Part:BBa_K5382140
Cas9 ribonucleoproteins(gRNA HSP70)-Co-expression and self-assembly of RNPs
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.
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.
Figure 1. one-step method used to prepare the RNP complex.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 1167
Illegal NheI site found at 4506 - 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 3446
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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