CN114657177B - Method for simultaneously realizing gene editing and transcriptional regulation by using I-type CRISPR-Cas system - Google Patents

Abstract

The present invention discloses a method for simultaneously realizing gene editing and transcriptional regulation using a type I CRISPR-Cas system. The method provided by the present invention comprises the following steps: using a type I CRISPR-Cas system A to perform transcriptional regulation on gene A and using a type I CRISPR-Cas system B to perform gene editing on gene B; the target gene of the type I CRISPR-Cas system A is gene A and is a modified type I CRISPR-Cas system; the modified type I CRISPR-Cas system refers to a type I CRISPR-Cas system after the gRNA of the type I CRISPR-Cas system in the prior art is modified; the modification of the gRNA is to replace the spacer in the gRNA of the type I CRISPR-Cas system in the prior art with a spacer truncate; the target gene of the type I CRISPR-Cas system B is gene B and is a type I CRISPR-Cas system in the prior art. The present invention can simultaneously realize gene editing and different degrees of transcriptional inhibition.

Description

A method for simultaneously achieving gene editing and transcriptional regulation using a type I CRISPR-Cas system

Technical Field

The present invention belongs to the field of biotechnology, and specifically relates to a method for simultaneously realizing gene editing and transcriptional regulation by using a type I CRISPR-Cas system.

Background technique

The CRISPR-Cas system has been widely used as a gene editing tool and has also been developed for transcriptional regulation. The type I CRISPR-Cas system is the most widely distributed. The principle of gene editing by the type I CRISPR-Cas system is that the gRNA binds to the Cascade effector, and after recognizing the target sequence, it recruits the Cas3 nuclease to cut the target, produce a double strand break (DSB), and activate the non-homologous end joining or homologous recombination repair mechanism, thereby introducing mutations in the target gene or precisely editing the target gene. To perform transcriptional regulation on the target gene, it is usually necessary to knock out the Cas3 protein or mutate its active site to prevent Cas3 from cutting the target gene. When the Cas3 endonuclease activity is lost, the gRNA and the Cascade effector will bind to the target DNA. Although the target cannot be cut and degraded, the binding of the effector will prevent the binding of RNA polymerase or transcription extension, thereby realizing the function of transcriptional regulation. Therefore, in the past, it was impossible to complete gene editing and transcriptional regulation at the same time using a CRISPR-Cas system.

Summary of the invention

The purpose of the present invention is to provide a method for simultaneously achieving gene editing and transcriptional regulation using a type I CRISPR-Cas system.

The present invention provides a method for transcriptionally regulating gene A of a target organism and performing gene editing on gene B thereof, comprising the following steps: using type I CRISPR-Cas system A to transcriptionally regulate gene A and using type I CRISPR-Cas system B to perform gene editing on gene B;

The target gene of Type I CRISPR-Cas system A is gene A and it is a modified Type I CRISPR-Cas system; the modified Type I CRISPR-Cas system refers to a Type I CRISPR-Cas system after the gRNA of the Type I CRISPR-Cas system in the prior art is modified; the modification of the gRNA is to replace the spacer in the gRNA of the Type I CRISPR-Cas system in the prior art with a spacer truncation;

The target gene of type I CRISPR-Cas system B is gene B and it is a type I CRISPR-Cas system in the prior art.

For ease of distinction, the gRNA of type I CRISPR-Cas system A is referred to as gRNA A, and the gRNA of type I CRISPR-Cas system B is referred to as gRNA B.

Type I CRISPR-Cas system A and type I CRISPR-Cas system B can share the Cascade effector and Cas3 nuclease.

The gene editing of gene B may be precise gene editing of gene B, such as mutation, insertion, deletion, etc.

Exemplarily, the organism is an organism with an endogenous type I CRISPR-Cas system. The method can be implemented by introducing a DNA molecule encoding gRNA A and a DNA molecule encoding gRNA B into the organism. The DNA molecule encoding gRNA A and the DNA molecule encoding gRNA B can be introduced into the organism through the same plasmid. The plasmid also has a Donor fragment required for gene editing of gene B.

Exemplarily, the organism is an organism that does not have an endogenous type I CRISPR-Cas system. The method can be implemented by introducing a DNA molecule encoding gRNA A, a DNA molecule encoding gRNA B, a DNA molecule encoding a Cascade effector, and a DNA molecule encoding a Cas3 endonuclease into the organism. Each DNA molecule can be introduced into the organism through the same plasmid, and can be introduced into the organism through multiple plasmids. The plasmid also has a Donor fragment required for gene editing of gene B.

The present invention also provides a kit for transcriptionally regulating gene A of a target organism and gene editing its gene B, comprising type I CRISPR-Cas system A and type I CRISPR-Cas system B;

The target gene of Type I CRISPR-Cas system A is gene A and it is a modified Type I CRISPR-Cas system; the modified Type I CRISPR-Cas system refers to a Type I CRISPR-Cas system after the gRNA of the Type I CRISPR-Cas system in the prior art is modified; the modification of the gRNA is to replace the spacer in the gRNA of the Type I CRISPR-Cas system in the prior art with a spacer truncation;

The target gene of type I CRISPR-Cas system B is gene B and it is a type I CRISPR-Cas system in the prior art.

For ease of distinction, the gRNA of type I CRISPR-Cas system A is referred to as gRNA A, and the gRNA of type I CRISPR-Cas system B is referred to as gRNA B.

Type I CRISPR-Cas system A and type I CRISPR-Cas system B can share the Cascade effector and Cas3 nuclease.

The gene editing of gene B may be precise gene editing of gene B, such as mutation, insertion, deletion, etc.

The kit includes gRNA A and gRNA B.

For another form, the kit includes biological materials for preparing gRNA A and gRNA B, such as a DNA molecule encoding gRNA A and a DNA molecule encoding gRNA B, such as a DNA molecule encoding gRNA A and gRNA B, such as a recombinant plasmid having a DNA molecule encoding gRNA A and a recombinant plasmid having a DNA molecule encoding gRNA B, such as a recombinant plasmid having a DNA molecule encoding gRNA A and gRNA B.

The kit may further include a Cascade effector or a DNA molecule encoding a Cascade effector or a recombinant plasmid having a DNA molecule encoding a Cascade effector.

The kit also includes Cas3 endonuclease or a DNA molecule encoding Cas3 endonuclease or a recombinant plasmid having a DNA molecule encoding Cas3 endonuclease.

The present invention also protects a method for transcriptionally regulating a target gene of a target organism, comprising the following steps:

Using the modified type I CRISPR-Cas system to regulate the transcription of target genes;

The modified type I CRISPR-Cas system refers to a type I CRISPR-Cas system after the gRNA of the type I CRISPR-Cas system in the prior art is modified; the modification of the gRNA is to replace the spacer in the gRNA of the type I CRISPR-Cas system in the prior art with a spacer truncation.

The modified gRNA of the type I CRISPR-Cas system is referred to as modified gRNA.

Exemplarily, the organism is an organism with an endogenous type I CRISPR-Cas system. The method can be achieved by introducing a DNA molecule encoding the modified gRNA into the organism.

Exemplarily, the organism is an organism that does not have an endogenous type I CRISPR-Cas system. The method can be implemented by introducing a DNA molecule encoding a modified gRNA, a DNA molecule encoding a Cascade effector, and a DNA molecule encoding a Cas3 endonuclease into the organism. Each DNA molecule can be introduced into the organism via the same plasmid, and can be introduced into the organism via multiple plasmids.

The present invention also protects a kit for transcriptional regulation of a target gene of a target organism, comprising a modified type I CRISPR-Cas system;

The modified type I CRISPR-Cas system refers to a type I CRISPR-Cas system after the gRNA of the type I CRISPR-Cas system in the prior art is modified; the modification of the gRNA is to replace the spacer in the gRNA of the type I CRISPR-Cas system in the prior art with a spacer truncation.

The modified gRNA of the type I CRISPR-Cas system is referred to as modified gRNA.

The kit includes the modified gRNA.

In another form, the kit includes biological materials for preparing the modified gRNA, such as a DNA molecule encoding the modified gRNA, such as a recombinant plasmid having a DNA molecule encoding the modified gRNA.

The kit may further include a Cascade effector or a DNA molecule encoding a Cascade effector or a recombinant plasmid having a DNA molecule encoding a Cascade effector.

The kit also includes Cas3 endonuclease or a DNA molecule encoding Cas3 endonuclease or a recombinant plasmid having a DNA molecule encoding Cas3 endonuclease.

The type I CRISPR-Cas system in the prior art consists of a Cascade effector, a Cas3 endonuclease, and a gRNA. The gRNA binds to the Cascade effector, recognizes the target sequence, and recruits the Cas3 endonuclease to cut the target.

Spacer is the segment in gRNA that specifically binds to the target sequence.

Spacer is the segment of gRNA that specifically binds to the target sequence through complementary base pairing.

The target sequence is a sequence adjacent to and downstream of the PAM in the target gene.

Any of the above-mentioned spacer truncations is obtained by truncating the distal part of the spacer targeting PAM in the gRNA of the type I CRISPR-Cas system in the prior art.

The transcriptional regulation is to inhibit gene expression.

Exemplarily, the type I CRISPR-Cas system may be a type I-B CRISPR-Cas system.

Accordingly, the length of the spacer is 31-72 nt. Accordingly, the length of the spacer truncation is 16-28 nt. Preferably, the length of the spacer truncation is 20-28 nt.

Exemplarily, any of the above modified gRNAs include the following segments: an upstream repeat segment, a spacer truncation, and a downstream repeat segment. Exemplarily, any of the above modified gRNAs consist of the following segments: an upstream repeat segment, a spacer truncation, and a downstream repeat segment. Exemplarily, the upstream repeat segment is the RNA corresponding to the DNA shown in nucleotides 81-88 in Sequence 2 of the sequence table. Exemplarily, the downstream repeat segment is the RNA corresponding to the DNA shown in nucleotides 105-126 in Sequence 2 of the sequence table.

Exemplarily, any of the above gRNA A comprises the following segments: an upstream repeat segment, a spacer truncation, and a downstream repeat segment. Exemplarily, any of the above gRNA A consists of the following segments: an upstream repeat segment, a spacer truncation, and a downstream repeat segment. Exemplarily, the upstream repeat segment is the RNA corresponding to the DNA shown in nucleotides 81-88 in Sequence 2 of the sequence table. Exemplarily, the downstream repeat segment is the RNA corresponding to the DNA shown in nucleotides 105-126 in Sequence 2 of the sequence table.

Exemplarily, any of the above gRNA B includes the following segments: an upstream repeat segment, a spacer, and a downstream repeat segment. Exemplarily, any of the above gRNA B consists of the following segments: an upstream repeat segment, a spacer, and a downstream repeat segment. Exemplarily, the upstream repeat segment is the RNA corresponding to the DNA shown in nucleotides 81-88 in Sequence 2 of the sequence table. Exemplarily, the downstream repeat segment is the RNA corresponding to the DNA shown in nucleotides 105-126 in Sequence 2 of the sequence table.

Exemplarily, any of the above described DNA molecules encoding the modified gRNA includes the following segments: repeat coding DNA, spacer truncated coding DNA and repeat coding DNA. Exemplarily, any of the above described DNA molecules encoding the modified gRNA consists of the following segments: repeat coding DNA, spacer truncated coding DNA and repeat coding DNA. Repeat coding DNA is shown in nucleotides 59-88 in sequence 2 of the sequence table.

Exemplarily, any of the above-described DNA molecules encoding gRNA A includes the following segments: repeat coding DNA, spacer truncated coding DNA, and repeat coding DNA. Exemplarily, any of the above-described DNA molecules encoding gRNA A consists of the following segments: repeat coding DNA, spacer truncated coding DNA, and repeat coding DNA. Repeat coding DNA is shown in nucleotides 59-88 in sequence 2 of the sequence table.

Exemplarily, any of the above-described DNA molecules encoding gRNA B includes the following segments: repeat encoding DNA, spacer encoding DNA, and repeat encoding DNA. Exemplarily, any of the above-described DNA molecules encoding gRNA B consists of the following segments: repeat encoding DNA, spacer encoding DNA, and repeat encoding DNA. Repeat encoding DNA is shown in nucleotides 59-88 in sequence 2 of the sequence table.

Exemplarily, the type I CRISPR-Cas system may be the CRISPR-Cas system of H. hispanica.

Exemplarily, the type I CRISPR-Cas system may be the CRISPR-Cas system of the Spanish salt box bacteria HH DLCR strain.

Exemplarily, the PAM may be TTC.

Exemplarily, any of the above-mentioned organisms may be microorganisms or plants.

For the embodiments of the present invention, the organism may specifically be Spanish salt box bacteria.

The present invention discloses for the first time a method for simultaneously realizing two functions of gene editing and transcriptional regulation using a type I CRISPR-Cas system. When the spacer in the gRNA is of wild-type length or longer, the CRISPR-Cas system can realize gene editing of the target; when the gRNA is modified to shorten the spacer, there is no need to knock out or mutate Cas3, and the CRISPR-Cas system can achieve different degrees of transcriptional inhibition of the target gene, but will not cut the target. In addition, since different target sites of the same gene also have different inhibition strengths, combining these two factors, a transcriptional inhibition library with richer gradients can be constructed. In the present invention, the system can also target multiple sites at a time, and by changing the corresponding spacer length, gene editing or different degrees of transcriptional inhibition can be achieved respectively.

In the present invention, there is no need to knock out or mutate Cas3. Gene editing and different degrees of transcription inhibition can be achieved simultaneously by simply changing the length of the spacer in the gRNA.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a schematic diagram of the structure of the recombinant plasmid.

FIG. 2 shows the results of step 3, step 4 and step 5 of Example 1.

FIG. 3 is the result of step six of Example 1.

FIG. 4 shows the results of Example 2.

Detailed ways

The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial channels. Each plasmid in the examples has been sequenced and verified.

Unless otherwise specified, the quantitative tests in the following examples were performed three times and the results were averaged.

The HH DLCR strain, whose full name is the Spanish salt box bacteria HH DLCR strain, is obtained by knocking out the pyrF gene and CRISPR sequence of the Spanish salt box bacteria (Haloarcula hispanica) ATCC 33960 strain. Knocking out the pyrF gene causes nutritional deficiency. The plasmid pWL502 (or its derivative plasmid) contains this gene, so that the successfully transformed strain is complemented to a non-nutritional deficiency strain; knocking out the CRISPR sequence can remove the background effect. Literature recording the HH DLCR strain (the HH DLCR strain is the △CR strain in the literature): Rui W, Ming L, Luyao G, et al. DNA motifs determining the accuracy of repeat duplication during CRISPR adaptation in Haloarcula hispanica [J]. Nucl C Acids Research, 2016 (9): 4266-4277.

Plasmid pWL502 (plasmid pWL502), plasmid pGK (pGK plasmid), plasmid pGKBE (pGKB Eplasmid) are all described in the following literature: Cheng, F., Gong, L., Zhao, D., Yang, H., Zhou, J., Li, M., and Xiang, H. (2017). Harnessing the native type I-B CRISPR-Cas for genome editing in a polyploid archaeon. J Genet Genomics 44, 541-548. Plasmid pGK and plasmid pGKBE are both derivative plasmids of plasmid pWL502.

30% artificial saline (30% SW): weigh 240 g of sodium chloride, 30 g of magnesium chloride hexahydrate, 35 g of magnesium sulfate heptahydrate, 7 g of potassium chloride, and 0.555 g of anhydrous calcium chloride (dissolved in 5 mL of distilled water); first add distilled water to each component and stir to dissolve, then slowly add calcium chloride solution and then adjust the volume to 1 L.

BSS-LS solution: weigh 5.58 g of sodium chloride, 0.201 g of potassium chloride, and 15 g of sucrose, add distilled water, stir to dissolve, and make up to 95 mL; sterilize with high-pressure steam at 115°C for 30 min; then add 5 mL of sterilized Tris-Cl (1 M, pH 8.2).

BSS-LS/Gly ⁺ solution: weigh 5.58 g of sodium chloride, 0.201 g of potassium chloride, 15 g of sucrose, and 15 mL of glycerol, add distilled water, stir to dissolve, and make up to 95 mL; sterilize with high-pressure steam at 115°C for 30 min; then add 5 mL of sterilized Tris-Cl (1 M, pH 8.2).

UBSS-LS solution: weigh 5.58 g of sodium chloride, 0.201 g of potassium chloride, and 15 g of sucrose, add distilled water, stir to dissolve, and make up to 100 mL; adjust the pH to 7.0-7.5 with Tris base or NaOH; sterilize by high-pressure steam at 115°C for 30 min.

60% PEG600 solution: Take 600 μL of PEG600 and mix thoroughly with 400 μL of UBSS-LS solution.

AS-168 culture medium: weigh 200g sodium chloride, 20g magnesium sulfate heptahydrate, 2g potassium chloride, 3g trisodium citrate, 5g acid hydrolyzed casein, 5g yeast extract, 1.8g sodium glutamate, trace amounts of ferrous sulfate heptahydrate and manganese chloride tetrahydrate, add distilled water, stir and dissolve, and dilute to 1L; adjust pH to 7.0-7.2 with NaOH; sterilize by high pressure steam at 115℃ for 30min. Add 1.2% agar powder to solid culture medium.

AS-168SY Medium: AS-168 medium formula minus the yeast extract component.

23% MGM recovery medium: Measure 76.7 mL of 30% artificial saline, weigh 0.1 g of yeast extract, 0.5 g of soy peptone, and 15 g of sucrose, add distilled water, stir to dissolve, and make up to 100 mL; adjust the pH to 7.5 with Tris base or NaOH; sterilize with high-pressure steam at 115°C for 30 min.

Embodiment 1,

The crtB gene of Spanish salt box bacteria, as shown in sequence 1 of the sequence table, encodes phytoene synthase (HAH_2563). Knocking out this gene will block the expression of carotenoids and change the bacteria from red to white.

Both the Cascade effector and the Cas3 endonuclease are endogenous to H.

In this example, for the crtB gene of Spanish salt box bacteria, its own type I-B CRISPR-Cas system was used, and gRNAs with spacers of different lengths were used to achieve different degrees of transcriptional inhibition of the crtB gene without cutting it.

1. Construction of plasmids for expressing gRNAs with different spacer lengths

1. Preparation of 7 PphaR-R-spacer-R fragments

In the early stage of this laboratory, the gRNA expression plasmid pWL502-PphaR-R-C1-R (i.e., plasmid pGK) targeting the crtB gene of the Spanish salt box bacteria DLCR strain has been constructed, which contains the PphaR-R-C1-R fragment. The PphaR-R-C1-R fragment is shown in Sequence 9 of the sequence table. In Sequence 9 of the sequence table, nucleotides 59-88 encode repeat, nucleotides 89 to 124 encode spacer, and nucleotides 125-154 encode repeat. The spacer length of the gRNA expressed by the PphaR-R-C1-R fragment is 36nt (targeting positions 269-304 of the crtB gene shown in sequence 1, and the PAM sequence is TTC).

Based on the PphaR-R-C1-R fragment, the spacer coding region was truncated to obtain 7 PphaR-R-spacer-R fragments, which were named PphaR-R-spacer16-R fragment (the spacer length of the expressed gRNA was 16bp), PphaR-R-spacer20-R fragment (the spacer length of the expressed gRNA was 20bp), PphaR-R-spacer24-R fragment (the spacer length of the expressed gRNA was 24bp), PphaR-R-spacer28-R fragment (the spacer length of the expressed gRNA was 28bp), PphaR-R-spacer30-R fragment (the spacer length of the expressed gRNA was 30bp), PphaR-R-spacer31-R fragment (the spacer length of the expressed gRNA was 31bp), and PphaR-R-spacer32-R fragment (the spacer length of the expressed gRNA was 32p).

The PphaR-R-spacer16-R fragment is shown in Sequence 2 of the sequence list. In Sequence 2 of the sequence list, nucleotides 59-88 encode repeat, nucleotides 89 to 104 encode spacer, and nucleotides 105-134 encode repeat. The spacer length of the gRNA expressed by the PphaR-R-spacer16-R fragment is 16 nt (targeting positions 269-284 of the crtB gene shown in Sequence 1, and the PAM sequence is TTC).

The PphaR-R-spacer20-R fragment is shown in Sequence 3 of the sequence list. In Sequence 3 of the sequence list, nucleotides 59-88 encode repeat, nucleotides 89 to 108 encode spacer, and nucleotides 109-138 encode repeat. The spacer length of the gRNA expressed by the PphaR-R-spacer20-R fragment is 20 nt (targeting positions 269-288 of the crtB gene shown in sequence 1, and the PAM sequence is TTC).

The PphaR-R-spacer24-R fragment is shown in Sequence 4 of the sequence list. In Sequence 4 of the sequence list, nucleotides 59-88 encode repeat, nucleotides 89 to 112 encode spacer, and nucleotides 113-142 encode repeat. The spacer length of the gRNA expressed by the PphaR-R-spacer24-R fragment is 24 nt (targeting positions 269-292 of the crtB gene shown in sequence 1, and the PAM sequence is TTC).

The PphaR-R-spacer28-R fragment is shown in Sequence 5 of the sequence list. In Sequence 5 of the sequence list, nucleotides 59-88 encode repeat, nucleotides 89 to 116 encode spacer, and nucleotides 117-146 encode repeat. The spacer length of the gRNA expressed by the PphaR-R-spacer28-R fragment is 28 nt (targeting positions 269-296 of the crtB gene shown in sequence 1, and the PAM sequence is TTC).

The PphaR-R-spacer30-R fragment is shown in Sequence 6 of the sequence list. In Sequence 6 of the sequence list, nucleotides 59-88 encode repeat, nucleotides 89 to 118 encode spacer, and nucleotides 119-148 encode repeat. The spacer length of the gRNA expressed by the PphaR-R-spacer30-R fragment is 30 nt (targeting positions 269-298 of the crtB gene shown in sequence 1, and the PAM sequence is TTC).

The PphaR-R-spacer31-R fragment is shown in Sequence 7 of the sequence list. In Sequence 7 of the sequence list, nucleotides 59-88 encode repeat, nucleotides 89 to 119 encode spacer, and nucleotides 120-149 encode repeat. The spacer length of the gRNA expressed by the PphaR-R-spacer31-R fragment is 31 nt (targeting positions 269-299 of the crtB gene shown in sequence 1, and the PAM sequence is TTC).

The PphaR-R-spacer32-R fragment is shown in Sequence 8 of the sequence list. In Sequence 8 of the sequence list, nucleotides 59-88 encode repeat, nucleotides 89 to 120 encode spacer, and nucleotides 121-150 encode repeat. The spacer length of the gRNA expressed by the PphaR-R-spacer32-R fragment is 32 nt (targeting positions 269-300 of the crtB gene shown in sequence 1, and the PAM sequence is TTC).

2. Construction of recombinant plasmid

The PphaR-R-spacer16-R fragment was double-digested with BamHI and KpnI, and then ligated with the plasmid pWL502 double-digested with BamHI and KpnI to obtain the recombinant plasmid pWL502-PphaR-R-spacer16-R.

The PphaR-R-spacer20-R fragment was double-digested with BamHI and KpnI, and then ligated with the plasmid pWL502 double-digested with BamHI and KpnI to obtain the recombinant plasmid pWL502-PphaR-R-spacer20-R.

The PphaR-R-spacer24-R fragment was double-digested with BamHI and KpnI, and then ligated with the plasmid pWL502 double-digested with BamHI and KpnI to obtain the recombinant plasmid pWL502-PphaR-R-spacer24-R.

The PphaR-R-spacer28-R fragment was double-digested with BamHI and KpnI, and then ligated with the plasmid pWL502 double-digested with BamHI and KpnI to obtain the recombinant plasmid pWL502-PphaR-R-spacer28-R.

The PphaR-R-spacer30-R fragment was double-digested with BamHI and KpnI, and then ligated with the plasmid pWL502 double-digested with BamHI and KpnI to obtain the recombinant plasmid pWL502-PphaR-R-spacer30-R.

The PphaR-R-spacer31-R fragment was double-digested with BamHI and KpnI, and then ligated with the plasmid pWL502 double-digested with BamHI and KpnI to obtain the recombinant plasmid pWL502-PphaR-R-spacer31-R.

The PphaR-R-spacer32-R fragment was double-digested with BamHI and KpnI, and then ligated with the plasmid pWL502 double-digested with BamHI and KpnI to obtain the recombinant plasmid pWL502-PphaR-R-spacer32-R.

The above seven recombinant plasmids and plasmid pGK are collectively referred to as recombinant plasmid pWL502-PphaR-R-spacer-R. The schematic diagram of the structure of the recombinant plasmid pWL502-PphaR-R-spacer-R is shown in Figure 1A.

2. Transformation of HH DLCR strain

The plasmids to be transformed are recombinant plasmid pWL502-PphaR-R-spacer16-R, recombinant plasmid pWL502-PphaR-R-spacer20-R, recombinant plasmid pWL502-PphaR-R-spacer24-R, recombinant plasmid pWL502-PphaR-R-spacer28-R, recombinant plasmid pWL502-PphaR-R-spacer30-R, recombinant plasmid pWL502-PphaR-R-spacer31-R, recombinant plasmid pWL502-PphaR-R-spacer32-R, plasmid pGK or plasmid pWL502.

1. Cultivation and activation of bacteria

A single clone of the HH DLCR strain was picked from a solid AS-168 medium plate containing 50 mg/L uracil with a toothpick, inoculated into a liquid AS-168 medium containing 50 mg/L uracil, and cultured at 37°C and 200 rpm with shaking until the logarithmic phase (the bacteria were red), then transferred to a fresh liquid AS-168 medium containing 50 mg/L uracil at a volume ratio of 1:30, and cultured at 37°C and 200 rpm with shaking for 20-24 h (at this time, the bacteria were slightly red, and the OD _600nm value was about 1).

2. Conversion (all operations are carried out at room temperature)

After completing step 1, take 1 mL of bacterial solution into a sterilized EP tube, centrifuge at 6000 rpm for 3 min, and discard the supernatant; then add 200 μL of BSS-LS solution, blow gently with a pipette, centrifuge at 6000 rpm for 3 min, and discard the supernatant; then add 100 μL of BSS-LS/Gly ⁺ solution, blow gently with a pipette, then add 10 μL of pH 8.0, 0.5 M EDTA buffer, gently tap the EP tube wall with your hand, mix well and place at room temperature for 10 minutes (to form protoplasts); then add 5 μL of the plasmid to be transformed (DNA content is about 500 micrograms) (negative control plus an equal volume of ddH ₂ O), gently tap the EP tube wall with your hand, mix well and place at room temperature for 5 minutes; then add 115 μL 60% PEG600 solution, quickly invert the EP tube upside down several times until the solution becomes transparent and uniform, and then place it at room temperature for 20-30 minutes; then add 1mL of 23% MGM recovery medium, invert the EP tube upside down to mix the solution, centrifuge at 6000rmp for 3min, and discard the supernatant; then add 0.5mL of 23% MGM recovery medium to suspend the bacteria, and culture at 37℃ and 200rmp overnight.

3. Coating board

After completing step 2, take the bacterial solution and use 23% MGM recovery medium to perform 10-fold gradient dilution to obtain 10 ¹ -10 ³ -fold dilutions, and spread the original bacterial solution and each dilution on a solid AS-168SY medium plate.

4. Growth

(1) Place the plate prepared in step 3 at 37°C for 1 day. After the liquid is completely absorbed by the plate, place it in a sealed plastic bag and invert it at 42°C for 3-5 days. At this time, transformants can be seen and the transformation efficiency can be counted (see step 3 for results). If the target is cut, the chromosome is broken, and the recombinant bacteria die, no transformants can be observed.

(2) Streak the transformants onto solid AS-168SY medium plates and incubate them upside down in a 42°C incubator for 2 days. Then observe the color of the transformants (see step 4 for results) and perform molecular detection (see steps 5 and 6 for results).

3. Conversion efficiency statistics

Each plasmid to be transformed was treated with 3 replicates. The number of transformants on the corresponding plates at different dilution multiples was recorded, and the number of transformants between 30 and 300 was considered valid data. The transformation efficiency (number of transformants per microgram of plasmid) of each plasmid to be transformed was calculated, and a bar graph was made based on the statistical data.

The results are shown in Figure 2 A. In the figure, 16 bp corresponds to the recombinant plasmid pWL502-PphaR-R-spacer16-R, 20 bp corresponds to the recombinant plasmid pWL502-PphaR-R-spacer20-R, 24 bp corresponds to the recombinant plasmid pWL502-PphaR-R-spacer24-R, 28 bp corresponds to the recombinant plasmid pWL502-PphaR-R-spacer28-R, 30 bp corresponds to the recombinant plasmid pWL502-PphaR-R-spacer30-R, 31 bp corresponds to the recombinant plasmid pWL502-PphaR-R-spacer31-R, 32 bp corresponds to the recombinant plasmid pWL502-PphaR-R-spacer32-R, and 36 bp corresponds to the plasmid pGK.

The results showed that when the spacer length was 31bp, 32bp and 36bp, the interference ability of the spacer was strong and the number of transformants was very small; when the spacer length was 16bp, 20bp, 24bp, 28bp and 30bp, the transformation efficiency was at the same order of magnitude as the pWL502 plasmid. From the above results, it can be inferred that after shortening the spacer in the gRNA (the spacer length does not exceed 30bp), the CRISPR system did not cut the target.

4. Transformant Color Analysis

The transformants obtained by performing the above steps on each plasmid to be transformed were observed for color, and the results are shown in B of Figure 2 (the corresponding relationship is the same as A of Figure 2). The results show that when the spacer length is 16bp, the transformant color is a darker red, which is the same as pWL502, indicating that its crtB gene expression is almost not inhibited. When the spacer length is 20bp, 24bp, 28bp or 30bp, the transformant color gradually becomes lighter as the spacer shortens, and the 28bp and 30bp transformant colors are white, indicating that crtB gene expression is strongly inhibited. The above results show that the present invention achieves different degrees of transcriptional inhibition of the target gene.

5. Analysis of crtB gene mutation in transformants

In order to verify whether the crtB gene target site was edited by cleavage by Cas3 protein, the primer pair consisting of test-F1/test-R1 (the target sequence includes the crtB gene sequence) was used to perform PCR amplification on multiple transformants corresponding to plasmids with spacer lengths of 24 bp, 28 bp, and 30 bp, and the PCR products were subjected to agarose gel electrophoresis and sequencing.

test-F1: GCCCAGACGGGCGACATTCT;

test-R1: GACCATCGCGGTCTGCAAGA.

The results are shown in Figures 2C and 2D. In Figure 2C, - represents the HH DLCR strain, and 1-10 represent different transformants. The results show that for plasmids with a spacer length of 24bp or 28bp, the length of the crtB gene of each transformant did not change compared with the starting strain HH DLCR, and the sequencing results showed that the crtB gene sequence of each transformant did not mutate. For plasmids with a spacer length of 30bp, the crtB gene of some transformants had a deletion of about 1kb, indicating that the Cas3 protein had a certain degree of cutting of the target. The above results further show that by shortening the spacer length of the gRNA to no more than 28bp, the cutting of the target by the Cas3 protein can be prevented.

6. qRT-PCR quantitative analysis of crtB gene transcription in transformants

Take the transformant, extract total RNA, and reverse transcribe to obtain cDNA. Using cDNA as a template and 7S gene as an internal reference gene, perform real time PCR reaction on ViiATM7 Real-Time PCR System to detect the relative expression level of crtB gene. The primer pair used to detect crtB gene is a primer pair composed of crtB QF/crtB QR. The primer pair used to detect 7S gene is a primer pair composed of 7SF/7SR.

crtB QF (forward primer): CGATACAGCAGGAGACCG;

crtB QR (reverse primer): CATCGCGTCGATGAAGAC.

7SF (forward primer): TCGATGGTCCGCTGCTCAC;

7SR (reverse primer): GGGGGCGTCCGGTCTGA.

Reaction system: KAPA SYBR FAST qPCR master mix (2x) 10 μL, forward primer (10 μM) 1 μL, reverse primer (10 μM) 1 μL, template 5 μL (DNA content is about 3000 ng), Dye 0.4 μL, ddH ₂ O 2.6 μL.

Reaction conditions: 95°C for 5 min; 95°C for 15 s, 54°C for 25 s, 72°C for 35 s, 40 cycles.

The obtained data were statistically analyzed and a bar graph was drawn. The results are shown in Figure 3. In the figure, spacer-16bp corresponds to the recombinant plasmid pWL502-PphaR-R-spacer16-R, spacer-20bp corresponds to the recombinant plasmid pWL502-PphaR-R-spacer20-R, spacer-24bp corresponds to the recombinant plasmid pWL502-PphaR-R-spacer24-R, spacer-28bp corresponds to the recombinant plasmid pWL502-PphaR-R-spacer28-R, and spacer-30bp corresponds to the recombinant plasmid pWL502-PphaR-R-spacer30-R.

The results show that when the spacer length is 16bp, 20bp, 24bp, 28bp and 30bp, the expression of the crtB gene in the transformant strain changes to varying degrees, and as the spacer shortens, the inhibition strength weakens. Among them, when the spacer length is 24bp, 28bp and 30bp, the expression of the crtB gene is significantly downregulated by nearly one order of magnitude. It shows that with the change of the spacer length, the present invention achieves different degrees of inhibition of the target gene.

Based on the results in Example 1, the present invention can achieve different degrees of transcriptional regulation of the target gene (crtB gene) by using the Spanish salt box fungus HHDLCR strain I-B type CRISPR-Cas system and gRNA containing spacers of different lengths without knocking out the Cas3 protein; and does not change the sequence of the target site, that is, does not cut the target site.

Embodiment 2,

The cdc6E and crtB genes were edited and transcriptionally regulated simultaneously using the Spanish salt box bacteria HH DLCR strain I-B CRISPR-Cas system. The crtB gene is shown in sequence 1 of the sequence list. The cdc6E gene is shown in sequence 10 of the sequence list.

1. Construction of the editing and transcriptional regulation plasmid pGKBE-DonorE-SpacerBE

1. Preparation of DonorE-SpacerBE fragment

There is a knockout plasmid pGKBE targeting the cdc6E gene and crtB gene of the Spanish salt box bacteria DLCR strain, which expresses a gRNA with two spacers and contains the Donor sequence required for knocking out the crtB gene and the Donor sequence required for knocking out the cdc6E gene. The two spacers are the spacer ( _SB ) targeting the crtB gene and the spacer ( _SE ) targeting the cdc6E gene. In the gRNA expressed by the plasmid pGKBE, the length of _SB is 36nt (targeting the 269-304th position of the crtB gene shown in sequence 1, and the PAM sequence is TTC), and the length of _SE is 36nt (targeting the 385-420th position of the cdc6E gene shown in sequence 10, and the PAM sequence is TTC).

The DonorE-SpacerBE-18bp fragment is shown in Sequence 11 of the sequence table. In Sequence 11 of the sequence table, nucleotides 10-682 are the DonorE segment required for knocking out the cdc6E gene, nucleotides 732-761 encode repeats, nucleotides 762-779 encode spacers targeting the crtB gene, nucleotides 780-809 encode repeats, nucleotides 810-845 encode spacers targeting the cdc6E gene, and nucleotides 846-875 encode repeats. The length of the spacer targeting the crtB gene is 18 nt (targeting positions 269-286 of the crtB gene shown in sequence 1, and the PAM sequence is TTC), and the length of the spacer targeting the cdc6E gene is 36 nt (targeting positions 385-420 of the cdc6E gene shown in sequence 10, and the PAM sequence is TTC).

The DonorE-SpacerBE-24bp fragment is shown in Sequence 12 of the sequence table. In Sequence 12 of the sequence table, nucleotides 10-682 are the DonorE segment required for knocking out the cdc6E gene, nucleotides 732-761 encode repeats, nucleotides 762-785 encode spacers targeting the crtB gene, nucleotides 786-815 encode repeats, nucleotides 816-851 encode spacers targeting the cdc6E gene, and nucleotides 852-881 encode repeats. The length of the spacer targeting the crtB gene is 24 nt (targeting positions 269-292 of the crtB gene shown in sequence 1, and the PAM sequence is TTC), and the length of the spacer targeting the cdc6E gene is 36 nt (targeting positions 385-420 of the cdc6E gene shown in sequence 10, and the PAM sequence is TTC).

The DonorE-SpacerBE-28bp fragment is shown in Sequence 13 of the sequence table. In Sequence 13 of the sequence table, nucleotides 10-682 are the DonorE segment required for knocking out the cdc6E gene, nucleotides 732-761 encode repeats, nucleotides 762-789 encode spacers targeting the crtB gene, nucleotides 790-819 encode repeats, nucleotides 820-855 encode spacers targeting the cdc6E gene, and nucleotides 856-885 encode repeats. The length of the spacer targeting the crtB gene is 28 nt (targeting positions 269-296 of the crtB gene shown in sequence 1, and the PAM sequence is TTC), and the length of the spacer targeting the cdc6E gene is 36 nt (targeting positions 385-420 of the cdc6E gene shown in sequence 10, and the PAM sequence is TTC).

2. Construction of recombinant plasmid

The DonorE-SpacerBE-18bp fragment was double-digested with BamHI and KpnI, and then ligated with the plasmid pWL502 double-digested with BamHI and KpnI to obtain the recombinant plasmid pGKBE-DonorE-SpacerBE-18bp.

The DonorE-SpacerBE-24bp fragment was double-digested with BamHI and KpnI, and then ligated with the plasmid pWL502 double-digested with BamHI and KpnI to obtain the recombinant plasmid pGKBE-DonorE-SpacerBE-24bp.

The DonorE-SpacerBE-28bp fragment was double-digested with BamHI and KpnI, and then ligated with the plasmid pWL502 double-digested with BamHI and KpnI to obtain the recombinant plasmid pGKBE-DonorE-SpacerBE-28bp.

The above three recombinant plasmids and plasmid pGKBE are collectively referred to as recombinant plasmid pGKBE-DonorE-SpacerBE. The schematic diagram of the structure of the recombinant plasmid pGKBE-DonorE-SpacerBE is shown in B of Figure 1.

2. Transformation of HH DLCR strain

The plasmids to be transformed are respectively recombinant plasmid pGKBE-DonorE-SpacerBE-18bp, recombinant plasmid pGKBE-DonorE-SpacerBE-24bp, recombinant plasmid pGKBE-DonorE-SpacerBE-28bp, plasmid pGKBE or plasmid pWL502.

The method is the same as step 2 of Example 1.

3. Conversion efficiency analysis

Same as step 3 of Example 1.

The results are shown in Figure 4 A. In the figure, 18 bp corresponds to the recombinant plasmid pGKBE-DonorE-SpacerBE-18 bp, 24 bp corresponds to the recombinant plasmid pGKBE-DonorE-SpacerBE-24 bp, and 28 bp corresponds to the recombinant plasmid pGKBE-DonorE-SpacerBE-28 bp.

The results showed that the transformation efficiency of pGKBE-DonorE-SpacerBE plasmid was one order of magnitude higher than that of pGKBE plasmid, and there was no significant difference in transformation efficiency when the spacer length of crtB gene was 18bp, 24bp and 28bp.

4. Transformant Color Analysis

Same as step 4 of Example 1.

The results are shown in Figure 4B (corresponding to Figure 4A). The results show that when the spacer length is 18 bp, the transformant color is darker red, similar to pWL502, indicating that the expression of crtB gene is almost not inhibited. When the spacer length is 24 bp and 28 bp, the transformant color is white, indicating that the expression of crtB gene is strongly inhibited.

V. Analysis of cdc6E and crtB gene mutations in transformants

To verify whether the cdc6E and crtB gene target sites were edited, PCR amplification was performed on the corresponding transformants of the plasmids with a spacer length of 24 bp or 28 bp using the primer pair consisting of test-F1/test-R1 (the target sequence included the crtB gene sequence) or the primer pair consisting of test-F2/test-R2 (the target sequence included the cdc6E gene), and the PCR products were subjected to agarose gel electrophoresis and sequencing.

test-F2：CGTGCGCATTCCGGTCATCT;

test-R2: GGTCGACGAACGTCACTGTA.

The results are shown in Figure 4C. In Figure 4C, - represents the HH DLCR strain, + represents the transformants obtained from the pGKBE plasmid, and 1-10 represent different transformants obtained from the recombinant plasmid pGKBE-DonorE-SpacerBE-24bp or different transformants obtained from the recombinant plasmid pGKBE-DonorE-SpacerBE-28bp.

The results showed that the crtB gene length of each transformant did not change compared with the starting strain HH DLCR, and the sequencing results showed that the crtB gene sequence of the transformants obtained from each recombinant plasmid did not mutate, and the cdc6E gene length was shortened and successfully knocked out.

Based on the results in Example 2, the present invention can achieve gene editing and transcriptional regulation of two different genes simultaneously using the Spanish salt box bacteria HH DLCR strain I-B type CRISPR-Cas system without knocking out or mutating the Cas3 protein, and the efficiency is very high.

The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application is intended to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended claims.

Sequence Listing

<110> Institute of Microbiology, Chinese Academy of Sciences

<120> A method for simultaneously achieving gene editing and transcriptional regulation using a type I CRISPR-Cas system

<130> GNCYX203089

<160> 13

<170> SIPOSequenceListing 1.0

<210> 1

<211> 972

<212> DNA

<213> Haloarcula hispanica

<400> 1

atgcactccg ataacatcca aaccagcaaa tcgatacagc aggagaccgg ccggacgttt 60

cacctcgcga cgcgtctgct tccggagcgt atccgccacc cgacctacgt catgtacgcc 120

tttttccggg tcgcggacga ggtcgtcgac cagacggatg ggccgccccc gaccgtccag 180

cacgagcaac tggaggtaat ccgcgaggcc gcgctcggga acgtcgaccc ggccgagacc 240

gaccacgagg cggtcatggc ggcgtttcag gacctggccg agcgtcacga catctccgag 300

gagacgatca acgtcttcat cgacgcgatg gagatggaca tcgcacaggc ccgctacgag 360

acgttcgagg acctccgtga gtacatgggc ggctccgccg tcgctgtcgg ccacatgatg 420

acggaggtga tggacccgcc acagaaggcg gaggccctgc cccacgcgac ggcgctggcc 480

gaagcgttcc agctatcgaa cttcctgcgg gacgtccgcg aggacatcca cgactacggc 540

cgggtgtacc tcccacagga gacgctcgac cgccacggcg tcaccgagga gcaactggcc 600

gacgccgagg tcgacgacgc cttccgtgcc gtgatgcagg aggaactggc tcggaccgac 660

gaactgtacc gcgagggcgt cgccggcatc cgatacctcc cggaagactg ccagttcggc 720

gtgctgctgg ctgcggtcct gtacgctgac caccaccgac tcatccgcga ccgtggctac 780

gacgtgctga cggagacgcc ggaactcact cgtcgtcgcc ggctgtggtt gctcgcccgt 840

acgtggtggc actggcggcg caacggcgac cccgaagcga cgttctacac cgtgagcgct 900

gtctccgagc gcggccccgg cgagacgccg acggacgccc acggccacgg gcaacccgcg 960

tggcgtggat ga 972

<210> 2

<211> 151

<212> DNA

<213> Artificial Sequence

<400> 2

cgcggatccc gaagggaaca tatatgttac tgcaggtaca acaccgagtt aggagatggt 60

ttcagacgaa ccctcgtggg gttgaagcag gacctggccg agcggtttca gacgaaccct 120

cgtggggttg aagctttttt ttggtacccc g 151

<210> 3

<211> 155

<212> DNA

<213> Artificial Sequence

<400> 3

cgcggatccc gaagggaaca tatatgttac tgcaggtaca acaccgagtt aggagatggt 60

ttcagacgaa ccctcgtggg gttgaagcag gacctggccg agcgtcacgt ttcagacgaa 120

ccctcgtggg gttgaagctt ttttttggta ccccg 155

<210> 4

<211> 159

<212> DNA

<213> Artificial Sequence

<400> 4

cgcggatccc gaagggaaca tatatgttac tgcaggtaca acaccgagtt aggagatggt 60

ttcagacgaa ccctcgtggg gttgaagcag gacctggccg agcgtcacga cagtttcaga 120

cgaaccctcg tggggttgaa gctttttttt ggtaccccg 159

<210> 5

<211> 163

<212> DNA

<213> Artificial Sequence

<400> 5

cgcggatccc gaagggaaca tatatgttac tgcaggtaca acaccgagtt aggagatggt 60

ttcagacgaa ccctcgtggg gttgaagcag gacctggccg agcgtcacga catctcgttt 120

cagacgaacc ctcgtggggt tgaagctttt ttttggtacc ccg 163

<210> 6

<211> 165

<212> DNA

<213> Artificial Sequence

<400> 6

cgcggatccc gaagggaaca tatatgttac tgcaggtaca acaccgagtt aggagatggt 60

ttcagacgaa ccctcgtggg gttgaagcag gacctggccg agcgtcacga catctccggt 120

ttcagacgaa ccctcgtggg gttgaagctt ttttttggta ccccg 165

<210> 7

<211> 166

<212> DNA

<213> Artificial Sequence

<400> 7

cgcggatccc gaagggaaca tatatgttac tgcaggtaca acaccgagtt aggagatggt 60

ttcagacgaa ccctcgtggg gttgaagcag gacctggccg agcgtcacga catctccgag 120

tttcagacga accctcgtgg ggttgaagct tttttttggt accccg 166

<210> 8

<211> 167

<212> DNA

<213> Artificial Sequence

<400> 8

cgcggatccc gaagggaaca tatatgttac tgcaggtaca acaccgagtt aggagatggt 60

ttcagacgaa ccctcgtggg gttgaagcag gacctggccg agcgtcacga catctccgag 120

gtttcagacg aaccctcgtg gggttgaagc ttttttttgg taccccg 167

<210> 9

<211> 171

<212> DNA

<213> Artificial Sequence

<400> 9

cgcggatccc gaagggaaca tatatgttac tgcaggtaca acaccgagtt aggagatggt 60

ttcagacgaa ccctcgtggg gttgaagcag gacctggccg agcgtcacga catctccgag 120

gagagtttca gacgaaccct cgtggggttg aagctttttt ttggtacccc g 171

<210> 10

<211> 1329

<212> DNA

<213> Haloarcula hispanica

<400> 10

atggtcgacg tgaacgagaa cccgttcgat gggactgacg cgatcttcga acgaaagcaa 60

ccgctgaaaa aagacacgtt cacgccggat acgatctttc accgcgacga ggagatcgag 120

ttctacatca acgcgcttca ggacgtcatc gtcggccacg acccccaacaa cgtcttcgtc 180

tacggcccaa cgggggtcgg aaagactgcc gttacgaagt gggtacggga caaactcgaa 240

gagaaggccg aggccgagga catcccactc accgttgtcg gtccgataaa ctgccggaac 300

taccggtcgg cgtacgccct ggtcaacaca ctcgtcaacg agttccgaga tccggagaac 360

caactccccg agagcggcta cagcactgac agcgtcttcg agttcctcta cgaggagatc 420

gaagccgtgg gtgggaacgt cctcatcatc ctggacgaga tcgacaacat tccagcggac 480

gcgcggaacg acttcctgta tgaactgccg cgggccgaag cgaacgagaa cacgccgatc 540

accgatgcga aggtcggact catcgggatt tcgaacgacc tcaagttcgt cgacgtgctg 600

gaacccaaag tgaaatcgac gctcggggag cgagagatca agttcggtcc gtacgacgca 660

acggaactcc gagacattct cggctactac gccgacattg cgttccgaga ggacgtgctc 720

ggcgaagatg tcgtcccgct ggcggcggcc ttctcggcgc aggaacgggg cgacgttcgg 780

cagggactcc gaatcctcga aaaggccgga gagtacgcgc ggatggaggg cgcggacggt 840

gtgacagaag cacatacgcg acgggcgaca gacactatcg aaaccgacga actgctggat 900

tacttcgagc acgacctgag ttcacagcag gcgctgacgt acctcgcgac gacgcttgcg 960

ctcatcgaac cgaaacacga ggcgtcgaca aagcggattt acaacctcta ctcctcgatt 1020

gcggagtcga gtggccgccg cgtgaaatcc gaacgcaaga tctacgagtt cctcgaccag 1080

ctttccatgc aagggctagt ccgctcggcc gaacgcaacc tcggtcgcaa gggtgggcgc 1140

aagtacatct acgaggtcac cgacgacgcg accgatatca tcaacgccgc gctccagtcg 1200

tcctacagcg atgccgtgcc gagcaacgtc aacgggatac tcgaacacta tctggaagac 1260

gaggccaccg agtttgaggc accggacacg accgacgacg agcaacagaa cctctggcag 1320

ttcacgtag 1329

<210> 11

<211> 892

<212> DNA

<213> Artificial Sequence

<400> 11

cgcggatcct tcgaaacgga ataccgggag gctgtggtgg aaacggacgg acacaccggt 60

ctgcaagtgt tctacacact gcagtggagg gtaactgcca cataatcgct cggtagcact 120

ccgggttcac ggtctactgt tggcacacac acaccaccct gcaactgttc tggactgtac 180

aatagaggga ctgccactga acacacacac accaccgtgc aagtgttctg ccatgtgagt 240

cggtgggggg aggggagcaa atacgctggc tacgtgaact gccagaggtt ctgttgctcg 300

tcgtcggtca gcggttgctt tcgttcgaag atcgcgtcag tcccatcgaa cgggttctcg 360

ttcacgtcga ccatcgtacg ccaccttttg agtgactcta tataaaacca gtgaccaccg 420

tacaagtgtt tcaagtgttt tcacgccgaa cagtcacaca ccaccctgca agtgtttctt 480

cgatccctct ggaaaacagc cacgagcagt ctgctgcacc gacccttcat ctagaacact 540

tgagtcctgc gattctcgac tgtctcgttc tggaccgtat ctagaactag tagcactatt 600

atatatttta tggtaaaggc aataaaatca tattcaccat cgaacgtcca cctccataga 660

attcaagcta caagtgcttc agcgaaggga acatatatgt tactgcaggt acaacaccga 720

gttaggagat ggtttcagac gaaccctcgt ggggttgaag caggacctgg ccgagcgtcg 780

tttcagacga accctcgtgg ggttgaagcg atctcctcgt agaggaactc gaagacgctg 840

tcagtgtttc agacgaaccc tcgtggggtt gaagcttttt tttggtaccc cg 892

<210> 12

<211> 898

<212> DNA

<213> Artificial Sequence

<400> 12

cgcggatcct tcgaaacgga ataccgggag gctgtggtgg aaacggacgg acacaccggt 60

ctgcaagtgt tctacacact gcagtggagg gtaactgcca cataatcgct cggtagcact 120

ccgggttcac ggtctactgt tggcacacac acaccaccct gcaactgttc tggactgtac 180

aatagaggga ctgccactga acacacacac accaccgtgc aagtgttctg ccatgtgagt 240

cggtgggggg aggggagcaa atacgctggc tacgtgaact gccagaggtt ctgttgctcg 300

tcgtcggtca gcggttgctt tcgttcgaag atcgcgtcag tcccatcgaa cgggttctcg 360

ttcacgtcga ccatcgtacg ccaccttttg agtgactcta tataaaacca gtgaccaccg 420

tacaagtgtt tcaagtgttt tcacgccgaa cagtcacaca ccaccctgca agtgtttctt 480

cgatccctct ggaaaacagc cacgagcagt ctgctgcacc gacccttcat ctagaacact 540

tgagtcctgc gattctcgac tgtctcgttc tggaccgtat ctagaactag tagcactatt 600

atatatttta tggtaaaggc aataaaatca tattcaccat cgaacgtcca cctccataga 660

attcaagcta caagtgcttc agcgaaggga acatatatgt tactgcaggt acaacaccga 720

gttaggagat ggtttcagac gaaccctcgt ggggttgaag caggacctgg ccgagcgtca 780

cgacagtttc agacgaaccc tcgtggggtt gaagcgatct cctcgtagag gaactcgaag 840

acgctgtcag tgtttcagac gaaccctcgt ggggttgaag cttttttttg gtaccccg 898

<210> 13

<211> 902

<212> DNA

<213> Artificial Sequence

<400> 13

cgcggatcct tcgaaacgga ataccgggag gctgtggtgg aaacggacgg acacaccggt 60

ctgcaagtgt tctacacact gcagtggagg gtaactgcca cataatcgct cggtagcact 120

ccgggttcac ggtctactgt tggcacacac acaccaccct gcaactgttc tggactgtac 180

aatagaggga ctgccactga acacacacac accaccgtgc aagtgttctg ccatgtgagt 240

cggtgggggg aggggagcaa atacgctggc tacgtgaact gccagaggtt ctgttgctcg 300

tcgtcggtca gcggttgctt tcgttcgaag atcgcgtcag tcccatcgaa cgggttctcg 360

ttcacgtcga ccatcgtacg ccaccttttg agtgactcta tataaaacca gtgaccaccg 420

tacaagtgtt tcaagtgttt tcacgccgaa cagtcacaca ccaccctgca agtgtttctt 480

cgatccctct ggaaaacagc cacgagcagt ctgctgcacc gacccttcat ctagaacact 540

tgagtcctgc gattctcgac tgtctcgttc tggaccgtat ctagaactag tagcactatt 600

atatatttta tggtaaaggc aataaaatca tattcaccat cgaacgtcca cctccataga 660

attcaagcta caagtgcttc agcgaaggga acatatatgt tactgcaggt acaacaccga 720

gttaggagat ggtttcagac gaaccctcgt ggggttgaag caggacctgg ccgagcgtca 780

cgacatctcg tttcagacga accctcgtgg ggttgaagcg atctcctcgt agaggaactc 840

gaagacgctg tcagtgtttc agacgaaccc tcgtggggtt gaagcttttt tttggtaccc 900

cg 902

Claims (8)

1. A method for transcriptionally regulating gene A of a target organism and gene editing its gene B for non-therapeutic or diagnostic purposes, comprising the following steps: using type I CRISPR-Cas system A to transcriptionally regulate gene A and using type I CRISPR-Cas system B to gene edit gene B; The target gene of Type I CRISPR-Cas system A is gene A and it is a modified Type I CRISPR-Cas system; the modified Type I CRISPR-Cas system refers to a Type I CRISPR-Cas system after the gRNA of the Type I CRISPR-Cas system in the prior art is modified; the modification of the gRNA is to replace the spacer in the gRNA of the Type I CRISPR-Cas system in the prior art with a spacer truncation; The target gene of type I CRISPR-Cas system B is gene B and it is a type I CRISPR-Cas system in the prior art; The spacer is a segment in the gRNA that specifically binds to the target sequence; the target sequence is a sequence in the target gene that is adjacent to and downstream of the PAM; the spacer truncation is obtained by truncating the distal portion of the spacer in the gRNA of the type I CRISPR-Cas system in the prior art that targets the PAM; The type I CRISPR-Cas system is a type I-B CRISPR-Cas system; the length of the spacer is 31-72 nt; the length of the spacer truncation is 16-28 nt; The transcriptional regulation is to inhibit gene expression. 2. The method according to claim 1, characterized in that: the type I CRISPR-Cas system in the prior art consists of a Cascade effector, a Cas3 nuclease and a gRNA. 3. A kit for transcriptionally regulating gene A of a target organism and gene editing its gene B, comprising a type I CRISPR-Cas system A and a type I CRISPR-Cas system B; The target gene of Type I CRISPR-Cas system A is gene A and it is a modified Type I CRISPR-Cas system; the modified Type I CRISPR-Cas system refers to a Type I CRISPR-Cas system after the gRNA of the Type I CRISPR-Cas system in the prior art is modified; the modification of the gRNA is to replace the spacer in the gRNA of the Type I CRISPR-Cas system in the prior art with a spacer truncation; The target gene of type I CRISPR-Cas system B is gene B and it is a type I CRISPR-Cas system in the prior art; The spacer is a segment in the gRNA that specifically binds to the target sequence; the target sequence is a sequence in the target gene that is adjacent to and downstream of the PAM; the spacer truncation is obtained by truncating the distal portion of the spacer in the gRNA of the type I CRISPR-Cas system in the prior art that targets the PAM; The type I CRISPR-Cas system is a type I-B CRISPR-Cas system; the length of the spacer is 31-72 nt; the length of the spacer truncation is 16-28 nt; The transcriptional regulation is to inhibit gene expression. 4. The kit as claimed in claim 3, characterized in that: the type I CRISPR-Cas system in the prior art consists of a Cascade effector, a Cas3 nuclease and a gRNA. 5. A method for transcriptional regulation of a target gene of a target organism for non-therapeutic or diagnostic purposes, comprising the following steps: Using the modified type I CRISPR-Cas system to regulate the transcription of target genes; The modified type I CRISPR-Cas system refers to a type I CRISPR-Cas system after modifying the gRNA of the type I CRISPR-Cas system in the prior art; the modification of the gRNA is to replace the spacer in the gRNA of the type I CRISPR-Cas system in the prior art with a spacer truncation; The spacer is a segment in the gRNA that specifically binds to the target sequence; the target sequence is a sequence in the target gene that is adjacent to and downstream of the PAM; the spacer truncation is obtained by truncating the distal portion of the spacer in the gRNA of the type I CRISPR-Cas system in the prior art that targets the PAM; The type I CRISPR-Cas system is a type I-B CRISPR-Cas system; the length of the spacer is 31-72 nt; the length of the spacer truncation is 16-28 nt; The transcriptional regulation is to inhibit gene expression. 6. The method of claim 5, wherein the type I CRISPR-Cas system in the prior art consists of a Cascade effector, a Cas3 endonuclease and a gRNA. 7. A kit for transcriptional regulation of a target gene of a target organism, comprising a modified type I CRISPR-Cas system; The modified type I CRISPR-Cas system refers to a type I CRISPR-Cas system after modifying the gRNA of the type I CRISPR-Cas system in the prior art; the modification of the gRNA is to replace the spacer in the gRNA of the type I CRISPR-Cas system in the prior art with a spacer truncation; The spacer is a segment in the gRNA that specifically binds to the target sequence; the target sequence is a sequence in the target gene that is adjacent to and downstream of the PAM; the spacer truncation is obtained by truncating the distal portion of the spacer in the gRNA of the type I CRISPR-Cas system in the prior art that targets the PAM; The type I CRISPR-Cas system is a type I-B CRISPR-Cas system; the length of the spacer is 31-72 nt; the length of the spacer truncation is 16-28 nt; The transcriptional regulation is to inhibit gene expression. 8. The kit as claimed in claim 7, characterized in that: the type I CRISPR-Cas system in the prior art consists of a Cascade effector, a Cas3 nuclease and a gRNA.