Part:BBa_K4335012

pCrU6.3

pCrU6.3 is a previously characterized RNA polymerase III promoter from the chromosome 8 of Chlamydomonas reinhardtii.

The first prerequisite for CRISPR/Cas9 experiments is sgRNA transcription under the control of the RNA polymerase III promoter (RNAPII) . In general, the RNAPII transcription factor binding site is intrinsic to the transcriptional sequence and therefore not amenable to sgRNA transcription. U6snRNA is one of the few examples of sgRNAs with proximal RNAPII promoter elements. Therefore, we used previously characterized U6snRNA sequences from chromosome 8.

Usage

pCrU6.3 was constructed into plasmidand

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.

In addition, we found that the promoter had never appeared in the iGEM database before, so we will use the results in the literature to append to it for future reference. (the following results are from literature [2])

A) Schematic of DNA constructs. The promoter of heat shock protein HSP70A promotes the transcription of Chlamydomonas codon-adapted S. aureus (Sa Cas9) or S. pyogenes Cas9(Sp Cas9). Guide RNA transcription under the control of different CrU6-promoters (#1-4). Guide sequences were inserted into vectors containing the appropriate SaCas9 or SpCas9 scaffold. The coding sequence of aphVIII (PmR) is also located on the pCrU6-plasmids to enable antibiotic selection using paromomycin. NLS, nuclear localization signal; T, poly-thymine terminator.

(B) The phytoene synthase gene, PSY1, was chosen as a target gene. sgRNA transcription driven by Chlamydomonas U6 promoters (CrU6 #1-4) was assayed. Photographs of a selective agar plates from PSY1 inactivation experiments. If the cells were allowed to recover for 1 d before antibiotic selection on paromomycin-containing agar plates, mixed colonies of wild-type and PSY1-inactivated mutants were obtained. If the cells were allowed to recover for 2 d, pure white colonies with inactivated PSY1 were found. Recovery at 33°C for the first 24 h increased targeting frequencies for HS by approximately one-third. Targeting frequencies (targeted colonies/selected colonies) are the mean of three independent experiments. HS, HSP70A promoter; HR, HSP70A/RBCS2 promoter.

(C) Sequence alignment of amplified psy1 loci from white colonies. Dots indicate spacers, hyphens indicate deleted nucleotides. “PAM” (gray) and “Protospacer” sequences are indicated. PSY1, wild-type gene sequence.

Reference

[1Jakab, G., Mougin, A., Kis, M., Pollák, T., Antal, M., Branlant, C., and Solymosy, F. (1997). Chlamydomonas U2, U4 and U6 snRNAs. An evolutionary conserved putative third interaction between U4 and U6 snRNAs which has a counterpart in the U4atac-U6atac snRNA duplex. Biochimie 79: 387–395]

[2] 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:

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  Illegal PstI site found at 48
  Illegal PstI site found at 121
  
• 12
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  Illegal NotI site found at 453
  
• 21
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  Illegal PstI site found at 48
  Illegal PstI site found at 121
  
• 1000
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