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A cpf1-based DNA splicing method in vitro

A technology of D1-D2-D3 and motifs, applied in the field of DNA splicing in vitro based on Cpf1

Active Publication Date: 2021-08-27
CAS CENT FOR EXCELLENCE IN MOLECULAR PLANT SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Yeast in vivo splicing based on yeast recombination can further increase the upper limit of splicing fragment size, but like SLIC and Gibson splicing, they are all splicing based on homologous sequences and cannot do anything for splicing fragments with repetitive sequences

Method used

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  • A cpf1-based DNA splicing method in vitro
  • A cpf1-based DNA splicing method in vitro
  • A cpf1-based DNA splicing method in vitro

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0193] Example 1 The Apra resistance gene was seamlessly spliced ​​and cloned into pUC18.

[0194] 1) using pBC-Am as a template, using primers apr-cf (SEQ ID NO.14) and apr-cr (SEQ ID NO.15) to PCR amplify the Apra resistance gene apr (SEQ ID NO.3), And 17-bp Cpf1 recognition sequence and PAM (TTN) were introduced at both ends by primers.

[0195] 2) Using pUC18 (SEQ ID No.2) as a template, use primers pUC18-cf (SEQ ID NO.12) and pUC18-cr (SEQ ID NO.13) to amplify the linear vector by PCR, and introduce the primers at both ends 17-bp Cpf1 recognition sequence and PAM (TTN) and 5-bp linker sequence to create the same cohesive ends as apr (SEQ ID NO. 3).

[0196] 3) Recover and purify the above PCR products, mix them together in an equimolar ratio, add FnCpf1 (SEQ ID No. 1), crRNA1 (SEQ ID No. 4), and T4 DNA ligase, and react at 30° C. for 1 h.

[0197] 4) The above reaction product was inactivated at 65°C for 20 minutes, immediately placed on ice for 5 minutes, and transform...

Embodiment 2

[0200] Example 2 The promoter of the pathway-specific regulatory factor actII-orf4 in the actinhodine biosynthetic gene cluster was replaced by the constitutively expressed erythromycin promoter.

specific Embodiment approach

[0201] Such as image 3 As shown, through the mediation of 17-nt crRNA1 and crRNA2, FnCpf1 forms cohesive ends that are adapted at both ends on the HIW plasmid and pEASY-emp plasmid respectively, and actⅡ-orf4 (SEQ ID No.7) The promoter was replaced with the erythromycin promoter (SEQ ID No.8). The specific implementation is as follows:

[0202] 1) Using pBS-emp as a template, use primers emp-pf (SEQ ID No.17) and emp-pr (SEQ ID No.18) PCR amplification to obtain the erythromycin promoter (emp) (SEQ ID No.8 ), and cloned into pEASY-blunt (SEQ ID No.10) to obtain the subclone pEASY-emp, which was verified by Sanger sequencing. PAM (TTN), a 17-bp FnCpf1 recognition sequence, and upstream and downstream homologous sequences before the element to be replaced and the recognition sequence were introduced at both ends by primers.

[0203] 2) Actinhodine expression plasmids pHIW (SEQ ID No.9) and pEASY-emp were digested with crRNA2 (SEQ ID No.5) and crRNA3 (SEQ ID No.6) mediated by...

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Abstract

The invention relates to a method for splicing DNA in vitro based on Cpf1. The method guides Cpf1 to specifically cut double-stranded DNA at a specific position and generate preset sticky ends by designing and synthesizing a specific crRNA sequence that can be recognized by CRISPR-Cpf1. By using the method of the invention, predetermined cohesive ends can be obtained conveniently, quickly and accurately, and DNA can be spliced. DNA can be engineered, standardized, modularized or assembled using the method of the present invention.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular, the invention relates to a Cpf1-based DNA splicing method in vitro. The method of the invention can generate predetermined sticky ends, and can be used for DNA seamless splicing in vitro. Background technique [0002] In 2010, J. Craig Venter's laboratory completed the artificial construction of Mycoplasma mycoides "Synthia", setting off a storm of synthetic biology research. Compared with the traditional restriction endonuclease digestion-ligase ligation cloning method, seamless splicing has many advantages such as convenient design and good compatibility because it does not introduce additional sequences, and it plays an increasingly important role in the development of synthetic biology. role. [0003] The existing seamless splicing technologies mainly include splicing methods based on Type IIS restriction endonucleases, splicing methods based on specific base modifications, and s...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/63C12N15/10C12P19/34
CPCC12N15/1027C12N15/63C12N2800/80C12P19/34C12Q2521/301
Inventor 王金雷超赵国屏
Owner CAS CENT FOR EXCELLENCE IN MOLECULAR PLANT SCI
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