CN117987447A - Control method for eukaryotic cell continuous evolution and application thereof - Google Patents
Control method for eukaryotic cell continuous evolution and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of biomedicine, and discloses a control method for continuous evolution of eukaryotic cells and application thereof, wherein the method comprises the following steps: (1) stably transferring cell line construction; (2) continuing multiple rounds of evolution; (3) screening the mutants; (4) functional verification: and verifying endogenous genome loci of a plurality of cells, comparing editing conditions of the screened mutants relative to wild types, taking cells without transfection as negative controls, comparing editing efficiency and editing windows. The method, the mutant and the application thereof provided by the invention have the advantages that the cell survival rate of the base editing with higher expression editing efficiency and accuracy and lower off-target rate is higher, and the cells of the base editing device with higher expression editing efficiency and accuracy and lower off-target rate can be screened out after continuous multi-round evolution, so that the method and the mutant have wide application prospect.
Description
Technical Field
The invention belongs to the technical field of biomedicine, and particularly relates to a control method for eukaryotic cell continuous evolution, an obtained mutant and application thereof.
Background
Currently, optimizing gene editing systems or base editors is achieved mainly by protein engineering (Protein Engineering) and evolutionary methods. Protein engineering is based on the structure and function of the target protein, and is designed and modified artificially to optimize the molecular characteristics of the target protein. Protein engineering methods have been used many times to engineer base editing systems. The David r.liu team engineered the cytidine deaminase of CBE by protein engineering methods such that its editing window was reduced, but some reduction in editing efficiency was found. Mutation of the cytidine deaminase or adenine deaminase of CBE and ABE, respectively, by protein engineering techniques can reduce their RNA off-target effects, but at the same time a decrease in editing efficiency for some DNA sites is observed. Protein engineering techniques cannot completely avoid other regions or changes in overall structure and function due to modification of only local amino acid sites. Thus, there is still a potential for great improvement in base editors engineered by protein engineering techniques.
The evolution method optimizes a base editing system and can be divided into prokaryotic evolution and eukaryotic evolution. The prokaryotic evolution system has the advantage of short evolution period (e.g. about 20 minutes for breeding one generation and about 24 hours for mammalian cells), and the prokaryotic evolution platform has also been used earlier for optimization of base editing systems. The David r.liu team utilizes its phage-based prokaryotic evolution platform to evolve adenine deaminase (ecTadA) from escherichia coli into adenine deaminase that can function in mammalian cells. Then, the team of the human body again utilizes a phage-assisted continuous evolution method to continuously evolve a new generation of editor ABE8e, the editing window is obviously widened, and the off-target rate is also obviously improved. And (3) continuously evolving ABE8e by utilizing a eukaryotic evolution platform developed by us to obtain mutants with similar efficiencies and different window widths or moving windows. Due to the limitations of the microenvironment of the prokaryotic cell, it is very difficult to evolve some eukaryotic-derived proteins such as reverse transcriptase for PE base editing, or to maximize the parameters of the base editing or gene editing system, especially after transformation back into eukaryotic cells.
Direct evolution of base editing or gene editing systems with eukaryotic cells is one of the best evolutionary or optimization strategies. Currently only yeast-based evolution platforms have been successfully used to evolve gene editing systems, which are RNA editing systems. The existing base editing system (ABE, CBE, CGBE) has the problems of low editing efficiency, poor accuracy (including low purity of editing products and introduction of indels), off-target effect of DNA and RNA levels, and the like. However, a significant disadvantage of this evolution platform is that the yeast cells and mammalian cells still have a high genetic background difference and a long evolutionary distance, so that the application of this system does not guarantee that optimal results can be obtained. There is no report of continuous evolution of base editing systems or gene editing systems based on mammalian cells.
Disclosure of Invention
The invention aims to provide a control method for continuous evolution of eukaryotic cells, an obtained mutant and application thereof, and the expression editing efficiency and accuracy are improved by controlling continuous multi-round evolution so as to solve the problems.
In order to achieve the above object, the present invention provides the following technical solutions:
A method for controlling the sustained evolution of eukaryotic cells, comprising the steps of:
step one, stable transformation cell line construction: stably expressing a gene editing system to be evolved in a cell;
step two, continuous multi-round evolution: wherein each round of evolution comprises the steps of: 1) introducing a mutation treatment, 2) introducing a transiently transfected selection plasmid, 3) antibiotic or fluorescent marker selection mutant treatment;
step three, screening mutants: screening active mutants by using a fluorescent reporter gene or screening mutants by using common sequencing;
Step four, functional verification: and verifying endogenous genome loci of a plurality of cells, comparing editing conditions of the screened mutants relative to wild types, and comparing editing efficiency, editing windows, editing accuracy and off-target conditions of DNA and RNA levels by taking cells without transfection as negative controls.
The invention includes mutation and screening. The mutation system included plasmids expressing dCas9 (mixed with a low proportion of ABE8e, BE3, CGBE) and a gRNA-AID mix targeting the region to BE evolved. The screening system is a defective antibiotic expression plasmid, and can be correctly expressed after being repaired by the base editing system. The mutation system is used to introduce mutation into the functional gene of base editor, and then the antibiotic with proper concentration is used to screen the beneficial mutation. The base editor with high editing efficiency and high accuracy can repair the antibiotic genes which cannot be expressed normally more efficiently, so that the survival rate of the cells is improved. Off-target effects of the gRNA used to repair the antibiotic and the large number of grnas targeted to the base editor may lead to cell death and thus higher specificity of the base editor carried by the surviving cells screened. Therefore, the cell survival rate of the base editor with higher expression editing efficiency and accuracy and lower off-target rate is higher, and cells with higher expression editing efficiency and accuracy (including the editing window which is also narrowed, widened or moved according to the design) and lower off-target rate can be selected through continuous multi-round evolution.
In the above control method for continuous evolution of eukaryotic cells, the mutation introducing treatment in the second step comprises the following steps:
1) Paving cells;
2) The transfection mutant plasmids comprise dCS 9 expression plasmids and gRNA-AID mixture of target mutant genes.
In the above control method for the continuous evolution of eukaryotic cells, the dCas9 expression plasmid includes a low-ratio wide editing window mixed with a-to-G, C-to-T, C-to-G editors. The dCas9 expression plasmid includes a low ratio, wide editing window a to G, C to T, C to G editor to increase mutation rate and mutation species.
In the above control method for eukaryotic cell continuous evolution, the introduction of transiently transfected selection plasmid in step two comprises the steps of:
1) The cells are spread out and the cells are spread out,
2) Transfection of defective antibiotic expression plasmids.
In the above control method for continuous evolution of eukaryotic cells, the antibiotic selection mutant treatment in the second step comprises the following steps: and adding antibiotics during cell passage, setting 2-4 concentration gradients, gradually increasing the concentration of the added antibiotics, screening the surviving cells, and preferentially continuing to evolve the cells surviving at the higher concentration of the antibiotics.
In the control method for the continuous evolution of eukaryotic cells, puromycin with the initial concentration of 1 mug/ml is added during cell passage, 2-4 concentration gradients are set, the concentration of puromycin is gradually increased, surviving cells are screened, and surviving cells with higher antibiotic concentration are preferentially continued to evolve in the next round.
In the control method for the continuous evolution of eukaryotic cells, blasticidin with the initial concentration of 10ug/ml is added during cell passage, surviving cells continue to evolve in the next round, 2-4 concentration gradients are set, the concentration of the blasticidin is added to gradually rise, surviving cells are screened, and cells surviving at higher antibiotic concentration are preferentially subjected to the next round of evolution.
In the control method for the continuous evolution of eukaryotic cells, antibiotics are added after the cells are passaged, 2-4 concentration gradients are set, the concentration of the added antibiotics is gradually increased, the cells surviving at higher antibiotic concentration continue to evolve in the next round, the concentration of the added antibiotics is gradually increased in each round of evolution, and the concentration of the antibiotics reaches 300ug/ml at the maximum to screen the surviving cells.
In the above control method for continuous evolution of eukaryotic cells, the function verification in the fourth step includes the following steps: after multiple rounds of mutation, the cell genome is extracted, the pseudo-evolution region is amplified by PCR and connected to the expression vector plasmid before evolution, and the effective mutant is prepared by sequencing. The kit for extracting the genome of the cells is from GeneJET Genomic DNA purification kit, thermo. Amplification and sequencing of the base editor gene at various time points during evolution was confirmed, and it was expected that more and more nonsensical mutations and synonymous mutations were observed in multiple clones.
In the control method for the eukaryotic cell continuous evolution, when the screening concentration of antibiotics reaches 200 mug/ml to 300 mug/ml, cell genome is extracted, a pseudo-evolution region is amplified by PCR and is connected to an expression vector plasmid before evolution, and effective mutants are prepared by common sequencing, so that repeated, frame-shifting and mutation-free mutants are eliminated. After multiple rounds of mutation, the genome of the cell is extracted, and the pseudo-evolution region is amplified by PCR, and ordinary sequencing or deep sequencing is performed. The above steps need to be performed at multiple time points and care is taken to add positive and negative controls. Constructing an expression vector containing mutation sites, and verifying the base editing efficiency, accuracy and off-target rate of a wild strain before evolution at a plurality of sites of a plurality of endogenous genes of a plurality of cell lines under the same condition. The activity of the target mutant is determined primarily by ordinary sequencing, and then the targeted depth sequencing (Targeted deepsequencing) is used to compare whether the off-target rate is effectively increased or decreased, etc. If the lifting effect is not significant, the evolution can be continued.
In the above control method for continuous evolution of eukaryotic cells, after finishing the effective mutants, performing functional verification in the fourth step, wherein the functional verification comprises the following steps: constructing an expression vector containing mutation sites, and performing base editing efficiency test on the wild strain before evolution at a plurality of sites of a plurality of endogenous genes of a plurality of cell lines under the same condition, and detecting the expression of the mutant relative to the wild type by common sequencing or deep sequencing.
In the above control method for continuous evolution of eukaryotic cells, deep sequencing detection includes editing efficiency, editing window, editing accuracy and off-target rate at DNA and RNA levels. Here, the stage index is the observation of the presence or absence of non-synonymous mutations with higher mutation rates (generally more than 10% are defined), or synonymous mutations with a possible increase in codon efficiency.
After BE3 is evolved, the following mutants are selected according to the control method for eukaryotic cell continuous evolution, wherein the following mutants comprise 16 mutation sites, T140A, E146G, L P is mutated into T1, T (ACA) 80T (ACG), L (CTG) 104L (TTG) and Y120H are mutated into T6, E24G, T140A, E G is mutated into T16, S137P is mutated into T7, S (TCA) 137S (TCG) and V139M, R198G is mutated into T17, L134S is mutated into T8 and W153C is mutated into CBE18.
According to the mutant obtained by the eukaryotic cell continuous evolution control method, after ABEmax is evolved, the following mutants are screened: a1 comprises an amino acid mutation; a11 comprises an amino acid mutation; a19 includes Y9C, E24E, R25G, R38G, Y72C, T110A, N126D, Y207C, P251P, a259V, Y270Y, R295G; a26 comprises Y72C, M150V, K159E, a311T; a23 comprises N71S, Y72C; a14 comprises K157E, H211R, R295G, S368S, cas9n K R; m26 comprises E2G, Y9C, Y72C, Y270C; m10 comprises Y9C, N126D; m14 comprises R38G, N126D; m19 comprises R46G, N71S, Y72C, N126D, R149G, K159E; m25 comprises N126D, a141V; m4 comprises D118G, R220C; m5 comprises Y9C, Y72C, R97G, N126S; m1 comprises D118G, T187T, M258V; m7 comprises D118G, M258V; m8 comprises R149G; m9 comprises D118G; m13 comprises M258V; m11 comprises D118G, T187A, R223G; m22 comprises R149G, K159E; m17 comprises Y72C.
According to the mutant obtained by the eukaryotic cell continuous evolution control method, after ABE8e is evolved, the following mutants are screened: e3 comprises the amino acid mutation S171G, R152G, Y72C; e7 comprises N37D, L120P; e8 comprises N37G, L120P; e15 comprises N37G, R38G, R106G
S171G, G99C; e16 comprises L67P, L120P, N156D; e22 comprises N37S, N156D, Y80C; L120P, E24 comprises Y122C, M60I; e27 comprises E24G, R25G, Y122C; e28 comprises H127R, Y72C; e29 comprises R20G, L67P; e30 comprises N37D, L67P; e34 comprises N37D, Y122C, I98T, H51R, S170C, Q70R; e36 comprises E24G, R25G, N37G, R38G, L67P, S6G; e40 comprises N37G, R38G, L67P, R106G, N36S, cas 9T 38A; e44 comprises NLS K6E, R12G, N37G, R38G, R106G, V119A, Y122H; e48 comprises N37G, R38G, R106G, H51R, Q70R, N121D; e56 comprises L67P, H127R; e59 comprises R12G, N37D, L120P, Y122H; e60 comprises L120P, H127R; e65 comprises R20G, R106G; e67 contains R12G, E24G, R25G, L67P, N156D, linker S5G.
Application of the eukaryotic cell continuous evolution method described above, which is applicable at least to the following base editing systems: CBE, ABE, C to G gene editing system in CGBE and leader gene editing system in PE.
The application of the eukaryotic cell continuous evolution method can be used for evolving various CRISPR-Cas gene editing systems, at least comprising Cas9, cas12, cas13, cas14, cas3 and the like.
The application of the eukaryotic cell continuous evolution method can be used for evolving a gene editing or base editing system in mammalian cells and plant cells.
The application of the eukaryotic cell continuous evolution method can be at least used for evolving inactive Cas proteins, nickase active Cas proteins and nuclease active Cas proteins.
Application of the eukaryotic cell continuous evolution method described above, which can be used at least for evolving the currently known CRISPR-Cas system and similar gene editing or base editing systems.
The use of the eukaryotic cell sustained evolution method described above, wherein the similar gene editing or base editing system comprises IscB systems, prokaryotic Ago proteins (Prokaryotic Argonautes, pAgo), transcription activator-like effector nucleases (transcription activator-like (TAL) effector nucleases, TALEN) and zinc finger nucleases (Zinc Finger Nuclease, ZFN), etc.
The application of the eukaryotic cell continuous evolution method can be used for evolving gene editing and/or base editing systems in mammalian cells and plant cells.
Use of the eukaryotic cell sustained evolution method described above, which method observes that TadA or Apobec containing all amino acid mutations or nucleic acid mutations after evolution, can also be used in other gene editing and/or base editing systems such as, but not limited to, cas9, cas12, cas13, cas14, TALEN, ZFN.
Application of the eukaryotic cell continuous evolution method can be used for evolution of Cas proteins, functional proteins which can co-act with the Cas proteins can be evolved, including but not limited to cytosine deaminase (Apobec), adenosine deaminase (TadA), activation transcription elements (VP 16), reverse transcriptase and the like, and also can be used for evolution of guided RNA (including one or more of gRNA, sgRNA, crRNA, tracrRNA and pegRNA). Compared with the prior art, the invention has the beneficial effects that:
(1) The evolved base editor has higher editing efficiency and accuracy and lower off-target rate, and cells with higher expression editing efficiency and accuracy (including the editing window which is also narrowed or moved according to the design) and lower off-target rate are screened out through continuous multi-round evolution.
(2) The evolution process of the present invention includes the introduction of mutations and antibiotic selection. During cell passage, mutation is introduced by continuous transfection of mutant plasmids (including plasmids expressing dCas9 (mixed with low proportions of ABE8e, BE3, CGBE) and gRNA-AID targeting the mutant region), followed by transient transfection of selection plasmids (antibiotics expressing defects), and cells are passed through a suitable concentration of antibiotics to screen surviving cells 2 days after transfection, cells carrying the beneficial mutant base editor will have an increased proportion of surviving cells, cells carrying the neutral mutation or wild type editor will survive only, and cells carrying the deleterious mutation editor will have a reduced number or die. The beneficial mutant strains in the cells are ensured to have higher and higher proportion and the wild strains are fewer through multiple rounds of mutation and screening.
(3) The evolution process in the present invention includes the introduction of mutations and antibiotic selection. During passage of the cells, mutation is introduced by continuous transfection of mutant plasmids (including plasmids expressing dCas9 (mixed with a low proportion of ABE8e, BE3, CGBE) and gRNA-AID targeted to the mutated region), and then surviving cells are screened by adding an appropriate concentration of antibiotic, the proportion of cells carrying the beneficial mutant base editor will BE increased in surviving cells, cells carrying the neutral or wild-type editor will only survive, and cells carrying the deleterious mutant editor will BE reduced in number or die. The beneficial mutant strains in the cells are ensured to have higher and higher proportion and the wild strains are fewer through multiple rounds of mutation and screening.
(4) The eukaryotic sustained evolution in the invention can evolve some proteins or RNAs which are difficult to evolve in a prokaryotic environment, and has wide application range.
Drawings
FIG. 1 shows the results of ABEmax mutants after the first screening evolution in the examples of the present invention.
FIG. 2 shows the results of the second screening of the evolved ABEmax mutants according to the example of the present invention.
FIG. 3 shows the results of deep sequencing of the different sites of the 293T genome edited by the ABEmax mutant after evolution of the example of the present invention.
FIG. 4 shows the nucleotide substitutions A to G mediated by ABEmax and ABE8e at 293T cell genomic sites after evolution according to an embodiment of the present invention.
FIG. 5 shows the multi-site base substitution of BE4 after evolution compared to BE4 and YE1 before evolution in 293T cell genomes according to an embodiment of the present invention.
FIG. 6 is a schematic representation of transient transfection of the defect screening plasmids of examples 1-3 of the present invention.
Detailed Description
The technical scheme of the invention is further described in detail below with reference to the attached drawings and specific embodiments.
Example 1
Referring to fig. 1-6, the method for controlling the continuous evolution of eukaryotic cells provided in this embodiment comprises the following steps:
step one, stable transformation cell line construction: stably expressing a gene editing system to be evolved in a cell;
step two, continuous multi-round evolution: wherein each round of evolution comprises the steps of: 1) introducing a mutation treatment, 2) introducing a transiently transfected selection plasmid, 3) an antibiotic selection mutant treatment;
step three, screening mutants: screening active mutants by using a fluorescent reporter gene;
Step four, functional verification: and verifying endogenous genome loci of a plurality of cells, comparing editing conditions of the screened mutants relative to wild types, and comparing editing efficiency, editing windows and off-target conditions of DNA and RNA levels by taking cells without transfection as negative controls.
Specifically, the mutation-introducing treatment in the first step includes the steps of:
1) Paving cells;
2) Mutant plasmids, including dCas9 expression plasmids and gRNA mixtures targeting the mutant genes of interest, are transfected.
Wherein, the antibiotic screening mutant treatment in the second step comprises the following steps: and (3) adding blasticidin with an initial concentration of 10ug/ml during cell passage, continuing to evolve the surviving cells in the next round, setting 2 concentration gradients, gradually increasing the concentration of the blasticidin, screening the surviving cells after the concentration of the blasticidin reaches 300ug/ml, and continuing to evolve the surviving cells in the next round.
The transiently transfected selection plasmid in step two is shown in example 1 of FIG. 6, wherein after the first G-C base pair in the GTG is edited to an A-T base pair by the CBE base editor, the GTG is replaced with ATG, and ATG is used as a start codon to significantly improve the expression of blasticidin resistance gene, thereby enabling the survival of the cells.
The function verification in the fourth step comprises the following steps: when the screening concentration of antibiotics reaches 300ug/ml, extracting cell genome, amplifying the pseudo-evolution region by PCR and connecting to expression vector plasmid before evolution, and performing ordinary sequencing to obtain effective mutants, and eliminating repeated, frame-shifting, mutation-free and mutation-low mutants.
In addition, the deep sequencing detection comprises editing efficiency, editing window, editing accuracy and off-target rate.
After BE3 is evolved, the following mutants are selected according to the method for controlling the continuous evolution of eukaryotic cells, wherein the following mutants comprise 16 mutation sites, T140A, E146G, L P is mutated to T1, T80T, L104L, Y H is mutated to T6, flag D1G, E24G, T140A, E146G is mutated to T16, S137P is mutated to T7, S137S, V139M, R G is mutated to T17 and L134S is mutated to T8.
Example 2
The method for controlling continuous evolution of eukaryotic cells of this embodiment further comprises the following steps based on embodiment 1:
step one, stable transformation cell line construction: stably expressing a gene editing system to be evolved in a cell;
step two, continuous multi-round evolution: wherein each round of evolution comprises the steps of: 1) introducing a mutation treatment, 2) introducing a transiently transfected selection plasmid, 3) antibiotic or flow cytometer selection active mutant treatment;
step three, screening mutants: screening active mutants by using a fluorescent reporter gene;
Step four, functional verification: and verifying endogenous genome loci of a plurality of cells, comparing editing conditions of the screened mutants relative to wild types, and comparing editing efficiency, editing windows and off-target conditions of DNA and RNA levels by taking cells without transfection as negative controls.
Specifically, the mutation-introducing treatment in the first step includes the steps of:
1) Paving cells;
2) Mutant plasmids, including dCas9 expression plasmids and gRNA mixtures targeting the mutant genes of interest, are transfected.
Wherein, the treatment of the active mutant selected by the antibiotics in the second step comprises the following steps: and (3) adding puromycin with the initial concentration of 1 mug/ml during cell passage, continuously evolving the surviving cells in the next round, setting 2-4 concentration gradients, gradually increasing the concentration of the puromycin, screening the surviving cells after the concentration of the puromycin reaches 300 mug/ml, and continuously evolving the surviving cells in the next round.
Wherein, the flow cytometer screening active mutant treatment in the second step comprises the following steps: flow cytometry screened five to ten percent of the cells with the strongest fluorescence intensity before each screen.
The transiently transfected screening plasmid in step two is selected by replacing the first G-C base pair in the GTG with ATG after editing it with the CBE base editor as A-T base pair, which serves as the initiation codon to significantly increase puromycin resistance gene or green fluorescent protein gene expression, thereby allowing the surviving or increased fluorescent intensity of the located cells, as shown in example 1 of FIG. 6.
The function verification in the fourth step comprises the following steps: when the screening concentration of antibiotics reaches 300ug/ml, extracting cell genome, amplifying the pseudo-evolution region by PCR and connecting to expression vector plasmid before evolution, and performing ordinary sequencing to obtain effective mutants, and eliminating repeated, frame-shifting, mutation-free and mutation-low mutants.
In addition, the deep sequencing detection comprises editing efficiency, editing window, editing accuracy and off-target rate.
According to the mutant obtained by the eukaryotic cell continuous evolution control method, after BE3 is evolved, the following mutant is screened, and CBE18 contains W153C amino acid mutation.
Example 3
The method for controlling the continuous evolution of eukaryotic cells provided in this embodiment further comprises the following steps based on embodiments 1 and 2:
step one, stable transformation cell line construction: stably expressing a gene editing system to be evolved in a cell;
step two, continuous multi-round evolution: wherein each round of evolution comprises the steps of: 1) introducing a mutation treatment, 2) introducing a transiently transfected selection plasmid, 3) an antibiotic or a flow-through selection mutant treatment;
step three, screening mutants: screening active mutants by using a fluorescent reporter gene;
Step four, functional verification: and verifying endogenous genome loci of a plurality of cells, comparing editing conditions of the screened mutants relative to wild types, and comparing editing efficiency, editing windows and off-target conditions of DNA and RNA levels by taking cells without transfection as negative controls.
Specifically, the mutation-introducing treatment in the first step includes the steps of:
1) Paving cells;
2) Mutant plasmids, including dCas9 expression plasmids and gRNA mixtures targeting the mutant genes of interest, are transfected.
The function verification in the fourth step comprises the following steps: when the screening concentration of antibiotics reaches 300ug/ml, extracting cell genome, amplifying the pseudo-evolution region by PCR and connecting to expression vector plasmid before evolution, and performing ordinary sequencing to obtain effective mutants, and eliminating repeated, frame-shifting, mutation-free and mutation-low mutants.
The mutation introducing treatment in the second step comprises the following steps:
1) Transfection: transfecting the mutant plasmid, paving cells after 3 days, and transfecting and screening the plasmid;
2) And (3) passage: 2 days after transfection of the selection plasmid, the cells were passaged with puromycin at an initial concentration of 1ug/ml and surviving cells continued to evolve in the next round. Setting 2-4 puromycins with concentration gradient increase, screening living cells after the puromycins concentration reaches 300 mug/ml, and carrying out multiple rounds of mutation and screening.
Wherein, the treatment of the active mutant selected by the antibiotics in the second step comprises the following steps: and (3) adding puromycin with an initial concentration of 1ug/ml during cell passage, setting 2-4 concentration gradients, gradually increasing the concentration of the puromycin, and continuing the next round of evolution of the cells with the highest puromycin concentration, wherein the puromycin concentration reaches 300ug/ml, and stopping evolution.
According to the mutant obtained by the eukaryotic cell continuous evolution control method, after ABEmax is evolved, the following mutants are selected: a1 comprises an amino acid mutation; a11 comprises an amino acid mutation; a19 includes Y9C, E24E, R25G, R38G, Y72C, T110A, N126D, Y207C, P251P, a259V, Y270Y, R295G; a26 comprises Y72C, M150V, K159E, a311T; a23 comprises N71S, Y72C; a14 comprises K157E, H211R, R295G, S368S, cas9n K R; m26 comprises E2G, Y9C, Y72C, Y270C; m10 comprises Y9C, N126D; m14 comprises R38G, N126D; m19 comprises R46G, N71S, Y72C, N126D, R149G, K159E; m25 comprises N126D, a141V; m4 comprises D118G, R220C; m5 comprises Y9C, Y72C, R97G, N126S; m1 comprises D118G, T187T, M258V; m7 comprises D118G, M258V; m8 comprises R149G; m9 comprises D118G; m13 comprises M258V; m11 comprises D118G, T187A, R223G; m22 comprises R149G, K159E; m17 comprises Y72C.
Example 4
The method for controlling the continuous evolution of the eukaryotic cells provided by the embodiment further comprises the following steps on the basis of the embodiments 1-3:
step one, stable transformation cell line construction: stably expressing a gene editing system to be evolved in a cell;
step two, continuous multi-round evolution: wherein each round of evolution comprises the steps of: 1) introducing a mutation treatment, 2) introducing a transiently transfected selection plasmid, 3) a flow-through selection mutant treatment;
step three, screening mutants: screening active mutants by using a fluorescent reporter gene;
Step four, functional verification: and verifying endogenous genome loci of a plurality of cells, comparing editing conditions of the screened mutants relative to wild types, and comparing editing efficiency, editing windows and off-target conditions of DNA and RNA levels by taking cells without transfection as negative controls.
Specifically, the mutation-introducing treatment in the second step includes the steps of:
1) Paving cells;
2) The transfection mutant plasmids comprise dCS 9 expression plasmids and gRNA-AID mixture of target mutant genes.
In addition, the selection plasmid introduced with transient transfection in the second step comprises the steps of:
1) The cells are spread out and the cells are spread out,
2) Transfection of defective antibiotic expression plasmids.
Wherein, the dCAS9 expression plasmid comprises an editor mixed with A to G, C to T and C to G with low proportion and wide editing window.
Wherein, the treatment of the active mutant selected by the antibiotics in the second step comprises the following steps: and (3) adding puromycin with an initial concentration of 1 mug/ml during cell passage, setting 2-4 concentration gradients, gradually increasing the concentration of the puromycin, and continuing to evolve the cells with the highest puromycin concentration, wherein the concentration of the puromycin reaches 300 mug/ml, and stopping evolution.
The function verification in the fourth step comprises the following steps: after multiple rounds of mutation, the cell genome is extracted, the pseudo-evolution region is amplified by PCR and connected to the expression vector plasmid before evolution, and the effective mutant is prepared by sequencing.
According to the mutant obtained by the eukaryotic cell continuous evolution control method, after ABE8e is evolved, the following mutants are screened: e3 comprises the amino acid mutation S171G, R152G, Y72C; e7 comprises N37D, L120P; e8 comprises N37G, L120P; e15 comprises N37G, R38G, R106G, S171G, G99C; e16 comprises L67P, L120P, N156D; e22 comprises N37S, N156D, Y80C; L120P, E24 comprises Y122C, M60I; e27 comprises E24G, R25G, Y122C; e28 comprises H127R, Y72C; e29 comprises R20G, L67P; e30 comprises N37D, L67P; e34 comprises N37D, Y122C, I98T, H51R, S170C, Q70R; e36 comprises E24G, R25G, N37G, R38G, L67P, S6G; e40 comprises N37G, R38G, L67P, R106G, N36S, cas 9T 38A; e44 comprises NLS K6E, R12G, N37G, R38G, R106G, V119A, Y122H; e48 comprises N37G, R38G, R106G, H51R, Q70R, N121D; e56 comprises L67P, H127R; e59 comprises R12G, N37D, L120P, Y122H; e60 comprises L120P, H127R; e65 comprises R20G, R106G; e67 contains R12G, E24G, R25G, L67P, N156D, linker S5G. .
Example 5
The method for controlling the continuous evolution of the eukaryotic cells provided by the embodiment further comprises the following steps on the basis of the embodiments 1-4:
1) Transfection
Genome, PCR amplifying the quasi-evolution region and connecting to the expression vector plasmid before evolution, and ordinary sequencing to obtain effective mutant, eliminating repeated mutant with no mutation and low mutation rate.
Step three, screening mutants: screening active mutants by using a fluorescent reporter gene;
After finishing the effective mutant, performing function verification in the fourth step, wherein the function verification comprises the following steps: constructing an expression vector containing mutation sites, and performing base editing efficiency test on the wild strain before evolution at several sites of a plurality of endogenous genes of a plurality of cell lines under the same condition, and detecting the relative position of the mutants by deep sequencing
Transfection at evolution: HEK293T cells were plated into 12-well plates and transfected according to Lipofectamine 2000 instructions (zemoeid) when cell density reached about 70%.
Transfection of functional verification: HEK293T cells were plated onto 24-well plates, 1,500ng of base editor and 500ng of gRNA were transfected according to EZ Trans (Shanghai Li Ji Bio) instructions, and after 24 hours the medium was changed and 1. Mu.g/ml puromycin (Soy pal) and 10. Mu.g/ml blasticidin (Soy pal) were added. 5 days after transfection, cells were collected for general sequencing and deep sequencing.
2) Transduction and stable cell
Lentiviruses were prepared by transfection of lentiviral vectors and packaging plasmid psPAX and pVSV-g in 293T cells according to liposome 2000 (Siemens flight) instructions.
3) Genome extraction and PCR amplification
Genome extraction at the time of evolution experiment: genome extraction was performed according to genome extraction kit GeneJET Genomic DNA Purification Kit (zemoeid).
Genome extraction at functional verification: after cell harvest, 20. Mu.l of lysate (10 mM Tris-HCl (Tris-hydroxymethyl aminomethane hydrochloride) (pH 8.0), 50mM KCl (potassium chloride), 1.5mM MgCl2 (magnesium chloride), 0.5% Nonidet P-40 (ethylphenyl polyethylene glycol), 0.5% Tween-20 (Tween-20), and 100. Mu.g/ml proteinase K (proteinase K) (Roche)) was added, followed by PCR. Then, the mixture was centrifuged at 12,000rpm for 3 minutes, and the supernatant was subjected to PCR (95 ℃,5 minutes, 35 cycles (95 ℃,30 seconds; 56 ℃,30 seconds; 72 ℃,20 seconds), and 72 ℃ 5 minutes extension).
4) Deep sequencing and data analysis
The PCR products were purified using magnetic beads (Northey) and then library constructed for deep sequencing of Illumina Nova 6000 PE150 (An Nuoyou da). The results were analyzed using CRISPResso < 2 > (v.2.0.3).
Application example 1
1. Representation of the ABEmax and ABE8e editor after evolution
1) ABEmax mutants after first screening evolution
As shown in FIG. 1, the ABEmax mutant after evolution edited the performance of different sites of the 293T genome (common sequencing results). Post-evolution ABEmax mutants were mutated against pre-evolution amino acids as shown in table 1.
TABLE 1
| A19 | Y9C，E24E，R25G，R38G，Y72C，T110A，N126D，Y207C，P251P，A259V，Y270Y，R295G |
| A26 | Y72C，M150V，K159E，A311T |
| A23 | N71S，Y72C |
| A14 | K157E，H211R，R295G，S368S，Cas9n K30R， |
2) ABEmax mutant after second screening evolution
As shown in FIG. 2, the ABEmax mutant after evolution edited the performance of different sites of the 293T genome (common sequencing results). Post-evolution ABEmax mutants were mutated against pre-evolution amino acids as shown in table 2.
TABLE 2
3) Screening of evolved ABE8e mutants
As shown in FIG. 3, the evolved ABE8e mutants edited the performance of different sites of the 293T genome (deep sequencing results). The ABE8e mutants after evolution were mutated with respect to the amino acids before evolution as shown in table 3.
TABLE 3 Table 3
4) Evolved ABEmax, ABE8e editing features further screened in more 293T genomic locus studies
As shown in fig. 4, a number of mutants were generated after the evolution screening, from which parts were selected: m26 and M27 evolved from ABEmax, where M27 was the single TadA gene; e7, E16, E44, E15 and E65 evolved from ABE 8E. These mutants were further subjected to base editing experiments at more 293T cell genomic loci, and the editing effect of each mutant was detected by deep sequencing. M26 is still double TadA genes, so that the editing window is reduced, and higher editing efficiency is reserved. M27, E7, E16, E44 are single TadA genes, although the efficiency is reduced, the window is narrowed very narrow. In addition, when there are a plurality of editing sites within the editing window, the editing efficiency is severely degraded (fig. 4 a). E15 and E65 retain similar editing efficiencies to ABE8E, with editing window widths between ABE8E and ABEmax (FIG. 4 b). E60 then observes a window editing site shift (FIG. 4 c) that is consistent with the expectations.
As can be seen in FIG. 4, M26 and M27 evolved from ABEmax, and E7, E16, E44, E15 and E65 evolved from ABE 8E. As shown in FIG. 4a, the evolved mutants have a narrower editing window at these gene loci. As shown in FIG. 4b, E7, E15, E60 had narrower editing windows at these editing sites, whereas ABEmax and evolved M26 and M27 were inefficient at these gene sites. As shown in FIG. 4c, a shift to the right of the E60 editing window was observed at these gene loci. As shown in fig. 4d, the width of the E15, E60, E65 editing window was further studied. Amplicon depth sequencing, reads number at least 5 ten thousand. All edit efficiency experiments were 3 independent experiments, with the results equal to average plus SD.
2. Post-evolution CBE editor performance
The evolved BE editors were further screened, and the following four evolved editors (T1, T6, T16, T17) were further studied and compared in depth, including editing window width, index ratio of product, purity of product, DNA level off-target rate. YE1 has a narrower editing window than BE4 editor. By comparison of BE4, YE1, the evolved BE editor performed better (FIG. 5).
T6 has a narrower editing window than BE4 and YE1, and the editing activity is comparable or slightly lower. T1, T16, T17 have a somewhat narrower or comparable window edit width than YE1, but have a lower index ratio, higher product purity, and lower DNA off-target rate.
As shown in FIG. 5a, the evolved BE editor compares to BE4, YE1 in terms of edit window width, index ratio and purity of the product. As shown in FIG. 5b, the BE editors T7, T8, CBE18 after evolution have smaller editing windows than before evolution. This result was based on normal sequencing, using EditR to quantify the editing efficiency, and the result was three independent experiments. As shown in FIG. 5c, the BE editor CBE18 after evolution has a smaller editing window than before evolution. This result was based on normal sequencing, using EditR to quantify the editing efficiency, resulting in a secondary independent experiment. As shown in FIG. 5d, the evolved BE editor had a lower DNA miss rate than BE4 and YE 1. Amplicon depth sequencing, reads number at least 5 ten thousand. All edit efficiency experiments were at least 3 independent experiments, with the results equal to average plus SD.
As shown in Table 4, post-evolution CBE mutants were mutated with respect to pre-evolution amino acids.
TABLE 4 Table 4
| Post-evolution CBE mutants | Amino acid mutation site |
| T1 | T140A、E146G、L177P |
| T6 | T80T，L104L，Y120H |
| T16 | Flag D1G，E24G，T140A，E146G |
| T7 | S137P |
| T17 | S137S，V139M，R198G |
| T8 | L134S |
| CBE18 | W153C |
A11 editor functional protein TadA amino acid sequence:
amino acid sequence of M27 editor functional protein TadA:
The BE editor functional protein Apobec amino acid sequence:
ABEmax editor functional protein TadA amino acid sequence:
SEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGRHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGRVVFGARDAKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLSDFFRMRRQEIKAQKKAQSSTDSGGSSGGSSGSETPGTSESATPESSGGSSGGSSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTDSGGSSGGSSGSETPGTSESATPE.
Amino acid sequence of ABE8e editor functional protein TadA:
SEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGWRNSKRGAAGSLMNVLNYPGMNHRVEITEGILADECAALLCDFYRMPRQVFNAQKKAQSSINSGGSSGGSSGSETPGTSESATPE.
According to the method and the application provided by the embodiment of the invention, the cell survival rate of the base editing with higher expression editing efficiency and accuracy and lower off-target rate is higher, the cell of the base editing device with higher expression editing efficiency and accuracy and lower off-target rate can be accurately screened out after continuous multi-round evolution, and the method and the mutant can be popularized and applied in a larger range.
The foregoing description is only a preferred embodiment of the present invention, and is not intended to limit the embodiment of the present invention in any way, but any simple modification, equivalent variation and modification made according to the technical spirit of the embodiment of the present invention still fall within the scope of the technical solution of the embodiment of the present invention.
Claims (23)
1. A method for controlling the continuous evolution of eukaryotic cells, comprising the steps of:
step one, stable transformation cell line construction: stably expressing a gene editing system to be evolved in a cell;
Step two, continuous multi-round evolution: wherein each round of evolution comprises the steps of: 1) introducing a mutation treatment, 2) introducing a transiently transfected selection plasmid, 3) an antibiotic or fluorescent selection mutant treatment;
step three, screening mutants: screening active mutants by using a fluorescent reporter gene or screening mutants by using common sequencing;
Step four, functional verification: and verifying endogenous genome loci of a plurality of cells, comparing editing conditions of the screened mutants relative to wild types, and comparing editing efficiency, editing windows and off-target conditions of DNA and RNA levels by taking cells without transfection as negative controls.
2. The method for controlling the continuous evolution of eukaryotic cells according to claim 1, wherein said introducing mutation treatment in the second step comprises the steps of:
1) Paving cells;
2) The transfection mutant plasmids comprise dCS 9 expression plasmids and gRNA-AID mixture of target mutant genes.
3. The method of claim 2, wherein the dCas9 expression plasmid comprises a low ratio wide editing window of a to G, C to T, C to G editors.
4. The method for controlling the sustained evolution of eukaryotic cells according to claim 1, wherein said step two of introducing transiently transfected selection plasmids comprises the steps of:
1) The cells are spread out and the cells are spread out,
2) Transfection of defective antibiotic expression plasmids.
5. The method for controlling the sustained evolution of eukaryotic cells according to claim 1, wherein said antibiotic selection mutant treatment in step two comprises the steps of:
antibiotics are added during cell passage, 2-4 concentration gradients are set, the concentration of the antibiotics is gradually increased, surviving cells are screened, and surviving cells continue to evolve in the next round.
6. The method for controlling the continuous evolution of eukaryotic cells according to claim 5, wherein puromycin having an initial concentration of 1. Mu.g/ml is added at the time of cell passage, 2-4 concentration gradients are set and the concentration of puromycin added is gradually increased, and surviving cells are selected.
7. The method for controlling the continuous evolution of eukaryotic cells according to claim 5, wherein blasticidin having an initial concentration of 10. Mu.g/ml is added during passage of cells, surviving cells continue to evolve in the next round, 2-4 concentration gradients are set and the concentration of blasticidin added is gradually increased, and surviving cells are selected.
8. The method for controlling the continuous evolution of eukaryotic cells according to claim 5, wherein after the cell passage, antibiotics are added, 2-4 concentration gradients are set and the concentration of the added antibiotics is gradually increased, the surviving cells continue to evolve in the next round, the concentration of the added antibiotics is gradually increased in each round of evolution, and the concentration of the antibiotics reaches 300 mug/ml at most, so as to screen the surviving cells.
9. The method for controlling the continuous evolution of eukaryotic cells according to claim 1, wherein the functional verification in the fourth step comprises the steps of: after multiple rounds of mutation, the cell genome is extracted, the pseudo-evolution region is amplified by PCR and connected to the expression vector plasmid before evolution, and the effective mutant is prepared by sequencing.
10. The method for controlling the continuous evolution of eukaryotic cells according to claim 9, wherein when the antibiotic screening concentration reaches 200 μg/ml to 300 μg/ml, the genome of the cells is extracted, the region to be evolved is amplified by PCR and connected to the expression vector plasmid before evolution, and the effective mutants are prepared by ordinary sequencing, and the repeated, frame-shifted mutants and the wild type are eliminated.
11. The method for controlling the continuous evolution of eukaryotic cells according to claim 9, wherein the functional verification in the fourth step is performed after the effective mutants are prepared, wherein the functional verification comprises the steps of: constructing an expression vector containing mutation sites, and performing base editing efficiency test on the wild strain before evolution at a plurality of sites of a plurality of endogenous genes of a plurality of cell lines under the same condition, and detecting the expression of the mutant relative to the wild type by common sequencing or deep sequencing.
12. The method of claim 11, wherein the deep sequencing assay comprises editing efficiency, editing window, editing accuracy and off-target rate at DNA and RNA levels.
13. The method according to claim 1, wherein the mutant obtained in the second step is selected from a plurality of mutants comprising 16 mutation sites, wherein T140A, E146, 146G, L177P is mutated to T1, T (ACA) 80T (ACG), L (CTG) 104L (TTG), Y120H is mutated to T6, E24G, T A, E G is mutated to T16, S137P is mutated to T7, S (TCA) 137S (TCG), V139M, R198G is mutated to T17, L134S is mutated to T8, and W153C is mutated to CBE18.
14. The method for controlling the continuous evolution of eukaryotic cells according to claim 1, wherein the mutants obtained in said step two are selected from the following mutants after the evolution ABEmax: a1 comprises an amino acid mutation; a11 comprises an amino acid mutation; a19 includes Y9C, E24E, R25G, R38G, Y72C, T110A, N126D, Y207C, P251P, a259V, Y270Y, R295G; a26 comprises Y72C, M150V, K159E, a311T; a23 comprises N71S, Y72C; a14 comprises K157E, H211R, R295G, S368S, cas9n K R; m26 comprises E2G, Y9C, Y72C, Y270C; m10 comprises Y9C, N126D; m14 comprises R38G, N126D; m19 comprises R46G, N71S, Y72C, N126D, R149G, K159E; m25 comprises N126D, a141V; m4 comprises D118G, R220C; m5 comprises Y9C, Y72C, R97G, N126S; m1 comprises D118G, T187T, M258V; m7 comprises D118G, M258V; m8 comprises R149G; m9 comprises D118G; m13 comprises M258V; m11 comprises D118G, T187A, R223G; m22 comprises R149G, K159E; m17 comprises Y72C.
15. The method for controlling the continuous evolution of eukaryotic cells according to claim 1, wherein the mutants obtained in said step two are selected from the following mutants after evolution of ABE8 e: e3 comprises the amino acid mutation S171G, R152G, Y72C; e7 comprises N37D, L120P; e8 comprises N37G, L120P; e15 comprises N37G, R38G, R106G, S171G, G99C; e16 comprises L67P, L120P, N156D; e22 comprises N37S, N156D, Y80C; L120P, E24 comprises Y122C, M60I; e27 comprises E24G, R25G, Y122C; e28 comprises H127R, Y72C; e29 comprises R20G, L67P; e30 comprises N37D, L67P; e34 comprises N37D, Y122C, I98T, H51R, S170C, Q70R; e36 comprises E24G, R25G, N37G, R38G, L67P, S6G; e40 comprises N37G, R38G, L67P, R106G, N36S, cas 9T 38A; e44 comprises NLS K6E, R12G, N37G, R38G, R106G, V119A, Y122H; e48 comprises N37G, R38G, R106G, H51R, Q70R, N121D; e56 comprises L67P, H127R; e59 comprises R12G, N37D, L120P, Y122H; e60 comprises L120P, H127R; e65 comprises R20G, R106G; e67 contains R12G, E24G, R25G, L67P, N156D, linker S5G.
16. Use of a method for controlling the sustained evolution of eukaryotic cells according to any one of claims 1 to 15, characterized in that it is applicable at least to the following base editing systems: CBE, ABE, C to G gene editing system in CGBE and leader gene editing system in PE.
17. Use of a control method for the sustained evolution of eukaryotic cells according to any one of claims 1 to 15, characterized in that the method can be used to evolve a plurality of CRISPR-Cas gene editing systems, including at least Cas9, cas12, cas13, cas14 and Cas3.
18. Use of a control method for the sustained evolution of eukaryotic cells according to any one of claims 1 to 15, characterized in that the method is at least applicable for the evolution of inactive Cas proteins, nickase active Cas proteins and Cas proteins with nuclease activity.
19. Use of a method for controlling the sustained evolution of eukaryotic cells according to any one of claims 1 to 15, wherein the method is at least applicable for the evolution of currently known CRISPR-Cas systems and similar gene editing or base editing systems.
20. Use of a method for controlling the sustained evolution of eukaryotic cells according to any one of claims 16 to 19, wherein said similar gene editing or base editing system comprises IscB systems, prokaryotic Ago proteins, transcription activator-like effector nucleases, zinc finger nucleases.
21. Use of a method for controlling the sustained evolution of eukaryotic cells according to any one of claims 16 to 19, wherein said method is applicable to the evolution of gene editing and/or base editing systems in mammalian cells, plant cells.
22. Use of a control method for the sustained evolution of eukaryotic cells according to any one of claims 16 to 19, wherein TadA or Apobec comprising all amino acid mutations or nucleic acid mutations observed after the method has evolved is also useful for other gene editing and/or base editing systems such as, but not limited to, cas9, cas12, cas13, cas14, TALEN, ZFN.
23. Use of a method for controlling the sustained evolution of eukaryotic cells according to any one of claims 16 to 19, wherein the method is capable of evolving not only Cas proteins but also functional proteins co-acting with Cas proteins including but not limited to cytosine deaminase, adenosine deaminase, activating transcription elements, reverse transcriptase, and also directed RNAs including one or more of gRNA, sgRNA, crRNA, tracrRNA and pegRNA.
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