Part:BBa_K2330000

SaCas9 codon-optimised for E. coli

Usage and Biology

Staphylococcus aureus Cas9 is an endonuclease, which is programmable to cleave specific DNA sequence followed by the PAM (Protospacer Adjacent Motif) sequence 5’-NNGRRT-3’ [1]. One of the beneficial aspects of this protein is its small size compared to the SpCas9, the most widely used endonuclease. SaCas9 has only 1053 amino acids, against the 1368 of the SpCas9 [1]. Its small size is particularly advantageous when the SaCas9 is carried via adeno-associated virus (AAV) or bacteriophage, which have limitations of DNA packaging.

Does the part work?

Below is the description of the efficiency of our SaCas9 system, when it is delivered through P1 phage to E.coli. To check the efficiency of this SaCas9 system, we took advantage of different antibiotic resistant genes (Figure 1). Our E.coli testing platform (MoClo hereinafter) contains KanR plasmid with the regions targeted by the SaCas9. On the contrary, the SaCas9 is embedded in a ChlrR phagemid, which is delivered by the P1 phage (a plasmid contains phage replication machineries). Therefore, if the cleavage by SaCas9 occurs, it results in the loss of Kanamycin resistance in the MoClo E. coli, whilst in the acquisition of Chloramphenicol resistance. On the other hand, if the SaCas9 system doesn’t work, the host cells still displays Kanamycin resistance. (These cells should also have Chloramphenicol resistance, as the result of the phage infection.)

Figure 1: Strategy for checking the efficiency of SaCas9. After MoClo cells infected by phage with SaCas9 system are plated on Chloramphenicol plates (step1), several colonies were inoculated into Kanamycin plate (step2). Only ones which have successfully been cleaved their target sequence cannot grow on Kanamycin plates.

To test it, MoClo cells were mixed with phage and plated on LB plates (without any antibiotics/ with Kanamycin/ with Chloramphenicol) (step1). Subsequently, colonies from the Chloramphenicol plates (Those has been infected by phages.) were inoculated into the Kanamycin plate (step2) (Table 1, Figure 2).

The SaCas9 system targeting the KanR plasmid showed promising - albeit not consistent – results: only 1 out of 4 colonies for spacer Y (colony 6) did not grow on the Kanamycin plate, indicating that the SaCas9 might have cleaved the target KanR plasmid. However, the experiment was performed only once and further replicates are needed to confirm SaCas9 efficiency. In particular, one control colony (F, infected with control phage) failed to grow on Kanamycin plate, suggesting that extra care should be taken when performing control experiments.

Table 1: The efficiency of SaCas9 system (Step2). Colonies A-H and 1-8 are are the colonies of E.coli which has infected by negative control phages without spacers and with spacers, respectively.

Figure 2: Verification of efficiencies of the SaCas9 system. The result for Table 1 was obtained from these plates. X/Y MoClo cells refers to E.coli testing platform which contains sequence targeted by phage X/Y.

Reference

     [1] Ran, F. A., Cong, L., Yan, W. X., Scott, D. A., Gootenberg, J. S., Kriz, A. J., Zetsche, B.,  
     Shalem, O., Wu, X., Makarova, K. S., Koonin, E. V.  Sharp, P.A., Zhang, F. 2015. In vivo 
     genome editing using Staphylococcus aureus Cas9. Nature. 520 (7546). pp.186-191.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 627
  Illegal BglII site found at 780
  Illegal BglII site found at 926
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 200
  Illegal AgeI site found at 2756
  
• 1000
  COMPATIBLE WITH RFC[1000]