Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

In vivo plasmid editing system based on CRISPR/Cas and lambda-Red recombination system, and application thereof

An editing system and plasmid technology, applied in microorganism-based methods, DNA/RNA fragments, recombinant DNA technology, etc., can solve problems such as low efficiency and limitations of recombination, and achieve the effects of short time period, reduced cost, and expanded scope of use

Inactive Publication Date: 2019-05-03
INST OF PATHOGEN BIOLOGY CHINESE ACADEMY OF MEDICAL SCI
View PDF2 Cites 6 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the low recombination efficiency, selection markers or reverse selection elements are often used to screen for recombinants
Moreover, the formation of multimeric plasmids and the coexistence of recombinant and original plasmids in the same cell also limit the application of this method.

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • In vivo plasmid editing system based on CRISPR/Cas and lambda-Red recombination system, and application thereof
  • In vivo plasmid editing system based on CRISPR/Cas and lambda-Red recombination system, and application thereof
  • In vivo plasmid editing system based on CRISPR/Cas and lambda-Red recombination system, and application thereof

Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0057] 4. Preparation of Competent Cells

[0058] i. Preparation of competent electroporation: select a single clone and culture it in 4mL culture medium at 30°C for 16-18h. 1:25 was transferred to the saturated bacterial solution, and the inducer 20% arabinose was added when the OD was 0.2. Continue to cultivate to 0.4-0.5, heat shock in a water bath shaker at 42°C for 15 minutes, and immediately place it on ice for 10-15 minutes. Centrifuge at 6500g for 7 minutes at 4°C to collect the cells. ddH 2 O Wash the cells twice, and finally resuspend the cells in 1 / 100 of the initial culture volume in pre-cooled 10% glycerol, and then pack into 50 μL / EP tubes for use.

[0059] ii. Preparation of Transformation Competent: Conventional CaCl 2 method to prepare the competent state.

[0060] 5. Transformation and screening of recombinants

[0061] 50ng of target plasmid, 200ng of crRNA expression plasmid and 1μg of ssDNA (or 700ng of dsDNA) were mixed, placed on ice, and then tran...

Embodiment 1

[0066] Embodiment 1. Fragment replacement of plasmid pJV53-GFP

[0067] 1) Find the crRNA action sites of the GFP-encoded gene on the plasmid pJV53-GFP, the sequences are T1, T2, T3; for each site, synthesize forward and reverse oligos, the sequences of which are shown in Table 1 as SEQ ID No: 1 and 2, 4 and 5, 7 and 8. Forward and reverse oligos were phosphorylated with T4 polynucleotide kinase (NEB) (37°C, 30min) and then annealed (95°C, 5min / °C and slowly cooled to 25°C after 5min), ligated into pAC- crRNA vector, construct the corresponding crRNA expression plasmid.

[0068] 2) Allelic exchange substrates oligo 1, 2, 3 designed for replacement by homologous recombination, the sequences of which are listed in Table 1 as SEQ ID No: 3, 6, 9.

[0069] 3) Preparation of SY4539 electroporation competent cells.

[0070] 4) Transfer the above-mentioned crRNA expression plasmid, original target plasmid pJV53-GFP and allelic exchange substrate oligo into the competent cells prepa...

Embodiment 2

[0073] Embodiment 2. Fragment replacement of plasmid pJV53-GFPm back mutation

[0074] 1) For the above-mentioned plasmid pJV53-GFPm (the three sites correspond to plasmids pJV53-GFPm1, pJV53-GFPm2 and pJV53-GFPm3 respectively) that have been successfully replaced through verification, search for crRNA action sites, the sequences of which are RT1, RT2, RT3; For the forward and reverse oligos of each site, the sequences thereof are shown in Table 1 as SEQ ID Nos: 10 and 11, 13 and 14, and 16 and 17. Forward and reverse oligos were phosphorylated by T4 polynucleotide kinase (NEB) (37°C, 30min) and then annealed (95°C, 5min / °C after 5min, slowly cooled to 25°C), and then ligated into pAC- crRNA vector, construct the corresponding crRNA expression plasmid.

[0075] 2) Allelic exchange substrates oligo R1, R2, R3 designed for homologous recombination to achieve replacement, the sequences are shown in SEQ ID No: 12, 15, 18 in Table 1.

[0076]3) Preparation of SY4539 electroporati...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention discloses an in vivo plasmid editing system based on a CRISPR / Cas and lambda-Red recombination system as well as application thereof. The in vivo plasmid editing system based on the CRISPR / Cas and lambda-Red recombination system comprises the following components: an engineering strain of Escherichia coli with HsdR function deficiency as well as integrated lambda-Red recombinant protein gene capable of inducing expressing and Cas protein gene capable of inducing expressing in genome, a CrRNA expression plasmid inserted with a target sequence, an allelic exchange substrate, and atarget plasmid. By using a CRISPR / Cas system in integration with recombination engineering, a fast and traceless in vivo plasmid editing method is established by the invention; and the established invivo plasmid editing method enables convenient and quick gene deletion, point mutation and fragment insertion on plasmids in Escherichia coli, as well as establishment of a plasmid mutation library.

Description

technical field [0001] The present invention relates to a plasmid editing system, in particular to a CRISPR / Cas and λRed recombination system for transforming plasmids in vivo. Background technique [0002] Recombination engineering is a biotechnology based on the principle of nucleic acid homologous recombination, using phage recombination proteins to promote homologous recombination in vivo mediated by single-stranded DNA (ssDNA) or linearized double-stranded DNA (dsDNA). At present, it has been widely used in the transformation of genomic DNA, BAC and plasmid DNA of various prokaryotes, including mutation, deletion or insertion. However, this technology has the problem of low recombination efficiency. It is necessary to add a selection marker (such as antibiotics) to screen recombinants, and then remove the selection marker and then need to use a two-step method or introduce a sequence-specific recombinase to excise the resistance tag (resistance There are two short DNA ...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
IPC IPC(8): C12N1/21C12N15/70C12N15/90C12N15/65C12N15/113C40B50/06C12R1/19
Inventor 孙义成耿艺漫严海芹任改仙郭晓鹏钱朝晖赵振东金奇
Owner INST OF PATHOGEN BIOLOGY CHINESE ACADEMY OF MEDICAL SCI
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com