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Highly PAM-compatible truncated variant txcas9 of the endonuclease spcas9 and its application

A nuclease, compatibility technology, applied in the field of protein engineering, can solve problems such as bulky, and achieve the effect of precise editing

Active Publication Date: 2022-04-12
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although xCas9 has successfully improved the two shortcomings of SpCas9, its bulky problem still needs to be solved

Method used

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  • Highly PAM-compatible truncated variant txcas9 of the endonuclease spcas9 and its application
  • Highly PAM-compatible truncated variant txcas9 of the endonuclease spcas9 and its application
  • Highly PAM-compatible truncated variant txcas9 of the endonuclease spcas9 and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] Embodiment 1, construct the plasmid of txCas9 nuclease

[0062] 1. Design of variants

[0063] Using xCas9 (SEQ ID NO.3) as a template, cut off 180a.a.~299a.a., which is equivalent to the base sequence of SEQ ID NO.5 and the amino acid sequence of SEQ ID NO.6, and the bases in other positions remain unchanged. Recombine the rest and call it txCas9. Its transformation design idea is as follows: figure 1 As shown, the detailed steps are briefly described as follows:

[0064] First, use the restriction enzyme NotI (purchased from NEB) to digest xCas9, and then, pass AxyPrep TM Purification and recovery of DNA GelExtraction Kit. Finally, T4ligase (purchased from Takara) was used to connect the fragments recovered in the previous step to obtain the target product txCas9.

[0065] (1) Plasmid extraction kit

[0066] The TIANprep Mini Plasmid Kit used was ordered from Tiangen Biochemical Technology (Beijing) Co., Ltd. Extract the plasmid. For the extraction method, ref...

Embodiment 2

[0080] Embodiment 2, prepare txCas9 nuclease

[0081] 2. Protein expression and purification

[0082] 2.1 Protein expression

[0083] (1) Turn on the ultra-clean table, wipe the tabletop and various utensils and consumables with cotton balls containing 75% alcohol, turn on the ultraviolet lamp for 20 minutes, and start the fan for standby.

[0084] (2) Pipette 10 μl of txCas9-expressing Rosetta (DE3) (purchased from TIANGEN) bacterial solution into 6 ml of LB liquid medium containing double antibodies (Amp and Cm), and culture overnight at 37°C with shaking at 200 r / min.

[0085] (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, the OD value of the bacterial solution was detected at any time.

[0086] (4) When the OD value of the bacterial solution is close to 0.4-0.8, add t...

Embodiment 3

[0096] Embodiment 3, check txCas9 nuclease cleavage activity

[0097] 3. Detection of variant activity

[0098] The substrate DNA (SEQ ID NO.7) used is mainly amplified by conventional PCR using primers QG-F and QG-R, and then recovered and obtained by tapping rubber.

[0099](1) Purchasing amplification kits

[0100] The amplification kit Fast HiFidelity PCR Kit used was ordered from Tiangen Biochemical Technology (Beijing) Co., Ltd.

[0101] (2) Purchasing primers

[0102] All primers were ordered from Shanghai Sangon Bioengineering Co., Ltd. Their sequence is as follows:

[0103] QG-F: TAGTCCTGTCGGGTTTCG (SEQ ID NO. 8)

[0104] QG-R: TTCCATTCGCCATTCAGG (SEQ ID NO. 9)

[0105] The reaction system and amplification conditions are as follows:

[0106] The amplification system is as follows:

[0107]

[0108]

[0109] PCR reaction conditions:

[0110]

[0111] (3) Purchasing rubber tapping recovery kit

[0112] The rubber tapping recovery kit AxyPrep used TM...

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Abstract

The invention belongs to the technical field of protein engineering, and specifically relates to a highly PAM-compatible truncated variant txCas9 of CRISPR nuclease SpCas9 derived from Streptococcus pyogenes and its application. The high PAM compatibility truncated variant txCas9 nuclease in the present invention belongs to the CRISPR-Cas9 system. The txCas9 nuclease is recombined after truncating the 180th to 299th amino acids of the highly PAM compatible xCas9 nuclease. The highly PAM-compatible truncated variant txCas9 not only has gene editing activity comparable to wild-type CRISPR-Cas9 nuclease, but also can recognize NGN, GAA, and GAT PAMs, effectively broadening the targeting range of CRISPR-Cas9 nuclease . Importantly, the miniaturized Cas9 nuclease is suitable for in vivo transportation using adenoviral vectors with limited loading capacity, so it has greater application potential in in vivo precision editing of biomedicine.

Description

technical field [0001] The invention belongs to the technical field of protein engineering, and specifically relates to a highly PAM-compatible truncated variant txCas9 of CRISPR-Cas9 nuclease SpCas9 derived from Streptococcus pyogenes and its application. Background technique [0002] CRISPR-Cas9 is an immune system against foreign nucleic acids in bacteria and archaea, and has been developed as a gene editing technology to specifically cut DNA fragments (1). Among them, SpCas9 derived from Streptococcus pyogenes has become the most widely used CRISPR nuclease due to its simple construction, strong operability, and high efficiency (2). In recent years, this system has been widely used in the field of genetic engineering, for example, the use of CRISPR system for gene interference to elucidate gene functions (3); rapid establishment of cell or animal diseases by designing guide RNAs targeting disease-causing gene loci Model (4); fusion of inactivated Cas9 (dCas9) to an enha...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/20C12N15/55C12N15/70C12N1/21C12R1/19
CPCC12N9/20C12N15/70
Inventor 黄强朱海霞薛冬梅杜文豪
Owner FUDAN UNIV