阅读说明：本技术 一种真核生物CRISPR/Cas全基因组编辑载体文库及构建方法 (Eukaryotic organism CRISPR/Cas whole genome editing vector library and construction method ) 是由 马三垣 常珈菘 夏庆友 于 2020-05-07 设计创作，主要内容包括：本发明涉及一种真核生物CRISPR/Cas9全基因组编辑载体文库及构建方法,首先是构建一种piggyBac转座子系统介导的真核生物CRISPR/Cas9敲除骨架载体,然后综合运用搭桥PCR和酶切连接方法构建真核生物全基因组敲除突变体文库。本发明的特点是递送系统选用具有广泛生物适用性和超大外源基因承载能力的piggyBac转座子系统,构建方法选用搭桥PCR和酶切连接方法。构建的真核生物全基因组敲除载体文库效果好。(The invention relates to a eukaryotic organism CRISPR/Cas9 whole genome editing vector library and a construction method thereof, which comprises the steps of firstly constructing a piggyBac transposon system-mediated eukaryotic organism CRISPR/Cas9 knockout skeleton vector, and then comprehensively constructing a eukaryotic organism whole genome knockout mutant library by using a bypass PCR and enzyme digestion connection method. The invention is characterized in that the piggyBac transposon system with wide biological applicability and super-large exogenous gene bearing capacity is selected as the delivery system, and the bypass PCR and enzyme digestion connection method is selected as the construction method. The constructed eukaryotic whole genome knockout vector library has good effect.)

1. A construction method of a eukaryote CRISPR/Cas9 whole genome editing vector library is characterized by comprising the following specific steps:

(1) constructing a piggyBac transposon system mediated eukaryote CRISPR/Cas9 gene knockout vector skeleton, which is named as pB-CRISPRv2, and the nucleotide sequence of the vector skeleton is shown as SEQ ID NO. 1;

(2) constructing a vector with a U6 promoter nucleotide sequence, which is named as T-U6, and the nucleotide sequence is shown as SEQ ID NO. 2;

(3) constructing a vector with an sgRNA scaffold nucleotide sequence, which is named as T-sgRNA scaffold, wherein the nucleotide sequence is shown as SEQ ID NO. 3;

(4) designing a targeting site of all eukaryotic protein coding genes, designing flanking sequences of sgRNAs according to a nucleotide sequence of a U6 promoter and a nucleotide sequence of a sgRNA scaffold, synthesizing all sgRNA sequences containing flanking regions, and naming the sgRNA sequences as a single-stranded oligonucleotide library;

(5) amplifying a U6 promoter fragment by taking a vector T-U6 as a template, and naming the fragment as DGP-1; amplifying a sgRNA fragment by taking The synthesized pool of sgRNA oligonucleotides as a template, and naming The sgRNA fragment as DGP-2; amplifying an sgRNA scaffold fragment by taking the vector T-sgRNA scaffold as a template, and naming the sgRNA scaffold fragment as DGP-3; then, a mixture of DGP-1, DGP-2 and DGP-3 is used as a template to carry out a bridging PCR experiment, and a U6-sgRNA library fragment containing a sgRNA library is amplified and named as DGP-4;

(6) and (3) respectively carrying out enzyme digestion on the vector pB-CRISPRv2 and the fragment DGP-4, mixing and connecting, and building a library to obtain the vector library.

2. The method of claim 1, wherein in step (1), pB-CRISPRv2 comprises: piggyBac transposon arm, a screening marker Zeocin resistance gene expression frame, a Cas9 protein expression frame and an escherichia coli lethal gene ccDB expression frame.

3. The construction method according to claim 1, wherein the specific method of step (4) is: according to the spCas9 action rule, by combining with eukaryotic protein encoding genes, targeting sites of all eukaryotic protein encoding genes are designed, the number of the targeting sites of most proteins is 6, and the nucleotides have the following rules: 5 '-NNNNNNNNNNNNNNNNNNNN-NGG-3', all the targeting sites are in the first half part of CDS sequence, and the 12bp nucleotide near the seed sequence part of PAM region can not have repeat region on the genome; flanking sequences of sgrnas were designed with the following rules: 5 '-TACAA AATAT CGTGC TCTACAAGTG NNNNN NNNNN NNNNN NNNNN GTTTT AGAGC TAGAA ATAGC AAGTT-3', wherein 'N' is sgRNA sequence, and the whole sgRNA sequence containing flanking region is synthesized by means of gene chip and named as single-strand oligonucleotide library, and the nucleotide sequence is shown in SEQ ID NO. 4.

4. The method of claim 1, wherein in step (5), the designing of the bypass PCR primer comprises:

>KU-1R，5’-TCGATGATGATGATCAATTGTGGCGCGCCAAGCTGTCCAAGGAATGCGT-3’，SEQ IDNO.5；

>DG-1R，5’-TTGTAGAGCACGATATTTTGTATAT-3’，SEQ ID NO.6；

>DP-2F，5’-TCAATAGTTTAGTTTTTTTAGGTATATATACAAAATATCGTGCTCTACAA-3’，SEQ IDNO.7；

>DP-2R，5’-AAGTTGATAACGGACTAGCCTTATTTTAACTTGCTATTTCTAGCTCTAA-3’，SEQ IDNO.8；

>DG-3F，5’-TTAGAGCTAGAAATAGCAAGTTAAA-3’，SEQ ID NO.9；

>KU-1F，5’-ACCGATCGATCCTAGGCGCTAGCTAATGAAAGATCTTTATCGATTTAGC-3’，SEQ IDNO.10。

5. the construction method according to claim 4, wherein DGP-1, DGP-2 and DGP-3 are synthesized by the following steps:

using a vector T-U6 as a template, and using primers KU-1R and DG-1R to amplify a U6 promoter fragment, which is named as DGP-1; so as to; a single-stranded oligonucleotide library is used as a template, and primers DP-2F and DP-2R are used for amplifying a sgRNA fragment, which is named as DGP-2; the sgRNA scaffold fragment was amplified using the vector T-sgRNA scaffold as a template and the primers DG-3F and KU-1F, and was named DGP-3.

6. The method according to claim 4, wherein a mixture of DGP-1, DGP-2 and DGP-3 is used as a template, and primers KU-1F and KU-1R are used to perform a bridge PCR experiment, so that a U6-sgRNA library fragment containing the sgRNA library is amplified and named DGP-4.

7. The method of claim 1, wherein in step (5), DGP-1, DGP-2 and DGP-3 are mixed in a molar ratio of 1:1:1 and mixing.

8. The method of claim 1, wherein in step (6), the vector pB-CRISPRv2 and the fragment DGP-4 are cleaved by AscI/NheI.

9. The method for constructing a recombinant vector of claim 1, wherein in step (6), the cleaved vector pB-CRISPRv2 and the cleaved fragment DGP-4 are present in a molar ratio of 1:10 mixing, connecting by DNA ligase, establishing a library by an electrotransformation mode, extracting plasmids, and completing the library establishment, wherein the coverage of the constructed library is more than 100 x.

10. A eukaryotic CRISPA/Cas whole genome editing vector library constructed by the method of any one of claims 1-9.

Technical Field

The invention belongs to the technical field of gene editing, and relates to a eukaryotic organism CRISPR/Cas whole genome editing vector library and a construction method thereof.

Background

With the development of sequencing technology, more and more organisms complete whole genome sequencing, and functional genome research increasingly becomes an important field of life science research. In the face of massive functional genome data, the traditional research means is time-consuming and labor-consuming, and is not free. How to research functional genomes efficiently and rapidly becomes a concern of life scientists. The gene editing technology is a genetic manipulation technology developed in recent years, and the CRISPR/Cas9 is the most efficient and economic technology in the gene editing technology. Besides achieving single gene editing, CRISRP/Cas9 can also be conveniently used to perform whole genome editing by designing and constructing a simple sgRNA library. The CRISPR/Cas9 whole genome editing library can conveniently execute functional genome screening, realizes drug target gene screening, antiviral target gene screening and the like in species such as human, mice and the like at present, and is an important means for researching functional genomes.

Disclosure of Invention

In view of the above, the present invention aims to provide a eukaryotic CRISPA/Cas whole genome editing vector library and a construction method thereof.

In order to achieve the purpose, the invention provides the following technical scheme:

1. a construction method of a eukaryotic organism CRISPA/Cas whole genome editing vector library comprises the following specific steps:

(1) constructing a piggyBac transposon system mediated eukaryote CRISPR/Cas9 gene knockout vector skeleton, which is named as pB-CRISPRv2, and the nucleotide sequence of the vector skeleton is shown as SEQ ID NO. 1;

(2) constructing a vector with a U6 promoter nucleotide sequence, which is named as T-U6, and the nucleotide sequence is shown as SEQ ID NO. 2;

(3) constructing a vector with an sgRNA scaffold nucleotide sequence, which is named as T-sgRNA scaffold, wherein the nucleotide sequence is shown as SEQ ID NO. 3;

(4) designing a targeting site of all eukaryotic protein encoding genes, then designing flanking sequences of sgrnas according to a nucleotide sequence of a U6 promoter and a nucleotide sequence of sgRNA scaffold, synthesizing all sgRNA sequences containing flanking regions, and naming The sgRNA sequences as single-stranded oligonucleotide libraries (The pool of sgRNA oligonucleotides);

(5) amplifying a U6 promoter fragment by taking a vector T-U6 as a template, and naming the fragment as DGP-1; amplifying a sgRNA fragment by taking The synthesized pool of sgRNA oligonucleotides as a template, and naming The sgRNA fragment as DGP-2; amplifying an sgRNA scaffold fragment by taking the vector T-sgRNA scaffold as a template, and naming the sgRNA scaffold fragment as DGP-3; then, a mixture of DGP-1, DGP-2 and DGP-3 is used as a template to carry out a bridging PCR experiment, and a U6-sgRNA library fragment containing a sgRNA library is amplified and named as DGP-4;

(6) and (3) respectively carrying out enzyme digestion on the vector pB-CRISPRv2 and the fragment DGP-4, mixing and connecting, and building a library to obtain the vector library.

As one of the preferable technical schemes, in the step (1), pB-CRISPRv2 comprises: a piggyBac transposon arm (containing Inverted Terminal Repeat (ITR) of piggyBac transposon), a selection marker Zeocin resistance gene expression frame, a Cas9 protein expression frame and an escherichia coli lethal gene ccDB expression frame.

As one of the preferable technical proposal, the specific method of the step (4) is as follows: according to the spCas9 action rule, by combining with eukaryotic protein encoding genes, targeting sites of all eukaryotic protein encoding genes are designed, the number of the targeting sites of most proteins is 6, and the nucleotides have the following rules: 5 '-NNNNNNNNNNNNNNNNNNNN-NGG-3', all the targeting sites are in the first half part of CDS sequence, and the 12bp nucleotide near the seed sequence part of PAM region can not have repeat region on the genome; flanking sequences of sgrnas were designed with the following rules: 5 '-TACAA AATAT CGTGC TCTAC AAGTG NNNNNNNNNN NNNNN NNNNN GTTTT AGAGC TAGAA ATAGC AAGTT-3', wherein 'N' is sgRNA sequence, synthesizing The whole sgRNA sequence containing flanking region in gene chip mode, named as The pool of sgRNA oligonucleotides, and The nucleotide sequence is shown in SEQ ID NO. 4.

As one of the preferable technical schemes, in the step (5), the design of the bypass PCR primer comprises the following steps:

>KU-1R，5’-TCGATGATGATGATCAATTGTGGCGCGCCAAGCTGTCCAAGGAATGCGT-3’，SEQ IDNO.5；

>DG-1R，5’-TTGTAGAGCACGATATTTTGTATAT-3’，SEQ ID NO.6；

>DP-2F，5’-TCAATAGTTTAGTTTTTTTAGGTATATATACAAAATATCGTGCTCTACAA-3’，SEQID NO.7；

>DP-2R，5’-AAGTTGATAACGGACTAGCCTTATTTTAACTTGCTATTTCTAGCTCTAA-3’，SEQ IDNO.8；

>DG-3F，5’-TTAGAGCTAGAAATAGCAAGTTAAA-3’，SEQ ID NO.9；

>KU-1F，5’-ACCGATCGATCCTAGGCGCTAGCTAATGAAAGATCTTTATCGATTTAGC-3’，SEQ IDNO.10。

as a further preferred embodiment, the synthesis method of DGP-1, DGP-2 and DGP-3 is as follows:

using a vector T-U6 as a template, and using primers KU-1R and DG-1R to amplify a U6 promoter fragment, which is named as DGP-1; so as to; the pool of sgRNA oligonucleotides as template, using primers DP-2F and DP-2R to amplify The sgRNA fragment, named DGP-2; the sgRNA scaffold fragment was amplified using the vector T-sgRNA scaffold as a template and the primers DG-3F and KU-1F, and was named DGP-3.

As a further preferred embodiment, a bridge PCR experiment is performed using a mixture of DGP-1, DGP-2 and DGP-3 as a template and primers KU-1F and KU-1R, and a U6-sgRNA library fragment containing the sgRNA library is amplified and designated as DGP-4.

As one of the preferable technical schemes, in the step (5), DGP-1, DGP-2 and DGP-3 are mixed according to the molar ratio of 1:1:1 and mixing.

As one of the preferred technical schemes, in the step (6), the vector pB-CRISPRv2 and the fragment DGP-4 are subjected to double enzyme digestion by AscI/NheI.

As a further preferred technical scheme, the enzyme cutting condition of the vector pB-CRISPRv2 is a 50-L system, which comprises the following components: mu.g of vector, 1 mu L of each of AscI and NheI, and 50 mu L of double distilled water; the 12187bp scaffold was recovered.

As a further preferred technical scheme, the enzyme cutting condition of the segment DGP-4 is a 50 mu L system, which comprises the following steps: mu.g of fragment, 1. mu.L each of AscI and NheI, 50. mu.L of double distilled water.

As one of the preferable technical schemes, in the step (6), the carrier pB-CRISPRv2 after the enzyme digestion and the segment DGP-4 after the enzyme digestion are mixed according to the molar ratio of 1:10 mixing, connecting by DNA ligase, establishing a library by an electrotransformation mode, extracting plasmids, and completing the library establishment, wherein the coverage of the constructed library is more than 100 x.

2. The eukaryotic CRISPA/Cas whole genome editing vector library constructed by the method.

The invention has the beneficial effects that:

the invention firstly constructs a piggyBac transposon system mediated eukaryotic CRISPR/Cas9 knockout skeleton vector, and then constructs a eukaryotic whole genome knockout mutant library by comprehensively using a bypass PCR and enzyme digestion connection method. The invention is characterized in that the piggyBac transposon system with wide biological applicability and super-large exogenous gene bearing capacity is selected as the delivery system, and the bypass PCR and enzyme digestion connection method is selected as the construction method. In eukaryotes, the lentivirus system is widely used to deliver CRISPR whole genome editing libraries at present, but the lentivirus system has low efficiency in species other than mammals, and the carrying capacity is only a few kb, which limits its application, and the lentivirus system has low efficiency in non-mammalian cells. The eukaryotic whole genome knockout vector library constructed by the piggyBac transposon system disclosed by the invention has good effect.

Drawings

In order to make the object, technical scheme and beneficial effect of the invention more clear, the invention provides the following drawings for explanation:

FIG. 1 is a pB-CRISPRv2 vector map, comprising: piggyBacL/piggyBacR, piggyBac swivel arm; IE2, IE2 promoter; zeocin, Zeocin resistance gene; ser1PA, bombyx mori sericin 1(Ser1) gene polyA; ccDB, escherichia coli lethal gene ccDB; hr3-hsp 70; the Hr3 enhancer and hsp70 promoter; spCas9, spCas9 protein; SV40PA, SV40 polyA.

Fig. 2 shows the construction of a U6-sgRNA library fragment containing the entire sgRNA library using the bridge-approach PCR method.

FIG. 3 is a diagram of mass analysis of a vector library, in which 8% of the vector reads are present in 1-5, 4% of the vector reads are present in 5-10, 72% of the vector reads are present in 10-200, 12% of the vector reads are present in 200-500, and 3% of the vector reads are present in more than 500.

Detailed Description

Preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings.

All the following specific experimental methods, which are not indicated, are carried out according to accepted experimental methods and conditions, for example, according to the instructions provided by the manufacturers of reagents and consumables, or according to the classic laboratory book "molecular cloning guidelines" (third edition, J. SammBruke et al).