FokI and dCpf1 fusion protein expression vector and fixed-point gene editing method mediated by fusion protein

[0058] The experimental methods mentioned in the following examples are conventional methods unless otherwise specified; the implementation of the present invention is not limited thereto.

[0059] Construction and application of embodiment 1, pFok1-dCpf1 fusion protein expression vector

[0060] 1. Construction of pLB-dCpf1 vector

[0061] dCpf1 was obtained by PCR amplification from the commercial vector WN10151 (Addgene plasmid #53369) (such as figure 1 ), cloned onto the LB vector, and constructed the pLB-dCpf1 vector, and the plasmid DNA was detected by 1% agarose gel electrophoresis (such as figure 2 ).

[0062] 2. Construction of pFokI-dCpf1 fusion protein expression vector

[0063] The pLB-dCpf1 was obtained by cutting pLB-dCpf1 with BsmBI, and the fragment dCpf1 was cut back; the commercial vector pSQT1601 (4849bp, 5474bp) was double cut with Acc65I and Not1 enzymes, and the CAG framework 4849bp was recovered. T2A-NLS and FokI fragments amplified by PCR from laboratory-preserved vectors (primer sequences are shown in Table 1); T2A-NLS, FokI, dCpf1 fragments and CAG frameworks were ligated together using the Golden Gate cloning method, T7 ligase 25 Re...