A method for improving gene knockout efficiency and its application

Abstract

The present invention discloses a method for improving the efficiency of gene knockout and its application. The knockout technology provided by the present invention is based on CRISPR-Cas9 plasmid, and further adopts a "repair strategy" to knock out the target gene. The "repair strategy" described in the present invention is to express the target knockout gene in an exogenous plasmid after mutation, and then eliminate the repair plasmid after knocking out the target gene on the genome, so as to achieve the deletion of the target gene. The use of the "repair strategy" increases the knockout efficiency of the target gene from 0 to 8%.

Description

Technical Field

[0001] The invention belongs to the technical field of gene engineering, and specifically relates to a method for improving gene knockout efficiency and an application thereof. Background Art

[0002] Genetic engineering is a key technology for constructing chassis cells. The deletion of key genes in metabolic pathways or the overexpression of genes has become a conventional strategy. Currently, the more mature method is CRISPR-Cas9 technology.

[0003] CRISPR / Cas9 is the third generation of "genomic site-specific editing technology" after "zinc finger endonucleases (ZFNs)" and "transcription activator-like effector nucleases (TALENs)". With its advantages of low cost, easy operation and high efficiency, CRISPR / Cas9 has quickly become popular in laboratories around the world and has become a powerful helper for biological research.

[0004] Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) are short palindromic sequences found in bacteria that are regularly clustered and spaced. They can help bacteria resist the invasion of bacteriophages and are acquired immunity of bacteria against bacteriophages. The CRISPR sequence is composed of many short and conservative repeat sequences (repeat) and spacer sequences (spacer). The repeat sequence contains a palindromic sequence that can form a hairpin structure. The spacer region is special. They are foreign DNA sequences captured by bacteria. When these foreign genetic materials invade again, the CRISPR / Cas system will accurately strike them. The leader region upstream is considered to be the promoter of the CRISPR sequence. In addition, there is a polymorphic family gene upstream. The protein encoded by this gene can work together with the CRISPR sequence region. Therefore, this gene is named CRISPR associated gene (CRISPR associated, Cas). The Cas gene and the CRISPR sequence co-evolved to form a highly conservative CRISPR / Cas system in bacteria.

[0005] The principle of CRISPR / Cas9 system for gene knockout is that tracrRNA-crRNA is used to guide the action of Cas9 when it is fused into a single-stranded guide RNA (i.e., sgRNA). For the target gene, the artificially designed sgRNA (small guide RNA, sgRNA) is used to identify the target gene sequence and guide the Cas9 protease to effectively cut the DNA double strands to form a double-strand break of the DNA. Under normal circumstances, cells use an efficient non-homologous end joining method (Non-homologous End Joining, NHEJ) to repair the broken DNA. However, during the repair process, mismatches of base insertion or deletion usually occur, resulting in frameshift mutations, which make the target gene lose its function, thereby achieving gene knockout.

[0006] CRISPR / Cas9 is highly efficient in deleting meaningless genes, inserting target genes into neutral sites, and knocking out genes that have little effect on the normal metabolism of the strain. However, if the target gene to be knocked out has a significant impact on the normal growth of the strain, or if the constructed engineered strain needs to continuously knock out multiple genes in the same metabolic pathway, the efficiency of ordinary CRISPR-Cas9-mediated gene knockout is low or even zero. Summary of the invention

[0007] The purpose of this section is to summarize some aspects of embodiments of the present invention and briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this section and the specification abstract and the invention title of this application to avoid blurring the purpose of this section, the specification abstract and the invention title, and such simplifications or omissions cannot be used to limit the scope of the present invention.

[0008] In view of the above problems and / or the problems existing in the prior art, the present invention is proposed.

[0009] Therefore, the purpose of the present invention is to overcome the deficiencies in the prior art and provide a method for improving gene knockout efficiency.

[0010] To solve the above technical problems, the present invention provides the following technical solution: first construct an exogenous plasmid containing the gene fragment to be knocked out in the genome of the target strain, then introduce the exogenous plasmid into the target strain through seamless cloning technology, and then use CRISPR-Cas9 technology to knock out the target gene fragment on the strain genome, and finally gradually lose the exogenous plasmid through multiple passaging, thereby achieving the effect of knocking out the target gene.

[0011] As a preferred embodiment of the gene knockout method of the present invention, the exogenous plasmid is a plasmid containing a complementing gene expression cassette.

[0012] As a preferred embodiment of the gene knockout method of the present invention, the complemented gene after the synonymous mutation is not recognized by sgRNA.

[0013] As a preferred embodiment of the gene knockout method of the present invention, the gene complementation knockout plasmid is SX1628-71sgX, and its gene sequence is shown in SEQ ID NO:2.

[0014] As a preferred embodiment of the gene knockout method of the present invention, the gene to be knocked out is overexpressed using an exogenous plasmid.

[0015] As a preferred embodiment of the gene knockout method of the present invention, the gene knockout efficiency can reach 8%.

[0016] Another object of the present invention is to solve the deficiencies in the prior art and provide an application of a method for improving gene knockout efficiency.

[0017] In order to solve the above technical problems, the present invention provides the following technical solutions: the gene knockout method can be used to continuously knock out multiple genes in the same metabolic pathway.

[0018] As a preferred embodiment of the application of the gene knockout method of the present invention, the gene knockout method can be used to knock out a target gene that has a significant impact on the growth of the strain to be knocked out.

[0019] Beneficial effects of the present invention:

[0020] (1) The present invention is based on CRISPR-Cas9-mediated gene knockout and increases the efficiency of gene knockout through the "gene complementation strategy".

[0021] (2) Therefore, the present invention uses an exogenous plasmid to overexpress the gene to be knocked out, so that even if the gene is missing, it will not affect the normal metabolism of the strain. Subsequently, after knocking out the target gene, the exogenous plasmid is gradually lost through multiple passages, thereby achieving the effect of knocking out the target gene.

[0022] (3) The present invention uses the GS115-ΔAgt strain as the starting strain, and constructs a complementing knockout plasmid SX1628-71sgX for knocking out the Gly1 gene and a knockout plasmid SX1628-71sgXG without a complementing gene expression cassette. The results show that the efficiency of knocking out the Gly1 gene by the plasmid SX1628-71sgXG is 0, while the efficiency of knocking out the Gly1 gene by the plasmid SX1628-71sgX can reach 8%. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for describing the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative labor. Among them:

[0024] Figure 1 It is a schematic diagram of the structure of the artificially modified plasmid SX1628-71.

[0025] Figure 2 This is a schematic diagram of the structure of the artificially modified plasmid SX1628-71sgX.

[0026] Figure 3 This is the electrophoresis diagram of the fragments during the construction of plasmid SX1628-71.

[0027] Figure 4 This is the plate map of JM109-SX1628-71 transformants.

[0028] Figure 5 This is the electrophoresis diagram of the optimized fragment of gene Gly1.

[0029] Figure 6 These are the electrophoresis images of two fragments of SX1628-71 plasmid, a is SX1628-71 plasmid fragment 1, and b is SX1628-71 plasmid fragment 2.

[0030] Figure 7 This is a comparison chart of the sequencing results of SX1628-71sgX.

[0031] Figure 8 This is the electropherogram of the homology arm construction of Gly1 knockout.

[0032] Fig. 9 This is the electrophoresis diagram of SX1628-71sgX-mediated Gly1 knockout.

[0033] Fig.10 This is the sequencing result of SX1628-71sgXG.

[0034] Fig.11 This is the electrophoresis image of Gly1 knockout mediated by SX1628-71sgXG. DETAILED DESCRIPTION

[0035] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with the embodiments of the specification.

[0036] In the following description, many specific details are set forth to facilitate a full understanding of the present invention, but the present invention may also be implemented in other ways different from those described herein, and those skilled in the art may make similar generalizations without violating the connotation of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0037] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The term "in one embodiment" that appears in different places in this specification does not necessarily refer to the same embodiment, nor does it refer to a separate or selective embodiment that is mutually exclusive with other embodiments.

[0038] Sources of biological materials

[0039]

[0040] Culture medium

[0041] (1) LLBZ liquid medium (g / L): yeast extract powder 5, peptone 10, sodium chloride 5, bleomycin final concentration 25ug / ml, pH 7.0;

[0042] (2) LLBZ solid medium (g / L): yeast extract powder 5, peptone 10, sodium chloride 5, agar 20, bleomycin final concentration 25ug / ml, pH 7.0;

[0043] (3) YPDSZ liquid medium (g / L): yeast extract powder 10, peptone 20, sorbitol 182, glucose 20, bleomycin final concentration 100 ug / ml, pH 7.0;

[0044] (4) YPDSZ solid medium (g / L): yeast extract powder 10, peptone 20, sorbitol 182, glucose 20, agar 20, bleomycin final concentration 100 ug / ml, pH 7.0.

[0045] Embodiment 1:

[0046] Construction of SX1628-71 plasmid

[0047] The steps for the SX1628-71 plasmid used in this experiment are as follows:

[0048] 1) Using plasmid SX1628-7 as a template, primers were designed to construct plasmid SX1628-71 by PCR, and this plasmid can be used as a backbone to further construct knockout plasmids. Primers were designed to construct the pGAP-EGFP-RPS25Att expression cassette, which was then homologously recombined with SX1628-7 to construct plasmid SX1628-71.

[0049] The primers for constructing plasmid SX1628-71 are shown in Table 13, and the primers were synthesized at GENEWIZ. The PCR program for the plasmid construction process is shown in Table 1, and the PCR program is shown in Table 2.

[0050] Table 1 PCR reaction system

[0051]

[0052] Table 2 PCR reaction program

[0053]

[0054] Recover the agarose gel band with the correct molecular weight and determine its concentration before use; the electrophoresis diagram of the PCR amplification product is shown in the attached figure. Figure 3 As shown, SX1628-7 plasmid fragment (4979 bp), pGAP (477 bp), EGFP expression cassette (1012 bp), RPS25Att (485 bp), and Bleo expression cassette (1153 bp).

[0055] 2) Design primers to fuse the Bleo expression cassette with the RPS25Att expression cassette by PCR to obtain the RPS25Att+Bleo expression cassette fragment; Figure 3 As shown, the sequence is SEQ ID NO: 3;

[0056] 3) Design primers to perform overlapping PCR on the RPS25Att+Bleo expression cassette fragment, the EGFP expression cassette fragment and the pGAP fragment to obtain the fragment pGAP+EGFP expression cassette+RPS25Att+Bleo expression cassette, the sequence of which is shown in SEQ ID NO: 4;

[0057] 4) The pGAP+EGFP expression cassette+RPS25Att+Bleo expression cassette and plasmid fragment were connected by seamless cloning and transformed into JM109. After culturing for 1 hour, the bacterial solution was spread on LLBZ plates.

[0058] 5) Pick a single green colony on the plate and culture it at 37°C for 16 hours, then extract the SX1628-71 plasmid for later use. Figure 4 shown.

[0059] Embodiment 2:

[0060] Construction of SX1628-71sgX plasmid.

[0061] This example mainly provides the SX1628-71sgX plasmid and homology arms constructed by knocking out the gene Gly1. The specific steps are as follows:

[0062] That is, based on the SX1628-71 plasmid, the SX1628-71sgX plasmid containing the Gly1 optimized gene and the sg sequence was constructed.

[0063] 1) Use primers sgX-F1 and sgX-R1 to obtain the sg fragment; that is, use primers sgX-F1 and sgX-R1 as templates for PCR, and the system is shown in Table 3:

[0064] Table 3 sg fragment construction PCR system

[0065]

[0066] 2) Primers sgX-F2 and GLY1-sg2-yh-R1, primers GLY1-sg2-yh-F1 and sgX-R2 were used to perform sg optimization of the Gly1 gene. The Gly1 gene sequence before optimization is shown in SEQ ID NO: 5, and the Gly1 gene sequence after optimization is shown in SEQ ID NO: 6; the PCR program is shown in Table 2, and the Gly1 optimized gene fragment 1 and fragment 2 are fused by PCR to construct the Gly1 optimized gene complete fragment. The PCR system is shown in Tables 4 to 6

[0067] Table 4 PCR system for Gly1 optimized gene fragment 1

[0068]

[0069]

[0070] Table 5 PCR system for optimizing gene fragment 2 of Gly1

[0071]

[0072] Table 6 Gly1 optimized fusion system of gene fragment 1 and fragment 2

[0073]

[0074] Table 7 Gly1 optimized gene PCR system

[0075]

[0076] After the PCR of Gly1 optimized gene (1176 bp) was completed, agarose gel electrophoresis was performed. The electrophoresis diagram is shown in Figure 5 As shown, the glue is recovered and stored at low temperature for future use;

[0077] 3) Primers SX1628-71-F1 and SX1628-71-R1 were used to construct SX1628-71 plasmid fragment 1 (5403 bp), and primers SX1628-71-F2 and SX1628-71-R2 were used to construct SX1628-71 plasmid fragment 2 (1310 bp). The electrophoresis diagram is as follows: Figure 6 (a) and (b);

[0078] 4) The sg fragment, the complete fragment of the Gly1 optimized gene, the SX1628-71 plasmid fragment 1 and the SX1628-71 plasmid fragment 2 in Example 2 were seamlessly cloned and recombined to construct the SX1628-71sgX plasmid. The recombination system is shown in Table 8, and the recombination procedure is shown in Table 9; the recombinant product was transformed into Escherichia coli JM109, coated on LLBZ plates, and cultured at 37°C overnight.

[0079] Table 8 Homologous recombination system

[0080]

[0081] Table 9 Homologous recombination program

[0082]

[0083] 5) Pick a single colony from the plate and culture it in LLBZ liquid medium for 12 hours, then extract the plasmid for sequencing. The sequencing results are as follows: Figure 7 As shown, the plasmid with correct sequencing is plasmid SX1628-71sgX, and the plasmid is stored at -20°C for future use.

[0084] Embodiment 3:

[0085] Knockout of the Gly1 gene in the GS115-ΔAgt strain.

[0086] 1) Construction of knockout homology arms. Primers GLY1-TY-F1 and GLY1-TY-R11 were used to construct Gly1 knockout homology fragment 1, and primers GLY1-TY-F2 and GLY1-TY-R2 were used to construct Gly1 knockout homology fragment 2. The template was the GS115 genome. The PCR system was referred to Table 4-Table 6, and the PCR program was referred to Table 2. The nucleic acid electrophoresis diagrams of Gly1 knockout homology fragment 1 and Gly1 knockout homology fragment 2 are shown in FIG. Figure 8 (a) shows the final constructed Gly1 knockout homologous fragment nucleic acid electrophoresis diagram. Figure 8 As shown in (b); the Gly1 knockout homologous fragment was recovered by gel and then placed at low temperature for future use.

[0087] 2) Preparation of competent cells of GS115-Δagt strain. Take out the GS115-ΔAgt strain from the -80°C freezer, thaw it at room temperature, and place it on a clean bench. In a sterile environment, apply 200 μL of bacterial solution to YPDS medium and culture for 16 hours before preparing competent cells. The prepared competent cells are placed in a -80°C freezer for later use.

[0088] 3) 2 μg of the Gly1 knockout homologous fragment and the SX1628-71sgX plasmid constructed in Example 2 were transformed into GS115-ΔAgt competent cells, cultured at 30° C. for 1.5 h, and then spread on YPDSZ medium and cultured for 2 days.

[0089] 4) Pick a single colony from the plate in step 3 of Example 3 and culture it in YPDSZ liquid medium for 2 days, then extract the genome, and verify the deletion of the Gly1 gene by PCR. The homology arm verification primers are GLY1-TY-F1 and GLY1-TY-R2, and the Gly1 gene internal verification primers are GLY1-NBYZ-F and GLY1-NBYZ-R. The PCR system is shown in Table 10, the program is shown in Table 11, and the nucleic acid electrophoresis diagram is shown in Table 12. Fig. 9 shown.

[0090] Table 10Gly gene deletion verification PCR system

[0091]

[0092] Table 11 PCR reaction program

[0093]

[0094] Embodiment 4:

[0095] Calculation of the efficiency of knocking out the Gly gene using plasmid SX1628-71sgX.

[0096] 1) The knockout efficiency was calculated based on the nucleic acid electrophoresis diagram in step (4) of Example 3. The calculation formula is as follows:

[0097] Formula (1): Knockout efficiency = number of gene-deficient single colonies / number of picked single colonies; the knockout efficiency results are shown in Table 12.

[0098] Comparative Example 1:

[0099] The plasmid SX1628-71sgXG was used to knock out the Gly1 gene.

[0100] 1) Construction of plasmid SX1628-71sgXG. Plasmid SX1628-71sgX was used as a template and primers Bleo-F and SX1628-71sgX-CZ-R were used for PCR. The PCR system is shown in Table 1 and the PCR program is shown in Table 2. After the PCR was completed, the template plasmid was eliminated using DpnI and then transformed into JM109 competent cells. The cells were placed in a shaker at 37°C, 200 rpm and cultured for 1 hour. After that, the cells were spread on LLBZ plates and the plates were placed at 37°C overnight.

[0101] 2) Pick a single colony from the plate and culture it in 3 mL of LLBZ liquid medium. Place it in a shaker at 37°C and 200 rpm for 16 hours. Then extract the plasmid for sequencing. The sequencing results are as follows: Fig.10 As shown, the plasmid with correct sequencing was saved for future use.

[0102] 3) 2 μg of the Gly1 knockout homologous fragment and the SX1628-71sgXG plasmid constructed in step (2) of comparative example 1 were transformed into GS115-ΔAgt competent cells, cultured at 30° C. for 1.5 h, and then applied to YPDSZ medium and cultured for 2 days.

[0103] 4) Pick a single colony from the plate in step 3 of Example 3 and culture it in YPDSZ liquid medium for 2 days, then extract the genome, and verify the deletion of the Gly1 gene by PCR. The homology arm verification primers are GLY1-TY-F1 and GLY1-TY-R2, and the Gly1 gene internal verification primers are GLY1-NBYZ-F and GLY1-NBYZ-R. The PCR system is shown in Table 10, the program is shown in Table 11, and the nucleic acid electrophoresis diagram is shown in Table 12. Fig.11 shown.

[0104] Comparative Example 2:

[0105] Calculation of the efficiency of knocking out the Gly gene using plasmid SX1628-71sgXG.

[0106] 1) The knockout efficiency was calculated according to the nucleic acid electrophoresis diagram in step (4) of comparative example 1. The calculation formula is shown in formula (1). The knockout efficiency comparison is shown in Table 12.

[0107] Table 12 Comparison of knockout efficiency of complementation plasmid

[0108] Plasmid type SX1628-71sgXG SX1628-71sgX Knockout efficiency 0 8％

[0109] Table 13 Primer sequence list

[0110]

[0111]

[0112] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the claims of the present invention.

Claims

1. A method for improving gene knockout efficiency, characterized in that: It includes first constructing an exogenous plasmid containing the gene fragment to be knocked out in the genome of the target strain, then introducing the exogenous plasmid into the target strain through seamless cloning technology, then using CRISPR-Cas9 technology to knock out the target gene fragment on the genome of the strain, and finally gradually losing the exogenous plasmid through multiple passages; The exogenous plasmid is SX1628-71sgX, and its gene sequence is shown in SEQ ID NO: 2; The target strain is K.phaffi.GS115-ΔAgt; The knockout efficiency of the gene knockout method for knocking out the Gly gene is 8%.

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