Part:BBa_K4687024
CRISPR-MAD7 nuclease+recE/T recombinase:MADE/T
The recE/T system is responsible for facilitating homologous recombination, which enables the precise insertion of foreign DNA into the target bacterial genome. It creates single-stranded DNA breaks in the target genome and assists in the integration of desired genetic material by promoting recombination. The recE generates single-stranded DNA breaks, and recT assists in the recombination process by binding to these breaks and facilitating the exchange of genetic material. CRISPR-MAD7, a nuclease enzyme, is used to generate double-stranded breaks in the DNA at the desired location, initiating the genome editing process.The CRISPR-MAD7 system initiates the genome editing process by creating targeted DNA breaks, while the recE/T system ensures the accurate and efficient integration of foreign DNA sequences into the bacterial genome at those breaks. The recE/T system can be employed to create specific single-strand DNA breaks or overhangs at desired locations in the genome, while the MAD7 system can be used to target and cleave specific DNA sequences.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 1978
Illegal EcoRI site found at 6493
Illegal EcoRI site found at 6817
Illegal PstI site found at 3345
Illegal PstI site found at 4633
Illegal PstI site found at 5194
Illegal PstI site found at 6040
Illegal PstI site found at 6382
Illegal PstI site found at 7168 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 1978
Illegal EcoRI site found at 6493
Illegal EcoRI site found at 6817
Illegal PstI site found at 3345
Illegal PstI site found at 4633
Illegal PstI site found at 5194
Illegal PstI site found at 6040
Illegal PstI site found at 6382
Illegal PstI site found at 7168 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 1978
Illegal EcoRI site found at 6493
Illegal EcoRI site found at 6817
Illegal BglII site found at 3459
Illegal BglII site found at 4313
Illegal BglII site found at 4983
Illegal BglII site found at 6198
Illegal BglII site found at 6519
Illegal BglII site found at 6567 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 1978
Illegal EcoRI site found at 6493
Illegal EcoRI site found at 6817
Illegal PstI site found at 3345
Illegal PstI site found at 4633
Illegal PstI site found at 5194
Illegal PstI site found at 6040
Illegal PstI site found at 6382
Illegal PstI site found at 7168 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 1978
Illegal EcoRI site found at 6493
Illegal EcoRI site found at 6817
Illegal PstI site found at 3345
Illegal PstI site found at 4633
Illegal PstI site found at 5194
Illegal PstI site found at 6040
Illegal PstI site found at 6382
Illegal PstI site found at 7168
Illegal AgeI site found at 417
Illegal AgeI site found at 5416 - 1000COMPATIBLE WITH RFC[1000]
Contents
Introduction
In bacteria, precise editing of the targeted genome by CRISPR/Cas systems requires homology-directed repair (HDR) of the intended cleavage site and always requires the assistance of phage recombinase for efficient recombineering.
The recE/T system is responsible for facilitating homologous recombination, which enables the precise insertion of foreign DNA into the target bacterial genome. It creates single-stranded DNA breaks in the target genome and assists in the integration of desired genetic material by promoting recombination. The recE generates single-stranded DNA breaks, and recT assists in the recombination process by binding to these breaks and facilitating the exchange of genetic material.
MAD7, a nuclease enzyme, is used to generate double-stranded breaks in the DNA at the desired location, initiating the genome editing process.The CRISPR-MAD7 system initiates the genome editing process by creating targeted DNA breaks, while the recE/T system ensures the accurate and efficient integration of foreign DNA sequences into the bacterial genome at those breaks.
The RecE/T system can be employed to create specific single-strand DNA breaks or overhangs at desired locations in the genome, while the MAD7 system can be used to target and cleave specific DNA sequences.
Usage and Biology
Gene editing in Corynebacterium glutamicum using CRISPR-MAD7 can be done with or without the RecE/T system, and the choice depends on the specific goals of the editing and the desired precision. Here's a description of both situations:
Gene Editing with CRISPR-MAD7 Alone (Without RecE/T System)
Design Guide RNA (gRNA): We design a guide RNA (gRNA) molecule that is complementary to the target DNA sequence they want to edit in the Corynebacterium glutamicum genome.
Introduce MAD7 Protein: MAD7 protein, derived from the CRISPR-Cas12 system, is introduced into the cells along with the designed gRNA.
Target DNA Cleavage: MAD7, guided by the gRNA, recognizes the specific DNA sequence in the Corynebacterium glutamicum genome and cleaves it.
Repair Mechanisms: The cell's natural DNA repair mechanisms come into play. These mechanisms can introduce errors during the repair process, leading to insertions, deletions, or substitutions in the target gene.
Gene Editing with CRISPR-MAD7 and RecE/T System
When the recE/T system is combined with CRISPR-MAD7, it provides an additional layer of control and precision to the gene editing process:
Design Guide RNA (gRNA): As in the previous scenario, researchers design a gRNA that targets the desired DNA sequence.
Introduce MAD7 Protein: MAD7 protein and the designed gRNA are introduced into the cells.
RecE/T-Mediated Single-Strand Overhangs: Here's where the recE/T system comes into play. It can be used to create specific single-strand DNA breaks or overhangs at the target site. This is achieved by RecE, which acts as an exonuclease to digest DNA and create overhangs.
MAD7-Mediated DNA Cleavage: MAD7, guided by the gRNA, can then cleave the target DNA with greater precision because the single-strand overhangs created by recE/T serve as landing pads for MAD7, improving its binding and cleavage efficiency.
By combining CRISPR-MAD7 with the recE/T system, researchers can achieve more controlled and targeted gene editing in Corynebacterium glutamicum, resulting in fewer off-target effects and a higher likelihood of obtaining the desired genetic modifications. This approach is particularly valuable for applications requiring precise genome editing, such as gene insertions, deletions, or replacements.[1]
Characterization
Agarose gel electrophoresis
Figure 1. Agarose gel electrophoresis validation of PCR results of E.coil Top10-pJYS1-recE/T colonies.
The first lane was loaded with DL5000 DNA ladder whose sizes were marked on the image. We chose 2 × Phanta Max Master Mix because it contains Phanta Max Super-Fidelity DNA Polymerase, dNTP, and an optimized buffer system, which allows amplification by adding only primers and templates, reducing pipetting and improving throughput and reproducibility of results. The protective agent in the product allows the 2 × Phanta Max Master Mix (Dye Plus) to maintain stable activity after repeated freezing and thawing; it also contains an electrophoretic indicator, which can be used to spot samples for electrophoresis directly after the PCR reaction is completed.The PCR reaction consisted of 50 μL 2 × Phanta Max Master Mix, 4 μL forward primer (10 mM), 4 μL reverse primer (10 mM), 42 μL H2O and 1 μL colony.
After PCR, the correct bacterial clones were sent for sequencing.Once verified, these clones would be used for further experiments.
We edited the genome of Corynebacterium glutamicum with the help of the recE/T system. After coating the plate, colony PCR was performed and the above glue map was obtained. Of the eleven colonies selected, ten were successfully edited.
Successful production of lycopene
Lycopene is accumulated by knocking out crtEb and crtR genes. In the image above, the wild-type Corynebacterium glutamicum on the left and the deletion of crtR and crtEb genes on the right allow lycopene to accumulate.
Lycopene is a naturally occurring red-orange pigment that belongs to a group of pigments known as carotenoids. It is found in various fruits and vegetables, most notably in tomatoes, watermelons, and pink grapefruits. Lycopene is a potent antioxidant and is known for its potential health benefits, including its role in reducing the risk of certain chronic diseases.
The biosynthesis of lycopene in microorganisms like Corynebacterium glutamicum involves several genes encoding enzymes responsible for each step of the pathway. Here are some of the key genes involved:
crtE: This gene encodes geranylgeranyl diphosphate synthase, which catalyzes the condensation of isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) to form geranylgeranyl pyrophosphate (GGPP).
crtB: Encodes phytoene synthase, which converts GGPP into phytoene.
crtI: This gene codes for phytoene desaturase, which introduces double bonds into phytoene, converting it into various carotenoid intermediates, including lycopene.
crtEb: This gene encodes a putative lycopene elongase, which can be involved in further modifying lycopene to produce longer carotenoid compounds.[2]
These genes are part of the carotenoid biosynthesis pathway and play a crucial role in the production of lycopene in microorganisms like Corynebacterium glutamicum. The enzymes encoded by these genes catalyze specific reactions that convert precursor molecules into lycopene, the final product.
Lycopene Accumulation: Lycopene is the red pigment produced in this pathway. It accumulates in the cells of Corynebacterium glutamicum, giving them a reddish-orange color.
Reference
- ↑ Nannan Z,Lu L,Guangjuan L, et al. Multiplex gene editing and large DNA fragment deletion by the CRISPR/Cpf1-RecE/T system in Corynebacterium glutamicum.[J]. Journal of industrial microbiology & biotechnology,2020,47(8).
- ↑ Christian M,Andreas U,Jung-Won Y, et al. Engineering of Corynebacterium glutamicum for growth and L-lysine and lycopene production from N-acetyl-glucosamine.[J]. Applied microbiology and biotechnology,2014,98(12).
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