Part:BBa_K5490032

Responsive to synthetic NFAT CasRx

There is 1) a type of promoter with high specificity to a particular type of transcription factor, in this case, the synthetic NFAT, it achieves this by having various upstream elements of a small DNA sequence that contains the TATA box where polymerase II will bind 2) downstream, it contains a CasRx gene , a HA tag, an IRES2 RBS and a mCherry fused with NLS .

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 1335
  Illegal EcoRI site found at 1755
  Illegal PstI site found at 4754
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 1335
  Illegal EcoRI site found at 1755
  Illegal PstI site found at 4754
  Illegal NotI site found at 141
  Illegal NotI site found at 211
  Illegal NotI site found at 506
  Illegal NotI site found at 576
  Illegal NotI site found at 3744
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 1335
  Illegal EcoRI site found at 1755
  Illegal BamHI site found at 3735
  Illegal BamHI site found at 3809
  Illegal XhoI site found at 806
  Illegal XhoI site found at 3210
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 1335
  Illegal EcoRI site found at 1755
  Illegal PstI site found at 4754
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 1335
  Illegal EcoRI site found at 1755
  Illegal PstI site found at 4754
  Illegal NgoMIV site found at 1023
  Illegal AgeI site found at 7
  Illegal AgeI site found at 347
  Illegal AgeI site found at 372
  Illegal AgeI site found at 712
  
• 1000
  COMPATIBLE WITH RFC[1000]

Usage and Biology

CasRx is a highly effective RNA-targeting molecule with nuclease activity specifically against single-stranded RNA, making it a versatile tool for gene silencing and antiviral therapies. One of its major advantages over RNA interference (RNAi) is its lower off-target activity, providing more precision in targeting specific RNA sequences. As a viral protein, CasRx operates independently of the host's cellular machinery, which reduces variability and enhances efficiency.

CasRx is the smallest member of the RNA-targeting CRISPR family, allowing for easier delivery into cells while maintaining high specificity and efficiency in cleaving target RNA. Its minimal off-target effects and high efficiency make it ideal for large-scale transcriptome screening, precise gene silencing, and targeting viral RNA in therapeutic applications.

A key advantage of CasRx over other CRISPR systems is its simplicity--it does not require a PAM sequence or a tracrRNA, making it one of the most straightforward CRISPR systems to implement. CasRx can target virtually any RNA sequence when paired with a guide RNA (gRNA) that is complementary to the target.

In the described system, an mCherry fluorescent protein is separately expressed and directed to the nucleus via a nuclear localization signal (NLS). The purpose of mCherry is to act as a reporter, enabling independent monitoring of the CasRx activity and target RNA levels. This separation of signals allows for precise tracking of the effector molecule (CasRx) and the concentration of the target RNA, enhancing the accuracy of the experiment.

By monitoring mCherry fluorescence in the nucleus, researchers can confirm the presence of CasRx without interference from the target RNA cleavage activity, ensuring clear and accurate results in gene silencing studies or antiviral applications.

Konermann S, Lotfy P, Brideau NJ, Oki J, Shokhirev MN, Hsu PD. Transcriptome Engineering with RNA-Targeting Type VI-D CRISPR Effectors. Cell. 2018 Apr 19;173(3):665-676.e14. doi: 10.1016/j.cell.2018.02.033. Epub 2018 Mar 15. PMID: 29551272; PMCID: PMC5910255.

Chuang YF, Wang PY, Kumar S, Lama S, Lin FL, Liu GS. Methods for in vitro CRISPR/CasRx-Mediated RNA Editing. Front Cell Dev Biol. 2021 Jun 11;9:667879. doi: 10.3389/fcell.2021.667879. PMID: 34178991; PMCID: PMC8226256.