CN110452930A - A kind of genome edit methods and purposes based on immunocyte - Google Patents
A kind of genome edit methods and purposes based on immunocyte Download PDFInfo
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- CN110452930A CN110452930A CN201910764807.5A CN201910764807A CN110452930A CN 110452930 A CN110452930 A CN 110452930A CN 201910764807 A CN201910764807 A CN 201910764807A CN 110452930 A CN110452930 A CN 110452930A
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
- C12N15/90—Stable introduction of foreign DNA into chromosome
- C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
- C12N15/907—Stable introduction of foreign DNA into chromosome using homologous recombination in mammalian cells
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Abstract
The invention discloses a kind of genome edit methods based on immunocyte, including designing the genome of immunocyte, the genome edit tool of specified site sequence, the gene editing of immunocyte and fixed point gene knock-in integration；The gene editing of the immunocyte refer to based on nickase generate DNA single stranded gaps technology, be aided with a set of homologous recombination factors RecOFAR realize it is efficient, accurate, fixed point human primary cultured cells genome editor.The present invention significantly improves the safety of genome editor, reduces error rate；Adjustable point gene knock-in.
Description
Technical field
The present invention relates to genome editing technique field, specially a kind of genome edit methods based on immunocyte and
Purposes.
Background technique
Gene editing technology refers to that the mankind is allowed to carry out " editor " to target gene, and specific DNA segment is struck in realization
It removes, be added.And CRISPR/Cas9 technology since the advent of the world, just there are other unrivaled advantages of gene editing technology, skill
After art is continuously improved, be more believed in living cells it is most effective, most easily " edit " any gene.
Gene editing technology carries out precise manipulation to target gene, realizes site-directed point mutation, insertion, deletion, straight with this
It connects starting, close certain genes, or even directly Disease-causing gene edited in molecular level, modified, thus to unknown function
Gene carries out the technology of research and gene therapy.The most commonly used gene editing technology is to identify to target based on Cas9 recombinase
SgRNA sequence determines specified gene location, the gene editing being then oriented.Direct editing dcc gene can be passed through at present
Make its normal expression in body cell, changing genetic defect patient can not cure, can only rely on the status taken medicine all the life.Currently,
Gene editing technology obtains certain progress in the diseases such as tyrosinemia, β-thalassemia, has successfully cured trouble
There is the adult mice of tyrosinemia, which in vivo demonstrates its curative effect for treating genetic deficiency diseases in Adult Mammals
And safety, it is an important breakthrough of human gene therapy.
In order to preferably be edited to genome, it is proposed that a kind of genome edit methods based on immunocyte.
Summary of the invention
The present invention provides a kind of genome edit methods based on immunocyte and can effectively solve in above-mentioned background technique
The problem of.
To achieve the above object, the invention provides the following technical scheme: a kind of genome editing side based on immunocyte
Method, including design the genome of immunocyte, the genome edit tool of specified site sequence, immunocyte gene editing and
Fixed point gene knock-in integration and etc.；The gene editing of the immunocyte, which refers to, generates DNA single stranded gaps with based on nickase
Technology, be aided with a set of homologous recombination factors RecOFAR realize it is efficient, accurate, fixed point human primary cultured cells' gene
Group editor.
Further, the genome edit tool is Cas9 system.
Further, a kind of genome edit methods based on immunocyte, comprising the following steps:
A, the preparation of wild type Cas9 and the preparation of sgRNA, but it is not limited to Cas9 enzyme, it further include other any DNA mono-
Chain nickase；
B, potential nicks at positioning integration target target site；
C, foreign donor DNA profiling is designed；
D, preparation and the recombinant factor group RecOFAR in system of the present invention is provided；
E, turn human primary cultured cells using electricity and carry out pinpoint gene editing, transformation.
Further, the preparation of the wild type Cas9 and sgRNA: wild type Cas9 sequence is separately added into nuclear localization sequence,
Then SP6 promoter sequence is added in the upstream of whole section of sequence, is capped using the kit of in-vitro transcription and adds polyA's
MRNA obtains Cas9mRNA, freezes after being mixed with various combination spare；SgRNA synthesizes upstream by DNA and has T7 promoter sequence
It arranges and there are the downstream sequence of partial complementarity with upstream, obtain double-stranded DNA by PCR amplification, utilize the kit of in-vitro transcription
It obtains.
Further, potential nicks at the positioning integration target target site: the selected site nick, integration site are selected in
On nucleotide at the site nick or between the collaboration site nick.
Further, the design foreign donor DNA profiling: in upstream, there is the homologous sequence i.e. upstream of 1kb in the upstream of nick
Homology arm, in downstream, there is the homologous sequence i.e. downstream homology arm of 1kb in the downstream of nick；It is added at integration site and knocks in element；
Extracting plasmid by amplification becomes foreign donor DNA.
Further, each recombinant factor sequence is separately added into nuclear localization sequence, is then added in the upstream of whole section of sequence
SP6 promoter sequence is capped using the kit of in-vitro transcription and is added the mRNA of polyA, after being mixed with various combination-
80 DEG C freeze it is spare.
Further, the gene editing human immune cells: using electric shifting method by compound used in gene targeting press than
Example requires to import people's primary immune cells.
Further, the immunocyte is set as lymphocyte, Dendritic Cells, Monocytes/Macrophages, granulocyte or fertilizer
Maxicell.
Compared with prior art, beneficial effects of the present invention: the present invention significantly improves the safety of genome editor, reduces
Error rate；Adjustable point gene knock-in.
Specific embodiment
It, below will be to the embodiment of the present invention to keep the purposes, technical schemes and advantages of embodiment of the present invention clearer
In technical solution be clearly and completely described, it is clear that described embodiments are only a part of the embodiments of the present invention,
Instead of all the embodiments.Based on the embodiments of the present invention, those of ordinary skill in the art are not making creative labor
Every other embodiment obtained under the premise of dynamic, shall fall within the protection scope of the present invention.
The present invention provides a kind of genome edit methods based on immunocyte, including design immunocyte genome,
The genome edit tool of specified site sequence, the gene editing of immunocyte and fixed point gene knock-in integration and etc.；It is described
The gene editing of immunocyte refer to based on nickase generate DNA single stranded gaps technology, be aided with a set of homologous recombination factors
RecOFAR come realize it is efficient, accurate, fixed point human primary cultured cells genome editor.
Further, the genome edit tool is Cas9 system.
Further, a kind of genome edit methods based on immunocyte, comprising the following steps:
A, the preparation of wild type Cas9 and the preparation of sgRNA, but it is not limited to Cas9 enzyme, it further include other any DNA mono-
Chain nickase；
B, potential nicks at positioning integration target target site；
C, foreign donor DNA profiling is designed；
D, preparation and the recombinant factor group RecOFAR in system of the present invention is provided；
E, turn human primary cultured cells using electricity and carry out pinpoint gene editing, transformation.
Further, the preparation of the wild type Cas9 and sgRNA: wild type Cas9 sequence is separately added into nuclear localization sequence,
Then SP6 promoter sequence is added in the upstream of whole section of sequence, is capped using the kit of in-vitro transcription and adds polyA's
MRNA obtains Cas9mRNA, freezes after being mixed with various combination spare；SgRNA synthesizes upstream by DNA and has T7 promoter sequence
It arranges and there are the downstream sequence of partial complementarity with upstream, obtain double-stranded DNA by PCR amplification, utilize the kit of in-vitro transcription
It obtains.
Further, potential nicks at the positioning integration target target site: the selected site nick, integration site are selected in
On nucleotide at the site nick or between the collaboration site nick.
Further, the design foreign donor DNA profiling: in upstream, there is the homologous sequence i.e. upstream of 1kb in the upstream of nick
Homology arm, in downstream, there is the homologous sequence i.e. downstream homology arm of 1kb in the downstream of nick；It is added at integration site and knocks in element；
Extracting plasmid by amplification becomes foreign donor DNA.
Further, each recombinant factor sequence is separately added into nuclear localization sequence, is then added in the upstream of whole section of sequence
SP6 promoter sequence is capped using the kit of in-vitro transcription and is added the mRNA of polyA, after being mixed with various combination-
80 DEG C freeze it is spare.
Further, the gene editing human immune cells: using electric shifting method by compound used in gene targeting press than
Example requires to import people's primary immune cells.
Further, the immunocyte is set as lymphocyte, Dendritic Cells, Monocytes/Macrophages, granulocyte or fertilizer
Maxicell.
Finally, it should be noted that being not intended to restrict the invention the foregoing is merely preferred embodiment of the invention, to the greatest extent
Present invention has been described in detail with reference to the aforementioned embodiments for pipe, for those skilled in the art, still can be with
It modifies the technical solutions described in the foregoing embodiments or equivalent replacement of some of the technical features.It is all
Within the spirit and principles in the present invention, any modification, equivalent replacement, improvement and so on should be included in guarantor of the invention
Within the scope of shield.
Claims (9)
1. a kind of genome edit methods based on immunocyte, which is characterized in that including designing the genome of immunocyte, referring to
The genome edit tool of positioning point sequence, the gene editing of immunocyte and fixed point gene knock-in integration and etc.；It is described to exempt from
The gene editing of epidemic disease cell refer to based on nickase generate DNA single stranded gaps technology, be aided with a set of homologous recombination factors
RecOFAR come realize it is efficient, accurate, fixed point human primary cultured cells genome editor.
2. a kind of genome edit methods based on immunocyte according to claim 1, it is characterised in that: the gene
Group edit tool is Cas9 system.
3. a kind of genome edit methods based on immunocyte according to claim 1, it is characterised in that: including following
Step:
A, the preparation of wild type Cas9 and the preparation of sgRNA, but be not limited to Cas9 enzyme, further include other any DNA is single-stranded cuts
Mouth enzyme；
B, potential nicks at positioning integration target target site；
C, foreign donor DNA profiling is designed；
D, preparation and the recombinant factor group RecOFAR in system of the present invention is provided；
E, turn human primary cultured cells using electricity and carry out pinpoint gene editing, transformation.
4. a kind of genome edit methods based on immunocyte according to claim 3, it is characterised in that: described wild
The preparation of type Cas9 and sgRNA: wild type Cas9 sequence is separately added into nuclear localization sequence, is then added in the upstream of whole section of sequence
SP6 promoter sequence is capped and is added using the kit of in-vitro transcription the mRNA of polyA, Cas9 mRNA obtained, with not
It is spare with being frozen after combined hybrid；SgRNA synthesizes upstream with T7 promoter sequence by DNA and there are partial complementarities with upstream
Downstream sequence, obtain double-stranded DNA by PCR amplification, obtained using the kit of in-vitro transcription.
5. a kind of genome edit methods based on immunocyte according to claim 4, it is characterised in that: the positioning
Potential nicks at conformity goal target site: the selected site nick, integration site are selected at the site nick or cooperate with the site nick
Between nucleotide on.
6. a kind of genome edit methods based on immunocyte according to claim 5, it is characterised in that: the design
Foreign donor DNA profiling: in upstream, there are the homologous sequence i.e. upstream homology arm of 1kb, the downstream of nick in downstream in the upstream of nick
There is the homologous sequence i.e. downstream homology arm of 1kb；It is added at integration site and knocks in element；Extracting plasmid by amplification becomes external source
Donor dna.
7. a kind of genome edit methods based on immunocyte according to claim 6, it is characterised in that: described each heavy
Group factor sequence is separately added into nuclear localization sequence, and SP6 promoter sequence then is added in the upstream of whole section of sequence, is turned using external
The kit of record be capped and add polyA mRNA, after being mixed with various combination -80 DEG C freeze it is spare.
8. a kind of genome edit methods based on immunocyte according to claim 6, it is characterised in that: the gene
Editor human immune cells: require importing people's primary immune thin in proportion compound used in gene targeting using electric shifting method
Born of the same parents.
9. a kind of genome edit methods based on immunocyte according to claim 1, it is characterised in that: described immune
Cell is set as lymphocyte, Dendritic Cells, Monocytes/Macrophages, granulocyte or mast cell.
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| CN201910764807.5A CN110452930A (en) | 2019-08-19 | 2019-08-19 | A kind of genome edit methods and purposes based on immunocyte |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108949830A (en) * | 2018-08-03 | 2018-12-07 | 福州大学 | A method of realizing genome editor, pinpoint gene knock-in in fish |
| CN108998406A (en) * | 2018-08-03 | 2018-12-14 | 福州大学 | A kind of human primary cultured cells' genome editor, fixed point gene knock-in method |
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2019
- 2019-08-19 CN CN201910764807.5A patent/CN110452930A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108949830A (en) * | 2018-08-03 | 2018-12-07 | 福州大学 | A method of realizing genome editor, pinpoint gene knock-in in fish |
| CN108998406A (en) * | 2018-08-03 | 2018-12-14 | 福州大学 | A kind of human primary cultured cells' genome editor, fixed point gene knock-in method |
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