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Basic group editing system as well as constructing and applying methods thereof

A base editing and base technology, applied in the field of genome editing, can solve problems such as reducing the efficiency of base editing, and achieve the effect of enhancing the efficiency, reducing the rate of base insertion or deletion, and improving the rate of C-T base editing.

Active Publication Date: 2017-07-04
SHANGHAI TECH UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Compared with BE2 containing dCas9, BE3 containing nCas9 has a higher C-T editing efficiency in the target sequence, but the base insertion or deletion (insertion / deletion, indel) rate of BE3 in the target sequence caused by the CRISPR system itself However, it is higher, which reduces the efficiency of base editing (the ratio of C-T base editing rate to base insertion or deletion rate, C-T editing rate / indel rate) and accuracy

Method used

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  • Basic group editing system as well as constructing and applying methods thereof
  • Basic group editing system as well as constructing and applying methods thereof
  • Basic group editing system as well as constructing and applying methods thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] A base editing system, including UGI expression plasmid (sequence: SEQ ID NO: 20) and BE3 expression plasmid (sequence: SEQ ID NO: 21), or UGI and BE3 co-expression plasmid. The co-expression plasmid of UGI and BE3 is enhanced CRISPR base editor eBEa expression plasmid (sequence is SEQ ID NO: 22) or enhanced CRISPR base editor eBEb expression plasmid (sequence is SEQ ID NO: 23).

[0065] The human genome FANCF site uses the above-mentioned base editing system to implement high-efficiency and high-precision base editing:

[0066] 1. Experimental materials

[0067] 1) Reagent

[0068] Primers were synthesized from Shanghai Boshang Biological Co., Ltd. according to conventional methods;

[0069] Restriction enzymes, DNA ligases, high-fidelity DNA polymerases Purchased from NEB Company;

[0070] Plasmid Recombination Kit Clone Purchased from Vazyme Company;

[0071] pCMV-BE3 was purchased from the addgene website, and the sequence is SEQ ID NO: 21;

[0072] DNA g...

Embodiment 2

[0110] The RNF2 site of the human genome uses the base editing system in Example 1 to implement high-efficiency and high-precision base editing:

[0111] 1. Experimental materials

[0112] 1) Reagent

[0113] With embodiment 1.

[0114] 2) Cell lines and vectors

[0115] With embodiment 1.

[0116] 2. Experimental method

[0117] 1. Construction of base editing system

[0118] 1.1 Construction of UGI separate expression plasmid

[0119] With embodiment 1.

[0120] 1.2 Construction of eBEa and eBEb expression plasmids

[0121] With embodiment 1.

[0122] 2. Application of base editing system

[0123] 2.1 Construction of sgRNA expression plasmid

[0124] Anneal the following primers 15 and 16, and use T4 DNA ligase (NEB, M0202L) to ligate the annealed product into the sgRNA expression vector pGL3-U6-sgRNA digested with the restriction endonuclease BsaI (NEB, R3535L) according to the reagent instructions - After PGK-puromycin (addgene, #51133), the sgRNA expression pla...

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Abstract

The invention provides a basic group editing system as well as constructing and applying methods of the basic group editing system. The basic group editing system is characterized y comprising a UGI expression vector and a BE3 expression vector, or a UGI and BE3 coexpression vector. According to the basic group editing system as well as the constructing and applying methods of the basic group editing system, for the first time, the C-T editing ratio generated by a CRISPR basic group editor is increased and the basic group insertion or loss ratio caused by the CRISPR system is reduced internationally, then the efficacies of the CRISPR basic group editor is enhanced, and thus a novel method and a novel thought are provided for the implementation of the accurate and safe basic group editing of the CRISPR basic group editor in the genomes of the various species.

Description

technical field [0001] The invention relates to the field of genome editing, in particular to an enhanced CRISPR base editing system to realize high-efficiency and high-precision base-level genome editing methods and applications thereof. Background technique [0002] Genome editing technology refers to a genetic engineering technology that uses designable nucleases (molecular scissors) to modify specific segments of the genome DNA of an organism through base insertion, deletion or substitution to achieve editing of the target gene. Using genome editing technology to genetically manipulate cells can be widely used in basic life science research, biotechnology development, agricultural technology development, and pharmaceutical research and development. For example: directly correcting the genetic mutations that cause genetic diseases in the body will be able to treat genetic diseases fundamentally; carry out precise genetic engineering on crops to increase their yield or res...

Claims

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Application Information

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IPC IPC(8): C12N15/90C12N15/113
CPCC12N15/113C12N15/902C12N15/907C12N2310/10
Inventor 陈佳杨力杨贝薛尉王丽洁
Owner SHANGHAI TECH UNIV
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