Method for targeted edition of ISS sequence with CRISPR (clustered regularly interspaced short palindromic repeat) recombinant plasmid constructed by nucleotide sequence

Abstract

The invention provides a method for targeted edition of an ISS sequence with a CRISPR (clustered regularly interspaced short palindromic repeat) recombinant plasmid constructed by a nucleotide sequence. The method comprises the following steps: performing targeted designing of an sgRNAqingqin oligo sequences, adding BsmBI plasmid digestion sites at both ends, constructing a recombinant plasmid, performing transformation, paving, monoclonal selection, plasmid extraction, sequencing and sequence comparison, and performing identification so as to achieve successful construction. By adopting the method, a CRISPR case 9 sgRNAqingqin recombinant plasmid is used, and the ISS sequence can be precisely edited (including deletion, insertion and mutation), an effect that a spliced silencer ISS sequence is ineffective or the effect is weakened can be also achieved, so that exon 7 inclusion of an SMN2 (survival of motor neuron 2) can be remarkably promoted; and in addition, the recombinant plasmidcan be expressed sustainably in cells to take effects into play, only one-time transfection is needed, and the method has the best potential in treating related genetic diseases.

Description

technical field [0001] The invention belongs to the field of biotechnology, and specifically designs a method for targeted editing of an ISS sequence by a CRISPR recombinant plasmid constructed from a sgRNAqingqin oligo nucleotide sequence. Background technique [0002] Importance of SMN2 gene exon 7 inclusion in spinal muscular atrophy (SMA) patients: studies suggest that SMA is due to a point mutation in the survival of motor neuron 1 (SMN1) gene on chromosome 5 Or deletion results in the inability of the gene to express a functional full-length SMN protein. Different from animals, humans have produced a parallel homologous gene of SMN1 called SMN2 due to the inverted duplication of chromosome 5q13 region; and the two genes contain 9 exons (1, 2a, 2b, 3, 4, 5, 6, 7, 8), encoding the exact same SMN protein of 294 amino acids. The key difference between the two is that the nucleotide at position 6 of exon 7 is changed from C in SMN1 to T in SMN2 (C6T), although it does not...

AI Technical Summary

Problems solved by technology

However, there is no relevant research report on this technology in the treatment of SMA mice, and there is an urgent need to explore new methods

[0004] There are two genes expressing SMN protein in the human body, SMN1 and SMN2, but after the SMN1 gene mutation that plays the main role loses its function, there are only a small amount of full-length SMN gene and a small amount of SMN due to most of exon 7 of the SMN2 gene being cleaved. For functional proteins, the most effective method is ASO antisense oligonucleotides, which precisely target the 10th-27th sequence of complementary intron 7. For example, the oligonucleotide drug Spinraza, which was launched in the United States in 2016, targets the closed inner Intron 7 splices the silencer ISS sequence, thereby significantly promoting the ratio of exon 7 inclusion, but ASO has a short half-life and must be administered continuously

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