Efficient CRISPR/Cas (Clustered Regulatory Interspaced Short Palindromic Repeats/CRISPR associated) 9 gene editing system for Kluyveromyces optimization

A Kluyveromyces, gene technology, applied in the field of CRISPR/Cas9 high-efficiency gene editing system, can solve the problems of Kluyveromyces cerevisiae industrial production safety hazards, inability to stably replicate and express, and achieve the effect of safe gene editing

Active Publication Date: 2018-01-12
KANGMA SHANGHAI BIOTECH LTD
View PDF1 Cites 49 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, usually the plasmid of Saccharomyces cerevisiae is a 2µ plasmid (including pCAS), which cannot be stably replicated and expressed in Kluyveromyces[11]
In the Kluyveromyces CRISPR / Cas9 transformation system reported so far, the Cas9 gene is directly inserted into the yeast genome, resulting in continuous expression of the Cas9 protein, which poses a certain safety hazard to the industrial production of Kluyveromyces[12]. An efficient and safe CRISPR / Cas9 system capable of stably replicating and expressing in Kluyveromyces is necessary for the development of biotechnology and will be a great advancement in the field of biomedicine

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
  • Efficient CRISPR/Cas (Clustered Regulatory Interspaced Short Palindromic Repeats/CRISPR associated) 9 gene editing system for Kluyveromyces optimization
  • Efficient CRISPR/Cas (Clustered Regulatory Interspaced Short Palindromic Repeats/CRISPR associated) 9 gene editing system for Kluyveromyces optimization
  • Efficient CRISPR/Cas (Clustered Regulatory Interspaced Short Palindromic Repeats/CRISPR associated) 9 gene editing system for Kluyveromyces optimization

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1—KL-CAS1.0 System Transformation

[0036] pKM-Cas9 / gRNA plasmid construction

[0037] In the prior art, the pCAS plasmid used in Saccharomyces cerevisiae has both Cas9 gene sequence and gRNA elements, which can realize efficient genome transformation in Saccharomyces cerevisiae through one transformation, but cannot stably replicate and express in Kluyveromyces cerevisiae, such as figure 1 shown. Through transformation, the present invention constructs a new safe and efficient CRISPR / Cas9 gene editing system that is dedicated to Kluyveromyces, can be stably replicated, expressed, and genetically modified in Kluyveromyces, and the gene editing system is named It is KL-CAS1.0. The Kluyveromyces of the present invention is illustrated by taking Kluyveromyces lactis as an example, but not limited thereto.

[0038] Insertion of KL-CAS1.0 efficient pKD1 stabilizing element (SE)

[0039] pKD1 is a commonly used plasmid for Kluyveromyces transformation [13]. In or...

Embodiment 2

[0049] 2. Example 2—Determination of target gene and gRNA sequence

[0050] 2A. Kluyveromyces lactis target gene identification

[0051] Sequence search at the website http: / / www.uniprot.org / , species" Kluyveromyceslactis ",Key words" Threoninet RNA synthetase "" ThrRS "" TRS ". Here, inserting a marker DNA at the tail of this gene is taken as an example. Other target genes or insertion positions and sequences can be operated in a similar manner.

[0052] i. There are two kinds of TRS in K. lactis, which exist in the cytoplasm and mitochondria respectively. After the protein sequence is retrieved, analyze it on the website https: / / ihg.gsf.de / ihg / mitoprot.htmL to determine whether the retrieved protein exists in the cytoplasm (without mitochondrial recognition sequence, eukaryotic homology) or in mitochondria (with mitochondria recognition sequence, prokaryotic homology), pick cytoplasmic TRS Gene (http: / / www.uniprot.org / uniprot / Q6CL41) as the target gene, and named ...

Embodiment 3

[0056] 3. Example 3—target sequence pKM-Cas9 plasmid construction

[0057] The present invention replaces the gRNA sequence in the original plasmid with the PCR-homologous recombination method Kl - TRS gRNA sequence. The specific steps are: using the pKM-CAS1.0 plasmid as a template, using primers pKM-Cas9-TRS-F1:CTTTCTGATAATGTCTTGGCTTAAGTTTTAGAGCTAGAAATAGCAAG and primers pKM-Cas9-TRS-R1:GCTCTAAAACTTAAGCCAAGACATTATCAGAAAGTCCCATTCGCCAC for amplification. Mix 17 µL of amplification product, 1 µL of LDpn I, 2 µL of 10×digestion buffer, 37 o C warm bath for 3 h. Add 10 µL of Dpn I-treated product to 100 µL DH5α competent cells, place on ice for 30 min, 42 o After heat shock at C for 45 s, add 1 mL of LB liquid medium37 o C cultured with shaking for 1 h, spread on Amp-resistant LB solid culture, 37 o C was cultured upside down until a single clone grew out. Pick 5 single clones and shake them in LB liquid medium. After the PCR test is positive and confirmed by sequencing, ext...

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 relates to a special safe and efficient CRISPR / Cas (Clustered Regulatory Interspaced Short Palindromic Repeats / CRISPR associated) 9 gene editing system for Kluyveromyces optimization, and belongs to the field of biotechnology. In the prior art, pCAS plasmids used in saccharomyces cerevisiae have Cas9 gene sequences and gRNA (guide RNA) elements at the same time, and efficient genomemodification in the saccharomyces cerevisiae can be realized through one-time transformation, but stable copying and expression cannot be realized in Kluyveromyces. According to the system, Cas9 / gRNAfusion plasmids are transformed in Kluyveromyces cells, the plasmids are targetedly delivered to endogenous DNA sequences of the Kluyveromyces cells, and double strand breaks are generated; and donorDNA sequences are transformed in the Kluyveromyces cells, the sequences generate homologous recombination at the double strand breaks and target sites, and Tag sequences are inserted in the target sites. Through the modification, the new safe and efficient CRISPR / Cas9 gene editing system which is specially used for the Kluyveromyces and can perform stable copying and expression in the Kluyveromyces and perform gene modification is established.

Description

technical field [0001] The invention relates to a CRISPR / Cas9 high-efficiency gene editing system specially optimized for Kluyveromyces, belonging to the field of biotechnology. Background technique [0002] Microbial genome modification mainly relies on two biological mechanisms: endogenous homologous recombination repair (endogenous homology-directed repair, HDR) and non-homologous end joining (non-homologous end joining, NHEJ) [1]. The basic process of HDR is that after double-strand breaks (DSBs) occur in the genome, the donor DNA (sequence homologous to the sequences on both sides of the break site) undergoes homologous recombination with the damaged genome sequence at the break site , to achieve repair, this process is not easy to introduce insertion or deletion mutations [2]. The basic process of NHEJ is that the two ends of the broken DNA are directly joined without the aid of homologous recombination mediated by the donor DNA. This process is prone to insertion or ...

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
Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/81C12N1/19C12R1/645
CPCC12N15/81C12N1/145C12R2001/645
Inventor 郭敏代田纯李海洋于雪
Owner KANGMA SHANGHAI BIOTECH LTD
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

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

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap