISPR-based circuit with HDR (homology-directed repair) template for knock-in, to improve characterization CMV enhancer, CMV promoter and T7 promoter at [BBa_K2217000 BBa_K2217006 BBa_K2217007] were ligated to the same circuit with cas9, while HDR was ligated on a separate plasmid to be transfected to HepG2 cells for circuit evaluation. herein, Gel electrophoresis bands were described to document Cas9 characterization, while culture plates were also documented to compare the activity of both non-coding RNA circuit and CRISPR circuit, characterization data could be found at BBa_K1218011:Experience. Information about Our Team results can be found at [http://2017.igem.org/Team:AFCM-Egypt/Results# Egypt-AFCM Team Results]

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