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Truncated variant of CRISPR nuclease SpCas9 of streptococcus pyogenes and application of truncated variant

A nuclease, nucleic acid technology, applied in the field of protein engineering, can solve the problems of small loading, complex preparation, low titer and so on

Active Publication Date: 2019-09-17
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the shortcomings of adenovirus (such as lack of efficient packaging cells, complex preparation, and low titer defects), especially the small load (less than 4.7kb) of its own characteristics, limit the SpCas9+sgRNA target with a coding region larger than 4kp Delivery to target locations in vivo and cells [25]

Method used

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  • Truncated variant of CRISPR nuclease SpCas9 of streptococcus pyogenes and application of truncated variant
  • Truncated variant of CRISPR nuclease SpCas9 of streptococcus pyogenes and application of truncated variant
  • Truncated variant of CRISPR nuclease SpCas9 of streptococcus pyogenes and application of truncated variant

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Example 1, constructing the plasmid of CRISPR-Cas9 nuclease (TSpCas9).

[0055] 1. Mutant Design.

[0056] Using the pet21-6His-TEV-SpCas9 plasmid, namely SEQ ID NO.5, as a template, truncated 180aa (5822bp) to 299aa (6181bp), which is equivalent to the base sequence of SEQ ID NO.8 and the amino acid sequence of SEQ ID NO.9, The remaining part was recombined into a truncated SpCas9, which was called TSpCas9. Its transformation design idea is as follows: figure 1 As shown, the detailed steps are briefly described as follows:

[0057] First, use primers F-F and F-R, S-F and S-R to amplify 5267~5821bp (equivalent to 1-538bp of SEQ ID NO.10) and 6182~9391bp (equivalent to 898-4104bp of SEQ ID NO.12), and passed AxyPrep TM DNA Gel Extraction Kit (purchased from Axygen) was purified and recovered, and they were called amplified fragments F and S;

[0058] Secondly, use restriction endonucleases NdeI and XhoI (purchased from NEB) to digest pet21-6His-TEV-SpCas9 and F amp...

Embodiment 2

[0096] Example 2, preparing CRISPR-Cas9 (TSpCas9) nuclease.

[0097] 2. Protein expression and purification.

[0098] 2.1 Protein expression

[0099] (1) Turn on the ultra-clean bench, wipe the table top and various utensils with a cotton ball containing 75% alcohol, turn on the ultraviolet light for 20 minutes, and start the fan for standby;

[0100] (2) Pipette 10 μl of Rosetta (DE3) (purchased from TIANGEN) bacterial solution expressing Pet21-6His-TEV-TSpCas9 into 6 ml of LB liquid medium containing double antibodies (Amp and Cm), 37°C, 200r / min shaking culture overnight;

[0101] (3) Transfer the overnight cultured bacterial solution to 500ml LB (purchased from Sanko) liquid medium containing double antibodies according to the volume ratio of 1:100, and cultivate at 37°C with shaking at 200r / min. During the cultivation process, detect the OD value of the bacterial solution at any time;

[0102] (4) When the OD value of the bacterial solution is close to 0.4-0.8, add t...

Embodiment 3

[0112] Example 3, testing CRISPR-Cas9 (TSpCas9) nuclease cleavage activity.

[0113] 3. Detection of mutant activity.

[0114] The substrate DNA (SEQ ID NO.20) used was mainly obtained by conventional PCR amplification using primers QG-F: TAGTCCTGTCGGGTTTCG (SEQ ID NO.17) and QG-R: TTCCATTCGCCATTCAGG (SEQ ID NO.18). The reaction system and amplification conditions are as follows:

[0115] The amplification system is as follows:

[0116]

[0117] PCR reaction conditions:

[0118]

[0119] (3) Procurement of rubber tapping recovery kits

[0120] The rubber tapping recovery kit AxyPrep used TM The DNA Gel Extraction Kit was ordered from Axygen Company, and the rubber tapping recovery operation was carried out according to its instructions, and a relatively pure substrate DNA (SEQ ID NO.20) could be obtained.

[0121] Cas9 and sgRNA are mixed in equimolar amounts, and the substrate DNA can be adjusted to 0.2-1 times the molar mass of Cas9 according to experimental need...

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Abstract

The invention belongs to the technical field of protein engineering, and particularly relates to a truncated variant of CRISPR nuclease SpCas9 from streptococcus pyogenes and an application of the truncated variant. According to the truncated variant disclosed by the invention, CRISPR-Cas9(TSpCas9) nuclease belongs to a CRISPR-Cas9 system, has shearing activity equivalent to that of wild type CRISPR-Cas9 nuclease, and is obtained through the steps of cutting out 120 amino acids from the 180th to the 299th in an amino acid sequence of the wild type CRISPR-Cas9 nuclease and performing recombination; and the amino acid sequence of the wild type CRISPR-Cas9 nuclease is as shown in SEQID NO.7, or the CRISPR-Cas9 nuclease contains 90% of an amino acid sequence as shown in the SEQID NO.7, namely SEQID NO.15. The truncated variant can be used for genome editing, gene targeting, genome engineering, apparent genome engineering, gene therapy and in vitro diagnosis.

Description

technical field [0001] The invention belongs to the technical field of protein engineering, in particular to the protein engineering of CRISPR nuclease SpCas9 derived from Streptococcus pyogenes, and its application in genome editing, genome targeting, epigenome engineering and in vitro diagnosis. Background technique [0002] The CRISPR-Cas9 system produced by the long-term evolution of the bacterial immune system is a revolutionary gene editing technology, known as "gene magic scissors", which can conveniently and efficiently cut and edit the DNA strands of specific genes in the genome. The medical field has great application potential, such as genome editing, genome targeting, epigenome engineering and in vitro diagnostics, etc. [1-11] . [0003] The CRISPR-Cas9 nuclease SpCas9 from Streptococcus pyogenes is currently the most widely used CRISPR nuclease [12] . So far, this technology has been widely used in many fields, including medical research and biotechnology, su...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/22C12N15/55C12N15/90
CPCC12N9/22C12N15/902
Inventor 黄强汤洪海杜文豪薛冬梅
Owner FUDAN UNIV
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