Difference between revisions of "Part:BBa K5382150"
 
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CRISPR/Cas system, Including CRISPR (Clustered regularly interspaced short palindromic repeats) array and CRISPR-associated protein (Cas) two parts. These two parts assemble to form the RNP complex with gene editing activity, which has a wide range of applications in the field of biotherapy. RNP complex consists of two parts, namely Cas protein nucleic acid cutting part and sgRNA guided targeting part. At present, traditional Cas9 RNP is mainly assembled and prepared in vitro by incubating the purified Cas9 protein and the transcribed or chemically synthesized sgRNA in vitro. However, whether it is transcribed in vitro or chemically modified to synthesize sgRNA, its acquisition cost is too high and requires a lot of time, which is not conducive to its application in clinical therapy. <br>
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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.<br>
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In this project, a one-step method was used to prepare the RNP complex (figure 1.), and Escherichia coli was used as a microbial factory for the expression of Cas9 protein and the transcription of sgRNA, and the "biological self-assembly" of the complex was completed in the cell. Compared with the traditional method, this method does not require additional preparation of crRNA, and can quickly and substantially prepare inexpensive Cas9 RNP complexes by one-step purification. The RNP prepared by this method has extremely high stability and can remain active in the absence of RNase for more than one year. The Cas9 RNP was delivered to human cells by liposomal transfection or nanoparticle packaging, and the results showed that it had excellent intracellular gene editing activity.<br>
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In this study, we employed a novel one-step method to prepare the Cas9 RNP (refer to Figure 1). Probiotics <i>Escherichia coli</i> 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.<br>
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https://static.igem.wiki/teams/5382/part-pictures/big-sp.png<br>'''Figure 1.'''  one-step method used to prepare the RNP  
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<center>https://static.igem.wiki/teams/5382/part-pictures/40.png</center><br><center>'''Figure 1.'''  One-step method for the high-yield production of Cas9 RNP.</center>
  
 
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Latest revision as of 13:37, 2 October 2024


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 Cas9 RNP (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.

40.png

Figure 1. One-step method for the high-yield production of Cas9 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]