CN105331627B - A method of prokaryotic gene group editor is carried out using endogenous CRISPR-Cas system - Google Patents
A method of prokaryotic gene group editor is carried out using endogenous CRISPR-Cas system Download PDFInfo
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Abstract
A kind of method for carrying out prokaryotic gene group editor using endogenous CRISPR-Cas system of the present invention.When carrying out genome editor to the prokaryotes containing endogenous CRISPR-Cas system, only one need to be constructed while carrying editor's plasmid of artificial CRISPR cluster and donor dna, genome is occurred in endogenous CRISPR system to reach by homologous recombination the editor to genome after DNA interferes.Its biggest advantage is: wide using host range, all bacteriums containing endogenous CRISPR-Cas system and archaeal can operate；It can be used for a variety of edit modes, missing, insertion and point mutation etc. can operate；Higher editorial efficiency, screening positive rate is high, and background is low；Process is simple, and the time cycle is short, mitigates the workload of prokaryotic gene group editor significantly.
Description
Technical field
The invention belongs to genomics and genetic engineerings and field of biotechnology, and in particular to a kind of using endogenous
The method of CRISPR-Cas system progress prokaryotic gene group editor.
Background technique
CRISPR-Cas system resists the acquired immune system of the external sources such as virus invasion nucleic acid as a kind of prokaryotes,
Be widely present in about 90% archaeal and 40% bacterium (Van der Oost J et al., 2014；Barrangou R
et al.,2014).CRISPR-Cas system is divided into three main Types: I type, II type and type III；They have one respectively
A significant albumen: Cas3, Cas9 and Cas10 (Makarova KS et al., 2011).II type CRISPR system only needs
Mono- albumen of Cas9 and crRNA and trans-acting RNA exercise DNA interference activity (Deltcheva E et al.,
2011；Gasiunas G et al.,2012).Therefore simple II type CRISPR system is developed to eukaryotic gene group volume
Volume tool (Jinek M et al., 2012；Wang H et al., 2013), and it is widely used in different eucaryote and thin
Bacterium (Doudna JA et al., 2014；Sander JD et al.,2014；Hsu PD et al.,2014；Selle K et
al.,2015)。
In the past ten years, each laboratory establishes effective genetic manipulation system in some archaeal type sepecies
With tool (Leigh JA et al., 2011).Nevertheless, the genetics research of archaeal is because of its unique growth conditions, growth
Very challenging (the Valentine DL et that still seems slowly and to the factors such as most antibiotics are insensitive
al.,2007).Therefore, application of the genome edit methods of CRISPR system in archaeal is occupy still to require study and open
Hair.
That utilized currently based on the genome editing of CRISPR system is the i.e. CRISPR/ of II type CRISPR system
Cas9 system, and there is also many limitations for CRISPR/Cas9 system.Firstly, CRISPR/Cas9 is there may be undershooting-effect,
Studies have found that Cas9 albumen may allow between crRNA and target sequence, there are a degree of mispairing, and these
Quantity and position that mispairing occurs would necessarily affect the specificity of gene editing.Secondly, utilizing the CRISPR/Cas9 system of external source
System needs to carry out Cas9 albumen the Optimizing Reconstruction suitable for host cell, and need simultaneously in the cell expression Cas9 albumen and
The effect of sgRNA (guide RNA) competence exertion, it is more complicated using program.In addition, in the extreme biology of some growth conditions,
Environment intracellular may influence whether the activity of Cas9 albumen, to limit CRISPR/Cas9 systematic difference.
The present invention is in view of the above problems, we have proposed do genome volume using the CRISPR system of prokaryotes itself
Volume.The DNA interference activity and homologous recombination of CRISPR system is utilized in this method simultaneously, therefore largely avoids and miss the target
Effect enhances editor's specificity.This method only needs to construct one while carrying the editor of artificial CRISPR cluster and donor dna
Plasmid, process is simple, and the operation cycle is short.What this method utilized is the CRISPR system of prokaryotes itself, all containing endogenous
The bacterium of CRISPR-Cas system and archaeal are applicable in.
Sulfolobus solfataricus is the model organism of CRISPR research, about sulfolobus solfataricus S.solfataricus P2 and
I type and type III CRISPR-Cas systematic research show that it all has DNA in Iceland sulfolobus solfataricus S.islandicusREY15A
And (or) RNA interference activity (Manica A et al., 2011；Manica A et al.,2013；Zebec Z et al.,
2014；Gudbergsdottir S.,2011).We have found that the I-A type in Iceland sulfolobus solfataricus S.islandicusREY15A
DNA interference activity (Deng L et al., 2013) is all had with III-B Cmr- α." CCN " and " TCN " is Iceland's sulfolobus solfataricus
Required two PAMs (protospacer-adjacent the motifs) (Lillestol of I-ACRISPR system DNA interference activity
R.K et al.,2009).And the DNA interference activity that III-B type CRISPR is mediated does not need PAM sequence, but it is relied on
In transcription, and need to ensure crRNA 5 ' hold repeat sequence and the mispairing of protospacer upstream sequence (Deng L et al.,
2013)。
Summary of the invention
The purpose of the present invention is to provide the prokaryotes containing CRISPR-Cas system in endogenous editor's prokaryotes base
Because of the application in group, the prokaryotes be containing endogenous I type or type III CRISPR-Cas system, or simultaneously contain endogenous I
The bacterium of type and type III CRISPR-Cas system.
Prokaryotic gene is carried out using endogenous CRISPR-Cas system it is another object of the present invention to provide a kind of
The method of group editor carries editor's plasmid of artificial CRISPR cluster and donor dna by building one, in endogenous CRISPR simultaneously
System is to the editor reached by homologous recombination after genome generation DNA interference to genome.
In order to achieve the above object, the present invention takes following technical measures:
The claimed content of the present invention includes that the prokaryotes containing CRISPR-Cas system are raw in endogenous editor's protokaryon
Application in object genome；Application of the endogenous CRISPR-Cas system in editor's prokaryotic gene group.
A method of prokaryotic gene group editor is carried out using endogenous CRISPR-Cas system, comprising the following steps:
1) genome editor plasmid is constructed:
Intend editing area in prokaryotic gene group and chooses one section of sequence as protospacer, that is, target site, root
The primer of two reverse complementals is designed according to protospacer, sequence is respectively forward primer: 5 '-AAAG-Nn- 3 ', reversely draw
Object: 5 '-TAGC-N 'n- 3 ', wherein NnAnd N 'nFor reverse complementary sequence, N and N ' indicate that base A, T, G or C, n are indicated
The base number of protospacer；Above-mentioned two primer annealings are formed to the double-stranded DNA with cohesive end, i.e. spacer piece
Section；Artificial CRISPR carrier pSe-Rp is handled by restriction enzyme BspMI digestion, then and with cohesive end
Spacer segment enzyme connects, and obtains the artificial CRISPR plasmid (pAC) that can generate mature crRNA；Again will comprising mutant nucleotide sequence and
Homologous donor DNA segment is inserted on above-mentioned pAC plasmid with target site both ends in host cell gene group, obtains genome
It edits plasmid (pGE)；
The donor DNA segment passes through SOE-PCR's by the mutant nucleotide sequence of two homologous fragments in left and right and medium design
Method expands.
2) acquisition of mutant strain: the artificial CRISPR cluster after pGE plasmid electricity is transferred to prokaryotes competent cell, on plasmid
Pre-crRNA is transcribed out, pre-crRNA is processed to mature crRNA in the cell；The CRISPR- of crRNA and cellular endogenous
Cas albumen forms crRNP complex, is matched by the target dna chain on crRNA and host cell gene group to identify target position
Point is cut；Homologous recombination occurs for donor DNA segment and target site two sides sequence on subsequent plasmid, and then obtains gene
Group editor's mutant strain.
In procedure described above, the prokaryotes be containing endogenous I type or type III CRISPR-Cas system, or
Contain bacterium or the archaeal of endogenous I type and type III CRISPR-Cas system simultaneously, including but not limited to Iceland's sulfolobus solfataricus is (excellent
Select protokaryon Iceland sulfolobus solfataricus S.islandicusREY15A), for all bacteriums containing endogenous CRISPR-Cas system and Gu
Bacterium, such as: Escherichia coli Escherichia coli, staphylococcus epidermis Staphylococcus epidermidis, corruption
Shiva bacterium Shewanellaputrefaciens, thermophilic bacteria Thermus thermophilus, streptococcus thermophilus
Streptococcus thermophilus, pyrococcus furiosus Pyrococcusfuriosus, sulfolobus acidocaldarius
Sulfolobusacidocaldarius, sulfolobus solfataricus Sulfolobussolfataricus, hot autotrophy methane hot rod
Bacterium Methanothermobacterthermautotrophicus, the richly endowed bacterium Haloferaxvolcanii of salt, black shank bacterium
Pectobacteriumatrosepticum, Bacterium diphtheriae Corynebacterium diphtheriae etc..
Above-described genome editor includes but is not limited to missing, insertion and point mutation.
Compared with prior art, the invention has the following advantages that
The method provided by the invention for carrying out prokaryotic gene group editor using endogenous CRISPR-Cas system, only needs structure
It builds one while carrying editor's plasmid of artificial CRISPR cluster and donor dna, DNA is occurred to genome in endogenous CRISPR system
The editor to genome is reached by homologous recombination after interference.Its biggest advantage is: using host range it is wide, it is all containing
The bacterium of endogenous CRISPR-Cas system and archaeal can operate；It can be used for a variety of edit modes, missing, insertion and point mutation etc.
It can operate；Higher editorial efficiency, screening positive rate is high, and background is low；Process is simple, and the time cycle is short, significantly mitigation protokaryon
The workload of biological genome editor.
Detailed description of the invention
Fig. 1 is that the present invention is shown using the principle for the method that endogenous CRISPR-Cas system carries out prokaryotic gene group editor
It is intended to.
A is using the endogenous CRISPR system of Iceland's sulfolobus solfataricus S.islandicusREY15A in embodiment 1 to it in Fig. 2
LacS gene carries out accurate deletion mutation design diagram；
B is that wild type and transformant schematic diagram are compared in X-gal staining analysis in embodiment 1 in Fig. 2；
C is that PCR detects whether transformant is 43bp deletion mutation strain schematic diagram in embodiment 1 in Fig. 2；
D is that wild type and saltant type lacS schematic diagram are compared in DNA sequencing analysis in embodiment 1 in Fig. 2.
A is that the endogenous III-B type of Iceland's sulfolobus solfataricus S.islandicusREY15A is utilized in the embodiment of the present invention 2 in Fig. 3
CRISPR system carries out 6 × His label insertion mutation design diagram to the C-terminal of its Cmr-2 α albumen；
B is that DNA sequencing compares wild type cmr-2 α and carries 6 × His label saltant type cmr-2 α in embodiment 2 in Fig. 3
Schematic diagram；
C is the Cmr-2 α albumen for carrying 6 × His label in embodiment 2 with the anti-western blot identification of His mouse in Fig. 3
Schematic diagram；
In Fig. 3 D be embodiment 2 in Cmr2 α-His bacterial strain with carrying 6 × His label Cmr-2 α albumen do it is pure altogether
Change schematic diagram.
Fig. 4 is that pGE-2 α-His electricity turns Δ cas3/cas7 and Δ cas3 competent cell in embodiment 2, and PCR is examined and turned
Beggar's schematic diagram；
A is the design diagram of PCR the primer in embodiment 2 in Fig. 4；
B is that each 12 transformant PCR of two deletion mycopremnas verify electrophoretogram in embodiment 2 in Fig. 4.
Fig. 5 is Cmr-2 α (SiRe-0894), Cmr-2 β (SiRe-0598) and their homologous egg in the embodiment of the present invention 3
White PyrococcusfuriosusPF1129 carries out N-terminal sequence alignment schematic diagram；
A is that four conservative amino acid are selected as quasi- mutational site schematic diagram in embodiment 3 in Fig. 5；
B is that the endogenous I-A type CRISPR system of Iceland's sulfolobus solfataricus S.islandicusREY15A is utilized in embodiment 3 in Fig. 5
System carries out multisite mutation design diagram to its Cmr-2 α albumen HD domain；
C is that DNA sequencing shows tri- kinds of saltant type schematic diagrames of Cmr-2 α albumen HD domain in embodiment 3 in Fig. 5.
Specific embodiment
The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention..If not specified, embodiment
In experimental implementation routinely experiment condition or according to material, the condition of reagent manufacturer specification suggestion.
Embodiment 1:
A method of prokaryotic gene group editor is carried out using endogenous CRISPR-Cas system, comprising the following steps:
The present embodiment utilizes Iceland's sulfolobus solfataricus S.islandicusREY15A endogenous (I-A type CRISPR system and III-B
Type CRISPR system) CRISPR system be illustrated for accurate deletion mutation to its lacS gene:
1. editing the building of plasmid
(1) in Iceland sulfolobus solfataricus S.islandicusREY15A beta-galactosidase gene lacS (SIRE_RS11295)
It is upper choose+933 to+972 totally 40 bases as protospacer, 5 '-AGTGTAGTAATTAAC of reverse complementary sequence
ACCAATCCAGTCTAACCTACCCCTT-3 ', close to a CCT-PAM (Protospacer Adjacent Motif), because
This can be by I-A type CRISPR system target；Simultaneously again since 5 ' terminal sequence of crRNA is wrong with corresponding target site sequence
Match, so also can be simultaneously by III-B type CRISPR system target.Two primers are designed based on this protospacer
(LacS-E-SpF/LacS-E-SpR) (table 1), two primers generate the spacer piece that cohesive end is contained at both ends by annealing
Section；
Primer sequence used in 1. present invention of table
Black underscore mark is restriction enzyme site in upper table, forms four base quilts of spacer cohesive end
Black overstriking is shown；The mutational site Cmr-2 α HD domain is identified by italic font.
(2) artificial CRISPR carrier pSe-Rp (Peng, 2015) is handled through BspMI digestion, digestion products with it is above-mentioned
Spacer segment enzyme connects, and obtains artificial CRISPR plasmid；
(3) two SOE PCR primers (LacS-E-SOEF/LacS-E-SOER) that 43bp is lacked on lacS gene are designed
With two primers (LacS-E-SalIF/LacS-E-NotIR) (table 1) for being respectively provided with SalI and NotI restriction enzyme site, SOE is used
The method of PCR obtains the donor DNA segment for homologous recombination；
(4) donor DNA segment and above-mentioned artificial CRISPR plasmid use SalI and NotI digestion respectively, and then enzyme even converts,
Obtain editor plasmid pGE-lacS1；
2. the screening of mutant strain
(1) 500ng pGE-lacS1 plasmid electricity is transferred to Iceland's sulfolobus solfataricus S.islandicusREY15A competent cell,
It is coated with SCV (0.2%sucrose, 0.2%casamino acids plus 1%vitamin solution) solid medium,
78 DEG C are cultivated 7 days, and picking transformant was in 5ml SCV fluid culture medium culture three days；
(2) 90 μ l bacterium solutions is respectively taken to add 78 DEG C of one hours of incubation of 10 μ l X-gal (20mg/ml), observing response body respectively
It is color；
(3) PCR detection then is carried out with (F1/R1 and F2/R2) (table 1) respectively, it is found that 17 transformants of picking contain
There is pGE-lacS1 plasmid, while wherein 43bp missing has occurred in the lacS gene of 16 transformants, 1 is escape mutants, and
By primer (LacS-Seq-F/LacS-Seq-R) (table 1) amplifying genom DNA sequence verification, the volume of this method as the result is shown
Efficiency is collected 90% or more.
Embodiment 2:
A method of prokaryotic gene group editor is carried out using endogenous CRISPR-Cas system, comprising the following steps:
The embodiment of the present invention is with the endogenous III-B type CRISPR system of Iceland sulfolobus solfataricus S.islandicusREY15A to it
For the C-terminal of Cmr-2 α albumen carries out 6 × His label insertion mutation, it is illustrated.
1. editing the building of plasmid
(1) cmr-2 α gene (SIRE_ is chosen on Iceland's sulfolobus solfataricus S.islandicusREY15A genome
RS04505 totally 32 bases are reversed as protospacer for preceding 7 bases of last 25 bases and cmr-3 α gene)
Complementary series is 5 '-AATACATGTTTGCTCACCTTAAGTAAGATACT-3 ', and the front does not have CCN-PAM, thus only has
The DNA of III-B type CRISPR interferes activity.Two primer (2 α-His-SpF/2 α-His- are designed based on this protospacer
SpR) (table 1), two primers generate the spacer segment that cohesive end is contained at both ends by annealing；
(2) artificial CRISPR carrier pSe-Rp is handled through BspMI digestion, and digestion products and above-mentioned spacer segment enzyme connect,
Obtain artificial CRISPR plasmid；
(3) two SOE PCR primers (2 α-His-SOEF/2 α-His-SOER) comprising six histidine coding are designed
With two primers (2 α-His-SalIF/2 α-His-NotIR) (table 1) for being respectively provided with SalI and NotI restriction enzyme site, SOE is used
The method of PCR obtains the donor DNA segment for homologous recombination；
(4) donor DNA segment and above-mentioned artificial CRISPR plasmid use SalI and NotI digestion respectively, and then enzyme even converts,
Obtain editor plasmid pGE-2 α-His；
2. the screening of mutant strain
(1) it is thin to be transferred to Iceland's sulfolobus solfataricus S.islandicusREY15A competence for 500ng pGE-2 α-His plasmid electricity
Born of the same parents are coated with SCV (0.2%sucrose, 0.2%casamino acids plus 1%vitamin solution) solid culture
Base, 78 DEG C are cultivated 7 days, and picking transformant was in 5ml SCV fluid culture medium culture three days；
(2) sequence verification is carried out with primer (2 α-Seq-F/2 α-Seq-R) (table 1) amplification cmr-2 α gene, as the result is shown
Transformant is the mutant strain for being successively inserted into 6 × His label；
(3) counter-selection is carried out to insertion mutation strain with the SCV culture medium containing 5-FOA, edits plasmid pGE-2 α-His to eliminate；
The copurification of 3.His tag fusion protein and detection
3L SCV+U (uracil) culture medium culture Cmr2 α-His bacterial strain, to OD600When reaching 0.4, collects thallus and lay equal stress on
It is suspended from 50mM PBS buffer solution, high speed centrifugation takes supernatant after ultrasonic disruption, and it is small to be sufficiently mixed one with 500 μ l Ni sepharose 4Bs
When after cross column, then wash column with 20mM imidazoles PBS, 60mM imidazoles PBS respectively, finally repeat to elute with 2ml 200mM imidazoles PBS
Ni column twice, analyze and do western blot detection with His mouse is anti-respectively after SDS-PAGE by silver staining.
7 transformants are had detected, 7 are insertion mutation strain.
4. we use editor plasmid pGE-2 in order to further exclude the influence of I-A type CRISPR system in the present embodiment
α-His electricity turns two and has lacked the active Iceland's sulfolobus solfataricus S.islandicusREY15A competent cell of I type CRIPSR: Δ
Cas3/cas7 and Δ cas3,12 transformants of each picking carry out PCR verifying, and obtained insertion mutation strain ratio is respectively 11/12
With 12/12 (Fig. 4), further illustrate that this method success rate reaches 90% or more.
Embodiment 3:
A method of prokaryotic gene group editor is carried out using endogenous CRISPR-Cas system, comprising the following steps:
The present embodiment is to utilize the endogenous I-A type CRISPR system of Iceland's sulfolobus solfataricus S.islandicusREY15A to it
For Cmr-2 α albumen HD domain carries out multisite mutation, it is illustrated.
1. editing the building of plasmid
(1) cmr-2 α gene N-terminal HD domain is chosen on Iceland's sulfolobus solfataricus S.islandicusREY15A genome
40 bases are chosen at four conserved amino acid H/D/K/I as protospacer, 5 '-CGACCCTCCTTGGA of sequence
AGGCATGGGTAATTACAAGGAATATT-3 ', the front have CCA-PAM.Two primers are designed based on this protospacer
(2 α-HDmut-SpF/2 α-HDmut-SpR), two primers generate the spacer segment that cohesive end is contained at both ends by annealing；
(2) artificial CRISPR carrier pSe-Rp is handled through BspMI digestion, and digestion products and above-mentioned spacer segment enzyme connect,
Obtain artificial CRISPR plasmid；
(3) two SOE PCR primer (2 α-HDmut-SOEF/2 α-comprising four mutating acid coding are designed
HDmut-SOER two primers (2 α-HDmut-SalIF/2 α-HDmut- of SalI and NotI restriction enzyme site are respectively provided with) and
NotIR) (table 1) obtains the donor DNA segment for homologous recombination with the method for SOE PCR；
(4) donor DNA segment and above-mentioned artificial CRISPR plasmid use SalI and NotI digestion respectively, and then enzyme even converts,
Obtain editor plasmid pGE-2 α HD；
2. the screening of mutant strain
(1) 500ng pGE-2 α HD plasmid electricity is transferred to Iceland's sulfolobus solfataricus S.islandicusREY15A competent cell,
It is coated with SCV (0.2%sucrose, 0.2%casamino acids plus 1%vitamin solution) solid medium,
78 DEG C are cultivated 7 days, and picking transformant was in 5ml SCV fluid culture medium culture three days；
(2) sequence verification is carried out with primer (2 α-Seq-F/2 α-Seq-R) (table 1) amplification cmr-2 α gene, 7 turns of picking
Beggar is mutant strain, illustrates that editorial efficiency is 100%, due to the diversity of homologous recombination position, as the result is shown out three kinds it is prominent
Become type, be respectively only mutated two amino acid of H/D, mutation tetra- amino acid of tri- amino acid of H/D/K and H/D/K/I are sent out
Raw mutation, ratio 1:2:4.
Embodiment 4:
Using the method, in Iceland sulfolobus solfataricus S.islandicusHVE10/4, (one is widely used in virus and grinds for we
The bacterial strain studied carefully) in using its I-A type CRISPR system be successfully made the editor of lacS gene.The building and operation of pGE plasmid
Step is same as above embodiment 1.It is worth noting that S.islandicusHVE10/4 since transformation efficiency is very low, is implementing traditional base
Because being difficult success when knockout technique, and endogenous CRISPR system gene group edit methods provided by the invention are utilized, picking turns
Beggar is mutant strain, and editorial efficiency has reached 100%.
The scope of the present invention is not limited to the above embodiments.
Although being described in detail above with generality explanation and specific embodiment to the present invention, in the present invention
On the basis of make some modifications or improvements, this knows at one sightIt can be seen at a glance to those skilled in the art, needless to say.Therefore, not
Deviate do on the basis of present subject matter more or less to modify or improve, fall within the scope of the claimed invention.
Claims (1)
1. application of the prokaryotes containing CRISPR-Cas system in endogenous editor's prokaryotic gene group；
The prokaryotes be containing endogenous I type or type III CRISPR-Cas system, or simultaneously contain endogenous I type and type III
The bacterium of CRISPR-Cas system or archaeal；
Its application method includes:
1) genome editor plasmid is constructed:
Intend editing area in prokaryotic gene group and chooses one section of sequence as protospacer, that is, target site, according to
Protospacer designs the primer of two reverse complementals, and sequence is respectively forward primer: 5 '-AAAG-Nn- 3 ', reversely draw
Object: 5 '-TAGC-N 'n- 3 ', wherein NnAnd N 'nFor reverse complementary sequence, N and N ' indicate that base A, T, G or C, n are indicated
The base number of protospacer；Above-mentioned two primer annealings are formed to the double-stranded DNA with cohesive end, i.e. spacer piece
Section；Artificial CRISPR carrier pSe-Rp is handled by restriction enzyme BspMI digestion, then and with cohesive end
Spacer segment enzyme connects, and obtains the artificial CRISPR plasmid that can generate mature crRNA；It again will include mutant nucleotide sequence and and host
The homologous donor DNA segment in target site both ends is inserted on above-mentioned artificial CRISPR plasmid in cellular genome, obtains gene
Group editor's plasmid；
2) acquisition of mutant strain: genome editor's plasmid electricity is transferred to prokaryotes competent cell, the artificial CRISPR on plasmid
Cluster transcribes out pre-crRNA, and pre-crRNA is processed to mature crRNA in the cell；CrRNA and cellular endogenous
CRISPR-Cas albumen forms crRNP complex, is matched by crRNA with the target dna chain in host cell gene group to know
Other target site is cut；Homologous recombination occurs for donor DNA segment and target site two sides sequence on plasmid, and then obtains
Genome editor's mutant strain.
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