Part:BBa_K5382150

Cas9 ribonucleoproteins(gRNA PRDX4)-Co-expression and self-assembly of RNPs

The CRISPR/Cas system comprises two integral components: the CRISPR array, which consists of clustered regularly interspaced short palindromic repeats, and the CRISPR-associated protein (Cas). Together, these elements form the ribonucleoprotein (RNP) complex, which possesses gene-editing capabilities and has been extensively utilized in biotherapeutic applications. The RNP complex is composed of two distinct segments: the Cas protein responsible for nucleic acid cleavage and the guide RNA (sgRNA) that directs the targeting process. Traditionally, the Cas9 RNP complex is assembled and prepared in vitro by combining purified Cas9 protein with either transcribed or chemically synthesized sgRNA. However, the production of sgRNA, whether through transcription or chemical synthesis, is costly and time-consuming, limiting its clinical application.
In this study, we employed a novel one-step method to prepare the RNP complex (refer to Figure 1). Probiotics Escherichia coli Nissle1917 was utilized as a host organism for the expression of Cas9 protein and the transcription of sgRNA, thereby enabling the intracellular "biological self-assembly" of the complex. This approach eliminates the need for separate crRNA preparation and allows for the rapid and cost-effective production of Cas9 RNP complexes through a single purification step. The resulting RNP complex exhibits exceptional stability, retaining its activity in the absence of RNase for over a year. The Cas9 RNP was introduced into human cells via liposomal transfection or nanoparticle encapsulation by outer membrane vesicles (OMVs) in this study, demonstrating remarkable intracellular gene-editing activity.

Figure 1. one-step method used to prepare the RNP

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 4514
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]