CN105331627A - Method for editing prokaryotic genomes using endogenic CRISPR-Cas (CRISPR-associated) system - Google Patents
Method for editing prokaryotic genomes using endogenic CRISPR-Cas (CRISPR-associated) system Download PDFInfo
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Abstract
The invention relates to a method for editing prokaryotic genomes using an endogenic CRISPR-Cas (CRISPR-associated) system. An editor plasmid carrying both an artificial CRISPR cluster and a donor DNA is required to be established for the genome editing of a prokaryote containing the endogenic CRISPR-Cas system, after NDA interference is caused by the endogenic CRISPR-Cas to the genome, and the genome is edited through homologous recombination. The method has the advantages that a range of applicable hosts is wide, all bacteria and archaea containing the endogenic CRISPR-Cas being available for operation; the method is applicable to operations in various editing manners, such as deletion, insertion and point mutation; the editing efficiency is higher, screening positive rage is high, and the background is low; a flow is simple, a short period of time is required, and the workload of prokaryotic genome editing is greatly reduced.
Description
Technical field
The invention belongs to genomics and genetically engineered and biological technical field, be specifically related to a kind ofly utilize endogenous CRISPR-Cas system to carry out the method for prokaryotic gene group editor.
Background technology
CRISPR-Cas system resists the acquired immune system of the external source invasion nucleic acid such as virus as a kind of prokaryotic organism, (VanderOostJetal., 2014 in the ancient bacterium being extensively present in about 90% and 40% bacterium; BarrangouRetal., 2014).CRISPR-Cas system is divided into three main Types: I type, II type and type III; They have a significant albumen respectively: Cas3, Cas9 and Cas10 (MakarovaKSetal., 2011).II type CRISPR system only needs Cas9 albumen and crRNA and trans-actingRNA to exercise DNA and interferes active (DeltchevaEetal., 2011; GasiunasGetal., 2012).Therefore simple II type CRISPR system is developed to eukaryotic gene group edit tool (JinekMetal., 2012; WangHetal., 2013), and be widely used in different eukaryotes and bacterium (DoudnaJAetal., 2014; SanderJDetal., 2014; HsuPDetal., 2014; SelleKetal., 2015).
In the past ten years, each laboratory establishes effective genetic manipulation system and instrument (LeighJAetal., 2011) in some ancient bacterium type species.However, the genetics research of ancient bacterium because of the growth conditions of its uniqueness, poor growth and to the factor such as most antibiotics is insensitive and still seem very challenging (ValentineDLetal., 2007).Therefore, the application of genome edit methods in ancient bacterium occuping CRISPR system still requires study and develops.
What the current genome editing based on CRISPR system utilized is all II type CRISPR system and CRISPR/Cas9 system, and CRISPR/Cas9 system also exists many limitation.First, may there is effect of missing the target in CRISPR/Cas9, studies have found that Cas9 albumen may allow the mispairing existed between crRNA and target sequence to a certain degree, and the quantity that these mispairing occur and position will inevitably have influence on the specificity of gene editing.Secondly, utilize the CRISPR/Cas9 system of external source, need Optimizing Reconstruction Cas9 albumen being applicable to host cell, and need simultaneously in cell inner expression Cas9 albumen and sgRNA (guide RNA) competence exertion effect, service routine more complicated.In addition, in the biology that some growth conditionss are extreme, born of the same parents' environment may have influence on the activity of Cas9 albumen, thus limits CRISPR/Cas9 systematic difference.
The present invention is directed to above problem, we have proposed and utilize the CRISPR system of prokaryotic organism self to be genome editor.The DNA that the method make use of CRISPR system simultaneously interferes active and homologous recombination, therefore avoids effect of missing the target largely, enhances editor's specificity.The method only needs structure one to carry editor's plasmid of artificial CRISPR bunch and donor dna simultaneously, and flow process is simple, and the operational cycle is short.What the method utilized is the CRISPR system of prokaryotic organism self, all bacteriums containing endogenous CRISPR-Cas system and ancient bacterium all applicable.
Sulfolobus solfataricus is the model animals of CRISPR research, show that it all has DNA and (or) RNA and interferes active (ManicaAetal., 2011 about I type and type III CRISPR-Cas systematic research in sulfolobus solfataricus S.solfataricusP2 and Iceland sulfolobus solfataricus S.islandicusREY15A; ManicaAetal., 2013; ZebecZetal., 2014; GudbergsdottirS., 2011).We find that I-A type and III-BCmr-α all have DNA interference active (DengLetal., 2013) in Iceland sulfolobus solfataricus S.islandicusREY15A." CCN " and " TCN " is two PAMs (protospacer-adjacentmotifs) (LillestolR.Ketal., 2009) that Iceland sulfolobus solfataricus I-ACRISPR system DNA interferes needed for activity.And DNA that III-B type CRISPR mediates interferes activity to be do not need PAM sequence, but it depends on and transcribes, and need guarantee that crRNA5 ' holds repeat sequence and protospacer upstream sequence mispairing (DengLetal., 2013).
Summary of the invention
The object of the present invention is to provide the application of the prokaryotic organism containing CRISPR-Cas system in endogenous editor's prokaryotic gene group, described prokaryotic organism for containing endogenous I type or type III CRISPR-Cas system, or contain the bacterium of endogenous I type and type III CRISPR-Cas system simultaneously.
Another object of the present invention is that providing a kind of utilizes endogenous CRISPR-Cas system to carry out the method for prokaryotic gene group editor, by building editor's plasmid that is carried artificial CRISPR bunch and donor dna simultaneously, occur to be reached genomic editor by homologous recombination after DNA interferes to genome in endogenous CRISPR system.
In order to achieve the above object, the present invention takes following technical measures:
The claimed content of the present invention comprises, the application of the prokaryotic organism containing CRISPR-Cas system in endogenous editor's prokaryotic gene group; The application of endogenous CRISPR-Cas system in editor's prokaryotic gene group.
Utilize endogenous CRISPR-Cas system to carry out a prokaryotic gene group editor's method, comprise the following steps:
1) genome editor plasmid is built:
Prokaryotic gene group is intended editing area and chooses one section of sequence as protospacer and target site, design the primer of two reverse complementals according to protospacer, its sequence is respectively forward primer: 5 '-AAAG-N
n-3 ', reverse primer: 5 '-TAGC-N '
n-3 ', wherein N
nand N '
nfor reverse complementary sequence, N and N ' represents base A, T, G or C, and n represents the base number of protospacer; Above-mentioned two primer annealings are formed the double-stranded DNA with sticky end, i.e. spacer fragment; Artificial CRISPR carrier pSe-Rp cuts process through restriction enzyme BspMI enzyme, then connects with the spacer fragment enzyme with sticky end, obtains the artificial CRISPR plasmid (pAC) that can produce ripe crRNA; Again by comprising mutant nucleotide sequence and being inserted on above-mentioned pAC plasmid with the donor DNA segment of two ends, target site homology in host cell gene group, obtain genome editor plasmid (pGE);
Described donor DNA segment to be increased by the method for SOE-PCR by the mutant nucleotide sequence of two, left and right homologous fragment and medium design and forms.
2) acquisition of mutant strain: after pGE plasmid electricity proceeds to prokaryotic organism competent cell, the artificial CRISPR bunch on plasmid transcribes out pre-crRNA, and pre-crRNA is processed to ripe crRNA in cell; The CRISPR-Cas albumen of crRNA and cellular endogenous forms crRNP complex body, matches to identify that target site is cut by the target dna chain on crRNA and host cell gene group; Donor DNA segment subsequently on plasmid and both sides, target site sequence generation homologous recombination, and then obtain genome editor mutant strain.
In above-described step, described prokaryotic organism are for containing endogenous I type or type III CRISPR-Cas system, or contain the bacterium of endogenous I type and type III CRISPR-Cas system or ancient bacterium simultaneously, include but not limited to Iceland's sulfolobus solfataricus (preferred protokaryon Iceland sulfolobus solfataricus S.islandicusREY15A), the bacterium of endogenous CRISPR-Cas system and ancient bacterium is contained for all, such as: intestinal bacteria Escherichiacoli, staphylococcus epidermidis Staphylococcusepidermidis, corrupt Shiva bacterium Shewanellaputrefaciens, thermophilic bacterium Thermusthermophilus, thermophilus streptococcus Streptococcusthermophilus, pyrococcus furiosus Pyrococcusfuriosus, sulfolobus acidocaldarius Sulfolobusacidocaldarius, sulfolobus solfataricus Sulfolobussolfataricus, hot autotrophy methane thermal bacillus Methanothermobacterthermautotrophicus, the richly endowed bacterium Haloferaxvolcanii of salt, black shank bacterium Pectobacteriumatrosepticum, diphtheria corynebacterium Corynebacteriumdiphtheriae etc.
Above-described genome editor includes but not limited to disappearance, inserts and point mutation.
Compared with prior art, the present invention has the following advantages:
Provided by the inventionly endogenous CRISPR-Cas system is utilized to carry out the method for prokaryotic gene group editor, only need build editor's plasmid that is carried artificial CRISPR bunch and donor dna simultaneously, occur to be reached genomic editor by homologous recombination after DNA interferes to genome in endogenous CRISPR system.Its maximum advantage is: application host range is wide, and all bacteriums containing endogenous CRISPR-Cas system and ancient bacterium all can operate; Can be used for multiple editing mode, disappearance, insertion and point mutation etc. all can operate; Higher editorial efficiency, screening positive rate is high, and background is low; Flow process is simple, and the time cycle is short, greatly alleviates the workload of prokaryotic gene group editor.
Accompanying drawing explanation
Fig. 1 is that the present invention utilizes endogenous CRISPR-Cas system to carry out the principle schematic of the method for prokaryotic gene group editor.
In Fig. 2, A utilizes Iceland sulfolobus solfataricus S.islandicusREY15A endogenous CRISPR system to carry out accurate deletion mutantion design diagram to its lacS gene in embodiment 1;
In Fig. 2, B is that in embodiment 1, wild-type and transformant schematic diagram are compared in X-gal staining analysis;
In Fig. 2, C is that in embodiment 1, PCR detects whether transformant is 43bp deletion mutantion strain schematic diagram;
In Fig. 2, D is DNA sequencing com-parison and analysis wild-type and saltant type lacS schematic diagram in embodiment 1.
In Fig. 3, A utilizes the C end of Iceland sulfolobus solfataricus S.islandicusREY15A endogenous III-B type CRISPR system to its Cmr-2 α albumen to carry out 6 × His label insertion mutation design diagram in the embodiment of the present invention 2;
In Fig. 3, B is that in embodiment 2, DNA sequencing compares wild-type cmr-2 α and carries 6 × His label saltant type cmr-2 α schematic diagram;
In Fig. 3, C is the Cmr-2 α proteose intention of carrying 6 × His label in embodiment 2 with His mouse-anti westernblot qualification;
In Fig. 3, D makees copurification schematic diagram with the Cmr-2 α albumen carrying 6 × His label in embodiment 2 in Cmr2 α-His bacterial strain.
Fig. 4 is that in embodiment 2, pGE-2 α-His electricity turns Δ cas3/cas7 and Δ cas3 competent cell, and PCR checks transformant schematic diagram;
In Fig. 4, A is the design diagram of PCR the primer in embodiment 2;
In Fig. 4, B is that in embodiment 2, two each 12 transformant PCR of deletion mycopremna verify electrophorogram.
Fig. 5 is that in the embodiment of the present invention 3, Cmr-2 α (SiRe-0894), Cmr-2 β (SiRe-0598) and their homologous protein PyrococcusfuriosusPF1129 carry out N terminal sequence comparison schematic diagram;
In Fig. 5, A is that in embodiment 3, four conservative amino acid are elected as and intended mutational site schematic diagram;
In Fig. 5, B utilizes Iceland sulfolobus solfataricus S.islandicusREY15A endogenous I-A type CRISPR system to carry out multisite mutation design diagram to its Cmr-2 α albumen HDdomain in embodiment 3;
In Fig. 5, C is DNA sequencing display Cmr-2 α albumen HDdomain tri-kinds of saltant type schematic diagram in embodiment 3.
Embodiment
Following examples for illustration of the present invention, but are not used for limiting the scope of the invention.If not specified, the experimental implementation in embodiment is experiment condition or the condition according to material, the specification sheets suggestion of reagent manufacturer all routinely.
Embodiment 1:
Utilize endogenous CRISPR-Cas system to carry out a prokaryotic gene group editor's method, comprise the following steps:
It is that example is described that the present embodiment utilizes Iceland sulfolobus solfataricus S.islandicusREY15A endogenous (I-A type CRISPR system and III-B type CRISPR system) CRISPR system to carry out accurate deletion mutantion to its lacS gene:
1. edit the structure of plasmid
(1) choose on Iceland sulfolobus solfataricus S.islandicusREY15A beta-galactosidase gene lacS (SIRE_RS11295)+933 to+972 totally 40 bases as protospacer, its reverse complementary sequence is 5 '-AGTGTAGTAATTAACACCAATCCAGTCTAACCTACCCCTT-3 ', it is close to a CCT-PAM (ProtospacerAdjacentMotif), therefore can by I-A type CRISPR system target; Simultaneously again due to crRNA5 ' terminal sequence and corresponding targetsite sequence mismatch, so also can simultaneously by III-B type CRISPR system target.Design two primers (LacS-E-SpF/LacS-E-SpR) (table 1) based on this protospacer, two primers generate the spacer fragment containing sticky end in two ends by annealing;
Primer sequence used by table 1. the present invention
In upper table, black underscore mark is restriction enzyme site, and four bases forming spacer sticky end are shown by black overstriking; Cmr-2 α HDdomain mutational site is identified by italic font.
(2) artificial CRISPR carrier pSe-Rp (Peng, 2015) cuts process through BspMI enzyme, and digestion products and above-mentioned spacer fragment enzyme connect, and obtain artificial CRISPR plasmid;
(3) design two SOEPCR primers (LacS-E-SOEF/LacS-E-SOER) lacking 43bp on lacS gene and respectively with two primers (LacS-E-SalIF/LacS-E-NotIR) (table 1) of SalI and NotI restriction enzyme site, obtain the donor DNA segment for homologous recombination by the method for SOEPCR;
(4) donor DNA segment and above-mentioned artificial CRISPR plasmid are cut with SalI and NotI enzyme respectively, and then enzyme connects conversion, obtain editor's plasmid pGE-lacS1;
2. the screening of mutant strain
(1) 500ngpGE-lacS1 plasmid electricity proceeds to Iceland sulfolobus solfataricus S.islandicusREY15A competent cell, coating SCV (0.2%sucrose, 0.2%casaminoacidsplus1%vitaminsolution) solid medium, cultivate 7 days for 78 DEG C, picking transformant cultivates three days in 5mlSCV liquid nutrient medium;
(2) respectively get 90 μ l bacterium liquid to add 10 μ lX-gal (20mg/ml) 78 DEG C respectively and hatch one hour, observing response system color;
(3) (F1/R1 and F2/R2) (table 1) is then used to carry out PCR detection respectively, find that 17 transformants of picking are all containing pGE-lacS1 plasmid, wherein the lacS gene of 16 transformants there occurs 43bp disappearance simultaneously, 1 is escape mutants, and by primer (LacS-Seq-F/LacS-Seq-R) (table 1) amplifying genom DNA sequence verification, result shows the editorial efficiency of the method more than 90%.
Embodiment 2:
Utilize endogenous CRISPR-Cas system to carry out a prokaryotic gene group editor's method, comprise the following steps:
The embodiment of the present invention carries out 6 × His label insertion mutation for the C end of Iceland sulfolobus solfataricus S.islandicusREY15A endogenous III-B type CRISPR system to its Cmr-2 α albumen, is described.
1. edit the structure of plasmid
(1) front 7 bases choosing last 25 bases of cmr-2 α gene (SIRE_RS04505) and cmr-3 α gene on Iceland sulfolobus solfataricus S.islandicusREY15A genome totally 32 bases as protospacer, its reverse complementary sequence is 5 '-AGTGTAGTAATTAACACCACAATCCAGTCTAACCTACCCCTT-3 ', do not have CCN-PAM before it, the DNA thus only with III-B type CRISPR interferes active.Design two primers (2 α-His-SpF/2 α-His-SpR) (table 1) based on this protospacer, two primers generate the spacer fragment containing sticky end in two ends by annealing;
(2) artificial CRISPR carrier pSe-Rp cuts process through BspMI enzyme, and digestion products and above-mentioned spacer fragment enzyme connect, and obtain artificial CRISPR plasmid;
(3) design two SOEPCR primers (2 α-His-SOEF/2 α-His-SOER) comprising six Histidines coding and respectively with two primers (2 α-His-SalIF/2 α-His-NotIR) (table 1) of SalI and NotI restriction enzyme site, obtain the donor DNA segment for homologous recombination by the method for SOEPCR;
(4) donor DNA segment and above-mentioned artificial CRISPR plasmid are cut with SalI and NotI enzyme respectively, and then enzyme connects conversion, obtain editor's plasmid pGE-2 α-His;
2. the screening of mutant strain
(1) 500ngpGE-2 α-His plasmid electricity proceeds to Iceland sulfolobus solfataricus S.islandicusREY15A competent cell, coating SCV (0.2%sucrose, 0.2%casaminoacidsplus1%vitaminsolution) solid medium, cultivate 7 days for 78 DEG C, picking transformant cultivates three days in 5mlSCV liquid nutrient medium;
(2) carry out sequence verification with primer (2 α-Seq-F/2 α-Seq-R) (table 1) amplification cmr-2 α gene, result display transformant is the mutant strain successfully inserting 6 × His label;
(3) sieve insertion mutation strain is counter with the SCV substratum containing 5-FOA, to eliminate editor's plasmid pGE-2 α-His;
The copurification of 3.His tag fusion protein and detection
3LSCV+U (uridylic) culture medium culturing Cmr2 α-His bacterial strain, treats OD
600when reaching 0.4, collect thalline and be resuspended in 50mMPBS damping fluid, after ultrasonic disruption, high speed centrifugation gets supernatant, post is crossed after fully mixing one hour with 500 μ lNi sepharose 4Bs, then 20mM imidazoles PBS is used respectively, 60mM imidazoles PBS washes post, and after finally using 2ml200mM imidazoles PBS to repeat wash-out Ni post twice, SDS-PAGE, silver dye is analyzed and done westernblot detection with His mouse-anti respectively.
Have detected 7 transformants, 7 are insertion mutation strain.
4. in order to get rid of the impact in the present embodiment of I-A type CRISPR system further, we turn with editor's plasmid pGE-2 α-His electricity Iceland sulfolobus solfataricus S.islandicusREY15A competent cell that two have lacked I type CRIPSR activity: Δ cas3/cas7 and Δ cas3, each picking 12 transformants carry out PCR checking, the insertion mutation strain ratio obtained is respectively 11/12 and 12/12 (Fig. 4), further illustrates the method success ratio and reaches more than 90%.
Embodiment 3:
Utilize endogenous CRISPR-Cas system to carry out a prokaryotic gene group editor's method, comprise the following steps:
The present embodiment carries out multisite mutation to utilize Iceland sulfolobus solfataricus S.islandicusREY15A endogenous I-A type CRISPR system to its Cmr-2 α albumen HDdomain, is described.
1. edit the structure of plasmid
(1) on Iceland sulfolobus solfataricus S.islandicusREY15A genome, choose cmr-2 α gene N holds HDdomain tetra-conserved amino acid H/D/K/I places to choose 40 bases as protospacer, its sequence is 5 '-CGACCCTCCTTGGAAGGCATGGGTAATTACAAGGAATATT-3 ', has CCA-PAM before it.Design two primers (2 α-HDmut-SpF/2 α-HDmut-SpR) based on this protospacer, two primers generate the spacer fragment containing sticky end in two ends by annealing;
(2) artificial CRISPR carrier pSe-Rp cuts process through BspMI enzyme, and digestion products and above-mentioned spacer fragment enzyme connect, and obtain artificial CRISPR plasmid;
(3) the SOEPCR primer that two comprise four mutating acid coding is designed
(2 α-HDmut-SOEF/2 α-HDmut-SOER) and respectively with two primers of SalI and NotI restriction enzyme site
(2 α-HDmut-SalIF/2 α-HDmut-NotIR) (table 1), obtains the donor DNA segment for homologous recombination by the method for SOEPCR;
(4) donor DNA segment and above-mentioned artificial CRISPR plasmid are cut with SalI and NotI enzyme respectively, and then enzyme connects conversion, obtain editor's plasmid pGE-2 α HD;
2. the screening of mutant strain
(1) 500ngpGE-2 α HD plasmid electricity proceeds to Iceland sulfolobus solfataricus S.islandicusREY15A competent cell, coating SCV (0.2%sucrose, 0.2%casaminoacidsplus1%vitaminsolution) solid medium, cultivate 7 days for 78 DEG C, picking transformant cultivates three days in 5mlSCV liquid nutrient medium;
(2) sequence verification is carried out with primer (2 α-Seq-F/2 α-Seq-R) (table 1) amplification cmr-2 α gene, 7 transformants of picking are mutant strain, illustrate that editorial efficiency is 100%, due to the diversity of homologous recombination position, result demonstrates three kinds of mutation types, be respectively H/D two amino acid that only suddenly change, sudden change H/D/K tri-amino acid and H/D/K/I tetra-amino acid are all undergone mutation, and ratio is 1:2:4.
Embodiment 4:
We utilize this method to utilize its I-A type CRISPR system to be successfully made the editor of lacS gene in Iceland sulfolobus solfataricus S.islandicusHVE10/4 bacterial strain of virus research (be widely used in).The structure of pGE plasmid and the same embodiment 1 of operation steps.It is worthy of note, S.islandicusHVE10/4 is very low due to transformation efficiency, is difficult to successfully, and utilizes endogenous CRISPR system gene group edit methods provided by the invention when implementing traditional gene knockout method, the transformant of picking is mutant strain, and editorial efficiency reaches 100%.
Scope is not limited to above-described embodiment.
Although above with general illustrate and specific embodiment to invention has been detailed description, basis of the present invention makes some modifications or improvements, and this knows at one sightIt can be seen at a glance to those skilled in the art, needless to say.Therefore, the amendment more or less that the basis of not departing from purport of the present invention is made or improvement, all belong to the scope of protection of present invention.
Claims (3)
1. the application of prokaryotic organism in endogenous editor's prokaryotic gene group containing CRISPR-Cas system.
2. application according to claim 1, described prokaryotic organism for containing endogenous I type or type III CRISPR-Cas system, or contain the bacterium of endogenous I type and type III CRISPR-Cas system or ancient bacterium simultaneously.
3. application according to claim 1, its method comprises:
1) genome editor plasmid is built:
Prokaryotic gene group is intended editing area and chooses one section of sequence as protospacer and target site, between its sequence clone to two CRISPR-Repeat sequences, obtain artificial CRISPR plasmid, again by comprising mutant nucleotide sequence and being inserted on above-mentioned artificial CRISPR plasmid with the donor DNA segment of two ends, target site homology in host cell gene group, obtain genome editor plasmid;
2) acquisition of mutant strain: pGE plasmid electricity proceeds to prokaryotic organism competent cell, after endogenous CRISPR-Cas system carries out DNA interference to target site, donor DNA segment on plasmid and both sides, target site sequence generation homologous recombination, and then obtain genome editor mutant strain.
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