Design method of sgRNA and lentivirus carrier formed by sgRNA and plasmids

A technology of lentiviral vector and design method, which is applied in the optimized design and application field of targeted knockout plasmid and lentiviral vector, and can solve problems such as inconvenient operation and increased system complexity

Active Publication Date: 2015-09-23
INST OF HEMATOLOGY & BLOOD DISEASES HOSPITAL CHINESE ACADEMY OF MEDICAL SCI & PEKING UNION MEDICAL COLLEGE
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Problems solved by technology

However, this method requires two sgRNAs to function, which increases the complexity of the system and makes it inconvenient to operate

Method used

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  • Design method of sgRNA and lentivirus carrier formed by sgRNA and plasmids
  • Design method of sgRNA and lentivirus carrier formed by sgRNA and plasmids
  • Design method of sgRNA and lentivirus carrier formed by sgRNA and plasmids

Examples

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preparation example Construction

[0047] Preparation of lentiviral vector:

[0048] First obtain the following fragments by enzyme digestion, PCR or direct synthesis:

[0049] Lentiviral backbone: Removal of EF1-GFP element (residual Wpre element) from the lentiviral vector by enzyme digestion overnight at 37°C: 2ul each of endonuclease NheI and PmeI, 5ug Lenti EF1-GFP-wpre plasmid, 5ul 10x buffer, 36ul of water. After running the gel at 70V for 2 hours, the correct band (6.1kb) was cut, and the DNA was extracted with a Qiagen DNA purification column.

[0050] U6-sgBBS: artificially synthesized (IDT), the sequence is shown in SEQ ID NO: 10; (U6 promoter-sgBbs1-sgRNA backbone).

[0051] EF1: PCR primer sequence EF1-F: GGCTCCGGTGCCCGTCA; EF1-R: GGCGATCGCTCACGACAC. The template is 5ng Lenti EF1-GFP-wpre plasmid. PCR was performed with KAPA HIFI polymerase. The conditions are: 98°C, 2min; 98°C 10sec, 60°C 30sec, 72°C 20sec, 30cycles. After the reaction, add 1ul endonuclease DpnI and incubate at 37°C for 30mi...

example 1

[0072] Example 1: When constructing sgRNA vectors, the minimum length of oligonucleotides is 17 bp to achieve effective knockout. When the length of sgRNA was 16bp, the knockdown efficiency was not different from control cells.

[0073]

example 2

[0074] Example 2: The knockout efficiency of the sgRNA with a length of 17 bp designed according to the optimization method is 80-98%.

[0075]

[0076]

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Abstract

The invention relates to a design method of an sgRNA and a lentivirus carrier formed by the sgRNA and plasmids. In the method, the design length of the sgRNA is 17bp or 18bp, and the tail end of the sgRNA is provided with a G or A; the target sequence is (N16)GNGG, (N16)ANGG, (N16)CNCC or (N16)TNCC; when a first basic group is A, C or T, g is additionally arranged in front of the first basic group. Whether a similar sequence exists in a gene group is searched in a TagScan database; when the sgRNA with the length of 17bp is additionally provided with a completely-matched sequence, the sgRNA is abandoned, and other appropriate sequences are tested; for the sgRNA which is 18bp long, when only one non-matched nucleotide is provided, the sgRNA is also abandoned, and other sequences with the designed sgRNA are continuously tested. The method for designing the sgRNA is optimized, the high knock-out efficiency and low target missing or no target missing can be realized.

Description

technical field [0001] The invention belongs to the field of gene editing and vector design, and in particular relates to a design method of sgRNA and the optimized design and application of a targeted knockout plasmid and a lentiviral vector containing a CRISPR / Cas9 system. Background technique [0002] CRISPR (Clustered regularly interspaced short palindromic repeats), known as regular clustered interspaced short palindromic repeats, is actually a gene editor, a system used by bacteria to protect themselves against phages, and a system to deal with attackers genetic weapon. A series of reports over the past few years indicate that it can be used to delete, add, activate or repress target genes in other organisms, including human, mouse, zebrafish, bacteria, fruit flies, yeast, nematodes and crop cells. gene. [0003] The CRISPR cluster is a special DNA repeat sequence family widely present in the genomes of bacteria and archaea, and its sequence consists of a leader (Lea...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/867C12N15/85
Inventor 张健萍张孝兵程涛
Owner INST OF HEMATOLOGY & BLOOD DISEASES HOSPITAL CHINESE ACADEMY OF MEDICAL SCI & PEKING UNION MEDICAL COLLEGE
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