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Dual-spacer sequence recognition and cleavage CRISPR-Cas9 vector construction, and application thereof in verrucosispora

A vector and sequence technology, applied in the field of genome editing, can solve the problems of long time, high cost, high off-target probability of CRISPR/Cas9 system, and achieve the effect of increasing stability

Active Publication Date: 2017-08-18
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI +1
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AI Technical Summary

Benefits of technology

This patented technique uses an improved way to create DNA sequences called chimeric dendritics (CDs) with specific functions like enzymatic activity or binding proteins attached thereto. By adding certain chemical groups at one end of each CD, it becomes possible to design new molecules containing these functionalities. These techniques allow scientists to study how organisms live and work better without relying solely upon expensive methods such as cloning them into vectors.

Problems solved by technology

The technical problem addressed in this patented patents relates to improving the efficient and accurate genomie editing technique used during bioremediation processes like microbial contaminations or cancer treatment procedures. Current methods require multiple steps involving rearranging large numbers of sequences at once, making them slow and prone to errors caused by external factors like pH changes or enzyme activities.

Method used

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  • Dual-spacer sequence recognition and cleavage CRISPR-Cas9 vector construction, and application thereof in verrucosispora
  • Dual-spacer sequence recognition and cleavage CRISPR-Cas9 vector construction, and application thereof in verrucosispora
  • Dual-spacer sequence recognition and cleavage CRISPR-Cas9 vector construction, and application thereof in verrucosispora

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Embodiment 1

[0076] Example 1. Construction of a double spacer sequence recognition and cutting CRISPR-Cas9 vector and its application in Verrucospora

[0077] 1. Construction of double spacer sequence recognition cutting CRISPR-Cas9 vector

[0078] 1. Transform the carrier pHelp for auxiliary cloning spacer

[0079] Transformation of the vector pHelp, which assists cloning and transcribing sgRNA, is carried out by a method including the following steps: Mutation assisting cloning expresses a spacer vector, adding a promoter psf and a terminator Term21 to it, and the promoter psf and terminator Term21 contain an element for transcribing sgRNA and enzyme digestion The recognition sites NcoI and XbaI can be inserted into the spacer sequence by means of sticky-end enzyme-cut ligation to obtain the transformed vector pHelp that assists in cloning the spacer (referred to as the transformed pHelp vector). The plasmid map is attached figure 1 , as can be seen from the figure: the modified pHelp...

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Abstract

The invention discloses a dual-spacer sequence recognition and cleavage CRISPR-Cas9 vector construction, and application thereof in verrucosispora. The invention provides a dual-spacer sequence recognition and cleavage CRISPR-Cas9 vector construction method. A dual-spacer sequence recognition and cleavage CRISPR-Cas9 vector for deleting an ocean verrucosispora abyssomicin gene cluster is successfully constructed by utilizing the method. The experiment proves that the dual-spacer sequence recognition and cleavage CRISPR-Cas9 vector for deleting the ocean verrucosispora abyssomicin gene cluster constructed by the invention can successfully delete the ocean verrucosispora abyssomicin gene cluster.

Description

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Claims

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Application Information

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Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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