CN104805118A - Method for targeted knockout of specific gene of Suqin yellow chicken embryonic stem cell - Google Patents
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Abstract
The invention discloses a method for targeted knockout of a specific gene of a Suqin yellow chicken embryonic stem cell. The method comprises the following steps: firstly, checking out an exon sequence of a gene, cloning the gene, sequencing to obtain a complete exon sequence, designing a CRISPR/Cas9 knockout target site on the sequence as gRNA, and constructing a CRISPR/Cas9 dual-promoter knockout vector; performing SSA activity detection on the constructed Cas9 vector, transfecting a control group with an empty vector, detecting a luciferase signal, and obtaining a result that the more the luciferase activity is increased relative to the control group, the higher the gRNA shearing activity is shown; transfecting the CRISPR/Cas9 vector which is high in SSA activity with ESCs, using flow cytometry to screen a GFP-positive cell, extracting genome DNA, and designing a primer.
Description
Technical field:
The present invention relates to Stem Cell Engineering field, particularly relate to and a kind ofly based on CRISPR/Cas9 technology, the method that target knocks out is carried out to the yellow chicken embryonic stem cells specific gene of Soviet Union fowl.
Technical background:
Current genome editing technique mainly contains three kinds of methods, Zinc finger nuclease (zinc finger endonuclease, ZFN), class activating transcription factor effector nuclease (Transcription Activator-Like EffectorNuclease, and the short palindrome tumor-necrosis factor glycoproteins of the regular intervals of cluster and genes involved (clusteredregularly interspaced short palindromic repeats/CRISPR-associated, CRISPR/Cas) TALEN).These three kinds of methods are all passed through to produce double-strand otch on external source DAN target site, thus induce homologous recombination repair and non-homologous end joining, and then realize genomic editor.
ZFN structure comprises zinc finger protein (Zinc finger protein, ZFP) structural domain and Fok I cutting structure territory, wherein Fok I cutting structure territory comes from a kind of IIS type restriction enzyme, when Fok I enzyme is Dimerized, play the target site of its nicking activity cutting DNA, form double-strand break (double-stranded breaks, DSB), thus the repair mechanism in inducing cell, i.e. homologous recombination repair (homology-directed repair, HDR) or nonhomologous end link (non-homologous end-joining, NHEJ), thus realize genomic target and knock out (knock-out) or knock in (knock-in), but also there is design suffering at present in ZFN, miss the target serious, the problems such as technical monopoly,
And TALEN structure comprises TALE (transcription activator-like effector) structural domain and Fok I endonuclease enzyme domains two parts, its mechanism of action is consistent with ZFN, also there is multinomial defect at present, such as cost is high, operability difficulty is large, cytotoxicity etc.;
Main based on typical Type II CRISPR/Cas in CRISPR/Cas system, structure is divided into three part tracrRNA be positioned at 5 ' end, CRISPR sequence is positioned at 3 ' end, middle is a series of Cas gene family, wherein Cas9 is core protein, and this II type system therefore can be claimed to be CRISPR/Cas9 system.When exogenous DNA array enter bacterium or archeobacteria containing in the cell of CRISPR, being similar to Cas1 nucleic acid binding protein and can mediating this complex body and exogenous DNA array combines in CRISPR complex body, then be similar to Cas2 endonuclease in this complex body to cut exogenous DNA array, form the small segment that numerous about 17-84bp is unequal, one of them fragment can be integrated between the leader sequence of CRISPR and first tumor-necrosis factor glycoproteins under the effect of associated protein, form a new intervening sequence, the sequence that invader is identical with intervening sequence is called protospacer.When foreign DNA is invaded again, the intervening sequence mated with its protospacer complementation, and the part tumor-necrosis factor glycoproteins on both sides transcribes formation precursor crRNA (pre-crRNA).After having transcribed, tracrRNA and pre-crRNA forms dimer, ribonucleoprotein complexes is formed again with Cas9 protein binding, be combined with foreign DNA, scanning, searching target sequence, the intervening sequence of crRNA and target sequence carry out complementary pairing, and the specific position of foreign DNA complementary pairing is sheared by nucleoprotein complex.
The target practice efficiency of CRISPR/Cas is higher, reaches as high as 80%, and target site flexible design, convenience, vector construction is simple; Be different from the mode of protein identification DNA in ZFN or TALEN, the identification of CRISPR/Cas system to target sequence is in the mode of RNA and DNA base pairing, can reduce the probability missed the target like this, reduce cytotoxicity, but is not that representative can not produce; In addition, the design comparatively in ZFN and TALEN, CRISPR/Cas is more simple, cheap, and generally common experiment also can Self-operating; Finally, multiple gene of can simultaneously practicing shooting a little exactly that CRISPR/Cas is maximum, and every many target sites only need many gRNA plasmids.
Have superiority and also must can there is limitation, such as CRISPR/Cas technology is also not yet ripe at present, need to design the higher gRNA plasmid of specificity, the serious effect and not building on the stem cell being without successful precedent etc. of missing the target, but believe the development along with CRISPR/Cas, these difficult problems can be solved at last.
Summary of the invention:
The invention discloses and a kind ofly based on CRISPR/Cas9 technology, the method that target knocks out is carried out to the yellow chicken embryonic stem cells specific gene of Soviet Union fowl: the exon sequence first inquiring gene, clone gene, order-checking, obtain complete exon sequence, and in this sequence, design CRISPR/Cas9 knock out target site as gRNA, build CRISPR/Cas9 double-promoter knockout carrier, wherein U6 promotor in fowl source starts gRNA expression, T7 promotor starts Cas9 genetic expression, and inserts GFP albumen in the carrier as reporter gene; The Cas9 carrier built carries out SSA Activity determination, terminator and target site are inserted in two fluorescence Luciferase carrier, after target site is positioned at terminator, cotransfection CRISPR/gRNA carrier, and the new luciferase reporter gene that builds and internal reference renilla plasmid, control group transfection empty carrier, detects luciferase signal, luciferase activity is more relative to control group growth, then prove that gRNA shear active is higher; The CRISPR/Cas9 carrier transfection ESCs of high SSA activity will be had, flow cytometry filters out GFP positive cell, extract genomic dna, design primer, clone the fragment of each 250bp before and after target site, use T7E1 enzyme to carry out Activity determination, the activated knockout carrier of tool is connected with carrier T, check order after shaking bacterium, calculate concrete activity value.
The present invention includes following steps:
(1) acquisition of goal gene
In ncbi database, the CDS sequence of goal gene in jungle fowl is inquired about according to gene order number, design Auele Specific Primer, the strand cDNA formed with the yellow chicken tissues of fowl of reviving or cell RNA reverse transcription clones for template and obtains the CDS sequence that in the yellow chicken of Soviet Union fowl, goal gene is complete, obtains complete goal gene exon sequence by comparison;
(3) gRNA design and synthesis
Find the PAM sequence in the exon sequence obtained, using the base of about 20bp before PAM sequence as target sequence, all target sequences, in full-length genome comparison, synthesize as gRNA without the target site of homology;
(4) CRISPR/Cas9 gene knockout carrier builds
Based on VK001-08 carrier, carrier carries out vector modification, starts the expression of gRNA using fowl source U6 as promotor, and T7 starts the expression starting Cas9 enzyme most, inserts GFP fluorogene as reporter gene simultaneously.
(5) CRISPR/Cas9 gene knockout carrier SSA Activity determination
Terminator and target site are inserted in Luciferase carrier, after target site is positioned at terminator, cotransfection CRISPR/gRNA carrier, and the new luciferase reporter gene that builds and internal reference renilla plasmid, control group transfection empty carrier, detect luciferase signal, luciferase activity is more relative to control group growth, then prove that gRNA shear active is higher;
(6) CRISPR/Cas9 gene knockout carrier endogenous activity detects
The s-generation ESC that the transfection of shear active higher CRISPR/Cas9 gene knockout carrier is in good condition, after 48 hours, flow cytometry filters out GFP positive cell, extract genomic dna, design primer, clone each 250bp before and after target site and amount to about 500bp fragment, use T7E1 enzyme to carry out Activity determination, knock out activity if exist, non-matching DNA fragmentation will be produced, can by non-matching restriction endonuclease--T7 endonuclease I shears; If do not undergo mutation, by generation pairing DNA fragmentation, and cannot by non-matching restriction endonuclease--T7 endonuclease I shears;
(7) CRISPR/Cas9 gene knockout carrier knocks out Efficiency testing
The s-generation ESC that the transfection of CRISPR/Cas9 gene knockout carrier is in good condition, after 48 hours, flow cytometry filters out GFP positive cell, extract genomic dna, design primer, clone each 250bp before and after target site and amount to about 500bp fragment, carry out TA clone, choose bacterium order-checking and contrast with original series, calculate the concrete efficiency of CRISPR/Cas9 gene knockout carrier, method of calculation: (bacterium liquid number/all order-checking bacterium liquid number * 100% of sequence mutates), notice that the bacterium liquid of order-checking needs abundant;
Superiority of the present invention is: present method is simple, repeatable strong, can carry out in common lab.Use the method can fast, the efficient checking completing gene gene function in chicken embryonic stem cells at short notice.
Embodiment
One carries out target knockout technique based on CRISPR/Cas9 technology to the yellow chicken embryonic stem cells specific gene of Soviet Union fowl, comprises the steps:
(1) acquisition of goal gene
In ncbi database, the CDS sequence of goal gene in jungle fowl is inquired about according to gene order number, design Auele Specific Primer, the strand CDNA formed with the RNA reverse transcription of the yellow chicken PGC of fowl of reviving clones for template and obtains the CDS sequence that in the yellow chicken of Soviet Union fowl, goal gene is complete, obtains complete goal gene exon sequence by comparison;
(4) gRNA design and synthesis
Find the PAM sequence in the exon sequence obtained, using the base of about 20bp before PAM sequence as target sequence, all target sequences, in full-length genome comparison, synthesize as gRNA without the target site of homology;
(5) CRISPR/Cas9 gene knockout carrier builds
Based on VK001-08 carrier, carrier carries out vector modification, starts the expression of gRNA using fowl source U6 as promotor, and T7 starts the expression starting Cas9 enzyme most, inserts GFP fluorogene as reporter gene simultaneously.
(6) CRISPR/Cas9 gene knockout carrier SSA Activity determination
Terminator and target site are inserted in Luciferase carrier, after target site is positioned at terminator, cotransfection CRISPR/gRNA carrier, and the new luciferase reporter gene that builds and internal reference renilla plasmid, control group transfection empty carrier, detect luciferase signal, luciferase activity is more relative to control group growth, then prove that gRNA shear active is higher;
(7) CRISPR/Cas9 gene knockout carrier endogenous activity detects
The s-generation ESC that the transfection of shear active higher CRISPR/Cas9 gene knockout carrier is in good condition, after 48 hours, flow cytometry filters out GFP positive cell, extract genomic dna, design primer, clone each 250bp before and after target site and amount to about 500bp fragment, use T7E1 enzyme to carry out Activity determination, knock out activity if exist, non-matching DNA fragmentation will be produced, can by non-matching restriction endonuclease--T7 endonuclease I shears; If do not undergo mutation, by generation pairing DNA fragmentation, and cannot by non-matching restriction endonuclease--T7 endonuclease I shears;
(8) CRISPR/Cas9 gene knockout carrier knocks out Efficiency testing
The s-generation ESC that the transfection of CRISPR/Cas9 gene knockout carrier is in good condition, after 48 hours, flow cytometry filters out GFP positive cell, extract genomic dna, design primer, clone each 250bp before and after target site and amount to about 500bp fragment, carry out TA clone, choose bacterium order-checking and contrast with original series, calculate the concrete efficiency of CRISPR/Cas9 gene knockout carrier, method of calculation: (bacterium liquid number/all order-checking bacterium liquid number * 100% of sequence mutates), notice that the bacterium liquid of order-checking needs abundant.
Claims (1)
1. a Soviet Union fowl yellow chicken embryonic stem cells specific gene target knockout technique, is characterized in that: comprise the steps:
(1) acquisition of goal gene
In ncbi database, the CDS sequence of goal gene in jungle fowl is inquired about according to gene order number, design Auele Specific Primer, the strand CDNA formed with the RNA reverse transcription of the yellow chicken PGC of fowl of reviving clones for template and obtains the CDS sequence that in the yellow chicken of Soviet Union fowl, goal gene is complete, obtains complete goal gene exon sequence by comparison;
(2) gRNA design and synthesis
Find the PAM sequence in the exon sequence obtained, using the base of about 20bp before PAM sequence as target sequence, all target sequences, in full-length genome comparison, synthesize as gRNA without the target site of homology;
(3) CRISPR/Cas9 gene knockout carrier builds
Based on VK001-08 carrier, carrier carries out vector modification, starts the expression of gRNA using fowl source U6 as promotor, and T7 starts the expression starting Cas9 enzyme most, inserts GFP fluorogene as reporter gene simultaneously.
(4) CRISPR/Cas9 gene knockout carrier SSA Activity determination
Terminator and target site are inserted in Luciferase carrier, after target site is positioned at terminator, cotransfection CRISPR/gRNA carrier, and the new luciferase reporter gene that builds and internal reference renilla plasmid, control group transfection empty carrier, detect luciferase signal, luciferase activity is more relative to control group growth, then prove that gRNA shear active is higher;
(5) CRISPR/Cas9 gene knockout carrier endogenous activity detects
The s-generation ESC that the transfection of shear active higher CRISPR/Cas9 gene knockout carrier is in good condition, after 48 hours, flow cytometry filters out GFP positive cell, extract genomic dna, design primer, clone each 250bp before and after target site and amount to about 500bp fragment, use T7E1 enzyme to carry out Activity determination, knock out activity if exist, non-matching DNA fragmentation will be produced, can by non-matching restriction endonuclease--T7 endonuclease I shears; If do not undergo mutation, by generation pairing DNA fragmentation, and cannot by non-matching restriction endonuclease--T7 endonuclease I shears;
(6) CRISPR/Cas9 gene knockout carrier knocks out Efficiency testing
The s-generation ESC that the transfection of CRISPR/Cas9 gene knockout carrier is in good condition, after 48 hours, flow cytometry filters out GFP positive cell, extract genomic dna, design primer, clone each 250bp before and after target site and amount to about 500bp fragment, carry out TA clone, choose bacterium order-checking and contrast with original series, calculate the concrete efficiency of CRISPR/Cas9 gene knockout carrier, method of calculation: (bacterium liquid number/all order-checking bacterium liquid number * 100% of sequence mutates), notice that the bacterium liquid of order-checking needs abundant.
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