Part:BBa_K2130013

SpCas9

This past is a human condon optimised SpCas9. SpCas9 recognises an 5'-NGG-3' PAM at the 3' end of the target sequence.

Example(PAM in brackets): 5'-NNNN...NNN(NGG)-3'

Evaluation studies from our team have compared the editing efficiency of this part compared to other Cas9/Cpf1.

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 341
  Illegal BglII site found at 1136
  Illegal BamHI site found at 1430
  Illegal XhoI site found at 1936
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 1168
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 259
  Illegal BsaI.rc site found at 1501
  Illegal BsaI.rc site found at 2857
  Illegal BsaI.rc site found at 3280
  Illegal BsaI.rc site found at 3292
  Illegal BsaI.rc site found at 4159
  Illegal SapI.rc site found at 2964
  Illegal SapI.rc site found at 3546
  Illegal SapI.rc site found at 3561
  

BNU-China 2020 - Contribution

We characterized this part by experiment, modeling and literature respectively, and the results are as follows:

Experiment: Characterization in Cryptococcus neoformans

We used this part and sgRNA(BBa_K3506050) in Cryptococcus neoformans. sgRNA was designed to target the ADE2 gene. A loss-of-function mutation in ADE2 results in an adenine auxotroph that forms pink colonies on culture plates that contain a low level of adenine, therefore enabling a visual evaluation of the action of CRISPR-Cas9. In our result, pink colonies grew on the YNBA plates, indicating that SpCas9(BBa_K2130013) successful targeted at the ADE2 locus in Cryptococcus neoformans.

Model：Cas9 expression time prediction

The DNA sequence of this part is 4227 bp. The rate of transcription in mammalian cells is nearly 1000 nucleotides per minute. It is estimated that this process can be done in about 4 minutes. The protein of Cas9 is 1450 amino acids. The rate of translation is nearly 140 amino acids per minute, so this process can be done in about 10 minutes. However, the folding time of Cas9 is unknown. We used deep neural networks (DNN) to predicted it and after repeated training, we estimated that Cas9 folding takes about 7.1~8.3ms.

Literature: The speed of Cas9 searching its target

Researchers from Uppsala University studied how fast Cas9 can find the target. They used dCas9 to find it out by using single molecule fluorescence microscopy and bulk restriction protection assays. They found that it takes six hours for a single fluorescently labeled dCas9 to find the correct target sequence, and determined the abundance of Cas9 in Streptococcus pyogenes by Western blotting to be almost twice that of the nonfused dCas9 strain where the time to bind a single target is 2 min, suggesting a search time of 1 min in Streptococcus pyogenes .Furthermore, the frequency of GG in the Streptococcus pyogenes genome is two-thirds that of E. coli, which can be expected to reduce the search time to 40 s.

Literature: A very fast Cas9 cutting method

In this article, investigators developed a caged RNA strategy cutting method which they called it very fast CRISPR (vfCRISPR). Cas9 could be expressed and bind on the DNA, but it could not cut until it was induced by light. Comparing with the former methods，their design overcame the shortages that the function of engineered proteins are compromised and cutting time is imprecise because Cas9 has to find the target after induction. 

The method of the design was based on the Streptococcus pyogenes Cas9 cleavage mechanism. Mismatches in the PAM-distal region prevent full unwinding of target DNA and conformational changes of the HNH domain required for cleavage. They use 6-nitropiperonyloxymethyl–modified deoxynucleotide thymine caged nucleotides which is light-sensitive to replace two or three uracils in PAM-distal to create a caged gRNA when hybridized to wild-type trans-activating CRISPR RNA (tracrRNA) to make Cas9 can bind but cannot cut until light stimulation at 365 or 405 nm is given.

In a word, vfCRISPR provides the highest spatial and temporal resolution to induce DSB at specific locations in living cells. The combination of cgRNA with other Cas9 based systems can promote the research of single strand breaks, basal excision or mismatch, and flap repair, respectively. Combining with vfCRISPR and subcellular light activation technology, it is possible to realize single allele specific precise genome editing and eliminate non targeted activity.

Reference

[1] Jones, D. L., Leroy, P., Unoson, C., Fange, D., Ćurić, V., Lawson, M. J., & Elf, J. (2017). Kinetics of dCas9 target search in Escherichia coli. Science (New York, N.Y.), 357(6358), 1420–1424. https://doi.org/10.1126/science.aah7084

[2] Liu Y, Zou RS, He S, Nihongaki Y, Li X, Razavi S, Wu B, Ha T. Very fast CRISPR on demand. Science. 2020 Jun 12;368(6496):1265-1269. doi: 10.1126/science.aay8204. PMID: 32527834.