SgRNA combination of targeted AHRR gene and application of sgRNA combination
A gene and targeting technology, applied in the field of gene editing, can solve problems such as difficulty in meeting the needs of obesity research, lack of unified modeling standards, and poor stability of obesity phenotypes, achieving stability and heritability, and high gene editing efficiency , the effect of extensive application value
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Embodiment 1
[0073] This embodiment provides a combination of sgRNAs targeting AHRR genes, the combination of sgRNAs targeting AHRR genes includes sgRNA1 and sgRNA2, the sgRNA1 includes the nucleic acid sequence shown in SEQ ID No.1, and the sgRNA2 includes SEQ ID No. The nucleic acid sequence shown in 2.
[0074] SEQ ID No. 1: GGAGATNTCGCCAAGTNCATGGG;
[0075] SEQ ID No. 2: GGCACATCTANCGTNATTATTGG;
[0076] Wherein, N represents any one of A, T, C or G.
[0077] The sgRNA1 specifically targets intron 5 of the AHRR gene, and the sgRNA2 specifically targets intron 8 of the AHRR gene, with good specificity, low off-target rate, and wide application value.
Embodiment 2
[0079] This embodiment provides an AHRR gene editing system, the AHRR gene editing system includes the sgRNA combination sgRNA1 and sgRNA2 targeting the AHRR gene and the mRNA of the Cas9 nuclease.
[0080] The AHRR gene editing system has the ability to knock out the AHRR gene in the genome. Through the cooperation of sgRNA combinations, it can reduce the off-target rate and the probability of non-specific editing, and has the ability to knock out large fragments, and the editing efficiency is higher; Cas9 is selected Nuclease mRNA is less toxic to cells, and the edited individuals are more likely to survive, reducing the difficulty of screening.
Embodiment 3
[0082] This embodiment provides a recombinant cell, which is a fertilized egg cell of a C57BL / 6 mouse whose genome has a mutation in the AHRR gene after being edited by the AHRR gene editing system in Embodiment 2.
[0083] The recombinant cells are constructed by the following method:
[0084] (1) Transcribe the mRNA of the Cas9 nuclease gene in vitro, mix it with the sgRNA targeting the AHRR gene, and obtain the AHRR gene editing system;
[0085] Ovulation induction was performed on C57BL / 6 mice, and fertilized eggs were cultured after in vitro fertilization;
[0086] (2) Microinjecting the AHRR gene editing system into the nucleus of a C57BL / 6 mouse fertilized egg cell to obtain a recombinant cell.
[0087] By directly injecting the mRNA of sgRNA1, sgRNA2 and Cas9 nuclease into the nucleus of the fertilized egg cell, the efficiency of gene editing is improved; the fertilized egg is selected to construct recombinant cells, and the sense mutation can be inherited through cel...
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