Part:BBa_K4335021

pCrU6.3+insert site+SpScaffold+polyT Term

BBa_ K4335021 consists of a terminator, a cloning site, SpScaffold and a terminator PCrU6.3 is a previously characterized RNA from chromosome 8 of Chlamydomonas reinhardtii [1]

Template of sgRNA. Use Golden Gate (BsaⅠ) to replace the template sequence with a ~20bp guide sequence. Add 'ACTT' on the 5'-terminal of your guide sequence and 'AAAC' on the reverse 5'-terminal.

sgRNA

Usage

pCrU6.3+insert site+SpScaffold+polyT Term is one of the core functional components of plasmid pTX2038 and pTX2040 Its main function is to express specific sgRNA, and combine with HSP70A Promoter+3×Flag+NLS+SpCas9+NLS+Rbcs2 Term [BBa_K4335020]works together to cut specific genes in the nuclear genome of Chlamydomonas reinhardtii, so as to knock out negative lipid related regulatory genes.

Result

Verification of the effect of Golden Gate inserting specific sgRNA

The following is the PCR verification electrophoresis diagram after we assembled various sgRNAs in Golden Gate Assembly.

Gel run of samples from colony PCR.Primer length ~660 bp. M: 2000 bp DNA Marker; 38: pTX2038 vector; 40: pTX2040 vector; sgRNA1-3: different sgRNAs is designed.

Figure 1 shows that the colony PCR has successfully amplified about 660 bp bands, which correspond to the vector corresponding to the sgRNA of each target gene (pTX2038/pTX2040). It preliminarily indicates that we have successfully inserted the sgRNA of the target gene into the vector (pTX2038/pTX2040), and then Sanger sequencing of each insert site has been carried out, which proves that we have successfully constructed.

Verification of Cas9 system functions

We selected PSY1 gene as the target gene to detect the sgRNA transcription driven by U6.3 promoter according to the method of [1] . The plant endo synthase gene (PSY1) is involved in chlorophyll synthesis, and disruption of PSY1 produces white colonies that are easy to detect and count.

PSY1 was selected as the target gene.Detection of chlamydia U6 promoter (CrU6.3) drive sgRNA transcription.Rhine chlamydomonas wild type and guide

The color of the algae transferred into PSY1 was significantly lighter than that of the wild type, and the individuals of white algae also appeared. We can confirm that Cas9 plays a role in Chlamydomonas Rhein and that the Cru6.3 promoter plays a normal role.

Due to the time factor, we can not further analyze the function of Cas9 system. We will prove that Cas9 protein has successfully synthesized and cleaved the specific gene sequence by protein purification, DNA extraction and sequencing in Chlamydomonas reinhardtii.

Reference

[1] Greiner, A. et al. Targeting of Photoreceptor Genes in Chlamydomonas reinhardtii via Zinc-Finger Nucleases and CRISPR/Cas9. Plant Cell 29, 2498–2518 (2017).

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 48
  Illegal PstI site found at 121
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal PstI site found at 48
  Illegal PstI site found at 121
  Illegal NotI site found at 453
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 48
  Illegal PstI site found at 121
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 48
  Illegal PstI site found at 121
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 551
  Illegal BsaI.rc site found at 533