CN106637421B

CN106637421B - Construction of double sgRNA library and method for applying double sgRNA library to high-throughput functional screening research

Info

Publication number: CN106637421B
Application number: CN201610969127.3A
Authority: CN
Inventors: 魏文胜; 朱诗优
Original assignee: Because Ji Boya (beijing) Biotechnology Co Ltd
Current assignee: Because Ji Boya (Beijing) Biotechnology Co. Ltd.
Priority date: 2016-10-28
Filing date: 2016-10-28
Publication date: 2019-12-27
Anticipated expiration: 2036-10-28
Also published as: CN106637421A

Abstract

The present invention provides a method for constructing a pgRNA expression plasmid library which, when introduced into a cell together with a Cas protein, can cause the cleavage of two sgRNA target sites on the genome, resulting in the knock-out of a target nucleic acid sequence, by which a functional nucleic acid sequence is screened. The invention also provides a method for constructing a nucleic acid sequence knockout library obtained by transferring the pgRNA library of the invention into a cell, which gene knockout library can be used for screening functional nucleic acid sequences. The invention also provides methods for high throughput screening of functional nucleic acid sequences. The invention is a high throughput CRISPR/Cas strategy that can use multiple pairs of grnas (pgrnas) to generate deletions of large amounts of nucleic acid sequences, enabling rapid identification of functional nucleic acid sequences.

Description

Construction of double sgRNA library and method for applying double sgRNA library to high-throughput functional screening research

Technical Field

The present invention relates to gene editing technology, and is especially method of screening functional nucleic acid sequence, especially functional non-coding element or functional paired gene.

Background

CRISPR/Cas systems [1] from bacteria and archaea have been developed as genome editing tools with a wide range of application values [2-4 ]. In addition to the editing of individual gene loci to study gene function one by one, high-throughput functional screening methods have also been reported to be developed for the function of coding genes and the high-throughput screening of key enhancer regions that regulate the expression of coding genes [3-4 ]. In these screens, the function of the targeted gene or regulatory element can be studied by selecting individual guide RNAs (sgRNAs) associated with a particular phenotype using cell growth or a particular marker as selection information [5-13 ].

Besides a plurality of single genes play a role in important biological processes, a plurality of physiological processes are completed by multi-gene multi-pathway cooperation, and how to obtain a group of genes simultaneously playing a certain important function and perform high-throughput screening is an important problem which is not solved, so that the method has great significance for further researching functions of the genes and providing a brand new mode. In addition, the vast majority (about 98%) of the mammalian genome is composed of non-coding regions, many of which have important regulatory roles. Functional analysis of non-coding regions has been challenging and effective high throughput screening strategies have been lacking to date. Although the CRISPR/Cas system has wide application in analyzing coding gene function, this strategy is based on disrupting the coding gene reading frame, which may not be useful for non-coding elements where indels caused by one gRNA are unlikely to produce a loss-of-function phenotype. Although the use of paired grnas (pgrnas) to create genomic deletions to study the function of individual lncrnas has been reported [14,15], high-throughput strategies for identifying functional non-coding elements have not been reported.

A method for constructing a paired gRNA CRISPR-Cas9 library has been previously reported (Joana a. visual et al, Rapid and effective one-step generation of paired gRNA CRISPR-Cas9libraries, Nature Communications,2015), but the method is cumbersome and inconvenient to operate, still requires a large amount of work when used for large-scale high-throughput screening, and has not been reported as an example for successfully constructing a high-throughput paired gRNA library by the method so far.

There remains a need in the art to develop a simple and feasible high throughput screening strategy for performing high throughput screening of functional nucleic acid sequences, such as functional non-coding elements or functional pair genes.

Disclosure of Invention

The present invention provides a method for constructing a pgRNA expression plasmid library which, when introduced into a cell together with a Cas protein, can cause the cleavage of two sgRNA target sites on the genome, resulting in the knock-out of a target nucleic acid sequence, by which a functional nucleic acid sequence is screened. For example, in the present invention, pgRNA can be targeted to the non-coding element, resulting in deletion of the region between the two sgRNA target sites on the genomic non-coding element, resulting in knock-out of the target non-coding element, for high-throughput screening of functional non-coding elements. For another example, in the present invention, two sgrnas contained in the pgRNA can also target two functionally related different genes, respectively, to simultaneously trigger the knockout of two genes in a cell, thereby performing high-throughput screening on functional pair genes. The invention also provides methods of constructing nucleic acid sequences, e.g., non-coding elements (e.g., lncRNA genes) or paired gene knockout libraries, obtained by transferring a pgRNA expression plasmid library of the invention into a cell, e.g., non-coding elements (e.g., lncRNA genes) or paired gene knockout libraries that can be used to screen for functional nucleic acid sequences, e.g., functional non-coding elements (e.g., lncRNA genes) or functional paired genes. The invention also provides methods of screening for functional nucleic acid sequences, such as functional non-coding elements (e.g., lncRNA genes) or functional pair genes.

According to one aspect of the present invention, there is provided a method of constructing a library of pgRNA expression plasmids, comprising:

(1) providing an initial plasmid comprising a first U6 promoter, a second gRNA backbone sequence coding sequence, and a transcription terminator, sequentially linked;

(2) inserting each of a plurality of 'first gRNA mate sequence coding sequence-spacer sequence-second gRNA mate sequence coding sequence' between a first U6 promoter on an initial plasmid and a second gRNA backbone sequence coding sequence, respectively, by a first ligation reaction, and then transforming competent cells to obtain a second plasmid mixture;

(3) the first gRNA backbone sequence, transcription terminator and second U6 promoter, which are sequentially ligated, are inserted between the two gRNA paired sequences in a second plasmid by a second ligation reaction, and competent cells are then transformed to obtain a pgRNA expression plasmid library.

In some embodiments, the first ligation reaction is a ligation reaction of an initial plasmid cleaved at the 3 'end of the first U6 promoter and the 5' end of the second gRNA backbone sequence encoding sequence with a mixture of a plurality of DNA oligonucleotide sequences.

In some embodiments, the initial plasmid is cleaved by a restriction endonuclease, preferably a Type II (Type IIs) restriction endonuclease, more preferably BsmBI.

In some embodiments, a suicide gene having restriction endonuclease cut sites at both ends thereof may be contained between the first U6 promoter and the second gRNA backbone sequence coding sequence in the initial plasmid. In a preferred embodiment, the suicide gene is the ccdB gene. Preferably, the restriction endonuclease used is a Type II (Type IIs) restriction endonuclease. More preferably, the restriction enzyme used is BsmBI.

In some embodiments, each of the plurality of DNA oligonucleotide sequences comprises two gRNA pairing sequences of one pgRNA, i.e., a first gRNA pairing sequence, a second gRNA pairing sequence, and comprises a sequence of "first gRNA pairing sequence coding sequence-spacer sequence-second gRNA pairing sequence coding sequence" that are sequentially linked.

In some embodiments, a cleavage site is included in the spacer sequence of the DNA oligonucleotide sequence to facilitate cleavage between the first gRNA mate sequence encoding sequence and the second gRNA mate sequence encoding sequence for the second ligation reaction. Preferably, the cleavage site is a restriction endonuclease cleavage site, preferably a Type II (Type IIs) restriction endonuclease cleavage site, more preferably a BsmBI cleavage site.

In some embodiments, the DNA oligonucleotide sequence may also have sequences at both ends that pair with primers to facilitate amplification of the DNA oligonucleotide sequence by a primer pairing-mediated amplification reaction.

In some embodiments, the DNA oligonucleotide sequence mixture is amplified prior to performing the first ligation reaction.

In some embodiments, the first ligation reaction is performed by the Gibson assembly method.

In some embodiments, there is a close proximity linkage in each second plasmid between the first U6 promoter and the first gRNA pairing sequence coding sequence, and between the second gRNA pairing sequence coding sequence and the second gRNA backbone sequence coding sequence.

In some embodiments, the second ligation reaction is a ligation reaction of a second plasmid mixture that is cleaved 3 'of the first gRNA pairing sequence coding sequence and 5' of the second gRNA pairing sequence coding sequence with a DNA fragment comprising a "first gRNA backbone sequence coding sequence-transcription terminator-second U6 promoter" sequence.

In some embodiments, the second plasmid mixture is cleaved by a restriction endonuclease, preferably a Type II (Type IIs) restriction endonuclease, more preferably BsmBI.

In some embodiments, the DNA fragment comprising the "first gRNA backbone sequence coding sequence-transcription terminator-second U6 promoter" sequence further comprises an adapter sequence between the transcription terminator and the second U6 promoter, which adapter sequence may comprise a primer binding region for amplifying a fragment containing the first gRNA pairing sequence coding sequence and/or the second gRNA pairing sequence coding sequence.

In some embodiments, there is a close linkage between the first U6 promoter and the first gRNA mate sequence coding sequence, and between the first gRNA mate sequence and the first gRNA backbone sequence coding sequence in each pgRNA expression plasmid.

In some embodiments, there is a close linkage between the second U6 promoter and the second gRNA mate sequence coding sequence, and between the second gRNA mate sequence and the second gRNA backbone sequence coding sequence in each pgRNA expression plasmid.

In some embodiments, the initial plasmid is a lentiviral vector plasmid.

In some embodiments, the first U6 promoter and the second U6 promoter can be the same or different.

In some embodiments, the first gRNA framework sequence and the second gRNA framework sequence can be the same or different.

In some embodiments, the initial plasmid further comprises a marker gene operably linked to the promoter.

In some embodiments, the marker gene is an antibiotic resistance gene or a fluorescent protein gene.

In some embodiments, the pgRNA targets a non-coding element.

In some embodiments, the pgRNA expression plasmid library targets more than 50 non-coding elements, preferably more than 100 non-coding elements, more than 200 non-coding elements, more than 500 non-coding elements, even more than 1000 non-coding elements.

In some embodiments, different pgrnas in the pgRNA expression plasmid library target different non-coding elements, or multiple pgrnas target the same non-coding element, or a combination of both.

In some embodiments, the non-coding element is a non-coding gene, preferably a incrna gene.

In some embodiments, the pgRNA targets a paired gene. That is, two grnas included in the pgRNA target two genes in the paired genes, respectively.

In some embodiments, the pgRNA expression plasmid library targets more than 50 pairs of paired genes, preferably more than 100 pairs of paired genes, more than 200 pairs of paired genes, more than 500 pairs of paired genes, even more than 1000 pairs of paired genes, or even more than 10000 pairs of paired genes.

In some embodiments, the paired genes are preferably paired encoding genes.

In some embodiments, the target site of the first gRNA pairing sequence or the second gRNA pairing sequence is independently located on the promoter or gene body of the target gene.

In some embodiments, the transcription terminator is polyT.

According to another aspect of the invention, there is provided a method of constructing a library of nucleic acid sequence knock-out cells comprising: constructing a pgRNA expression plasmid library by using the method, delivering the pgRNA expression plasmid library into a target cell, introducing Cas9 nuclease into the target cell, culturing the cell, and screening cells successfully transferred into the pgRNA expression plasmid library to obtain a nucleic acid sequence knockout cell library.

In some embodiments, the nucleic acid sequence is a non-coding element or a paired gene.

In some embodiments, the pgRNA expression plasmid library is delivered into the target cell by lentiviral infection. In some embodiments, the pgRNA expression plasmid library is co-transfected with a viral packaging plasmid into a host cell to produce a lentivirus containing the pgRNA expression plasmid library, and the target cell is infected with the lentivirus to deliver the pgRNA expression plasmid library into the target cell.

In some embodiments, the MOI of the lentiviral infection is less than or equal to 0.3.

In a preferred embodiment, the culture time between infection of cells and collection of infected cells is from about 48h to about 72 h.

In a preferred embodiment, infected cells are harvested by drug resistance or FACS methods.

According to another aspect of the present invention, there is provided a library of nucleic acid sequence knock-out cells obtained by the above-described method.

According to another aspect of the invention, there is provided a method of screening for a functional nucleic acid sequence comprising: and culturing the cell library or culturing the cell library under a specific screening condition, then extracting genomic DNA of a cell library mixture, amplifying DNA fragments containing any one or two gRNA pairing sequence coding sequences in the pgRNA pair, sequencing the amplified products by using a deep sequencing technology, and analyzing a sequencing result, thereby determining the function of the target nucleic acid sequence of the pgRNA.

In some embodiments, the incubation is for a time sufficient for the function of the nucleic acid sequence desired to be screened to be detected.

In some embodiments, a pgRNA with an increased proportion of its target nucleic acid sequence in the sequencing result as compared to the control indicates that the knockout of its target nucleic acid sequence is favorable for cell proliferation, and a pgRNA with a decreased proportion of its target nucleic acid sequence in the sequencing result as compared to the control indicates that the knockout of its target nucleic acid sequence is unfavorable for cell proliferation.

In some embodiments, the non-coding element is a incrna gene. In some embodiments, a pgRNA with an increased proportion of its target non-coding elements in the sequencing result compared to the control indicates that the knock-out of its target non-coding elements is favorable for cell proliferation, and a pgRNA with a decreased proportion of its target non-coding elements in the sequencing result compared to the control indicates that the knock-out of its target non-coding elements is unfavorable for cell proliferation.

In some embodiments, the paired genes are paired encoding genes.

In some embodiments, a pgRNA with an increased proportion of its target paired gene in the sequencing result compared to the control indicates that the knock-out of its target paired gene is favorable for cell proliferation, and a pgRNA with a decreased proportion of its target paired gene in the sequencing result compared to the control indicates that the knock-out of its target paired gene is unfavorable for cell proliferation.

The invention is a high-throughput CRISPR/Cas strategy that can use multiple pairs of grnas (pgrnas) to generate deletions of large amounts of nucleic acid sequences, enabling high-throughput functional studies of nucleic acid sequences in the genome. For example, the present invention can use pairs of grnas (pgrnas) to generate large deletions of large fragments of long non-coding rnas (lncrnas), enabling high-throughput functional studies of mammalian non-coding elements. The invention can also use multiple pairs of gRNAs (pgRNAs) to generate paired gene knockout, and obtain and research examples of important function played by multiple genes together through high-throughput screening. The methods of the invention can also be used to study other phenotypes and functions of interest other than simple growth, and can be more broadly applied to study other non-coding sequences, including microRNAs, cis-regulatory elements, and other elements of unknown function.

Drawings

Figure 1 is a lentivirus-delivered pgRNA to produce large fragment deletions with high efficiency in human cells stably expressing Cas 9. (a) Construction of lentiviral plasmids expressing paired guide RNA (pgRNA). The U6 promoter and gRNA coding sequence were cloned into the LL3.7 lentiviral backbone. The amplified DNA fragment encoding pgRNA was ligated by the gold Gate method (Golden Gate method) with two U6 promoters (U6)₂) Or only one common U6 promoter (U6)₁) In the lentivirus backbone of (a). (b, c) delivery of pgRNA vectors by lentivirusesInto human cells expressing Cas 9. The large fragment deletion induced by pgRNA targeting the CSPG4 gene was identified by PCR. Six pairs of gRNAs (b) were selected which produced large deletions of 2-4.5kb, and genomic PCR reactions (b, c) were performed using primers L1/R1. Enrichment of all infected Huh7.5 by FACS_OCCells were incubated for 6 days. Letter U6 in FIG. 1a₂And U6₁Representing two tandem approaches, the control was a pair of grnas, one targeting the CSPG4 locus and the other targeting the AAVS1 region. (d) Effect of culture time on efficiency of deletion of large fragments after infection of cells with pgRNA virus. pgRNA (3+ 3' in FIG. 1c, designed to generate a-3.5 kb deletion) was delivered to Huh7.5 by lentiviral infection_OCIn the cells, genomic DNA was extracted at the different time points indicated (upper panel of FIG. 1d), quantified using the primers L2/R2(b) corresponding to the sequences flanking the target site of the pgRNA, and normalized using the primers L3/R3(b) corresponding to the sequences distant from the target site of the pgRNA. See Table 12 below for primer sequences. Images were analyzed using ImageJ software and data were expressed as mean ± standard deviation (n ═ 3) (lower panel of fig. 1 d). (e) DNA sequencing analysis of large fragment deletions in pgRNA (3+ 3') targeted human CSPG4 locus in mixed cells 3 weeks after infection (fig. 1 d). The partial sequence of the target gene containing the target region of the two grnas is underlined, and the shaded nucleotides represent the PAM sequence. The dashed horizontal lines indicate deletion.

FIG. 2 is a large fragment deletion induced by lentivirus-mediated pgRNA. (a, b) 5 pairs of gRNAs were selected which induced large deletions of 1-5kb targeting MALAT1 gene with the respective U6 promoter (U6)₂) A genomic PCR reaction was performed using the primer L4/R4. Enrichment of all infected Huh7.5 by FACS_OCCells were incubated for 6 days. Controls were a pair of grnas, one targeting MALAT1 gene and the other targeting AAVS1 region. (c) Effect of culture time on efficiency of large fragment deletion after infection of cells with pgRNA virus targeting MALAT1 gene. pgRNA (2+ 2' in b, designed to produce a 4.3kb deletion) was delivered to Huh7.5 by lentivirus infection_OCIn the cells, genomic DNA was extracted at given different time points. Quantification was performed using primers L5/R5 corresponding to the sequences flanking the target site of the pgRNA. (d) Target of cell infectionThe effect of the incubation time after introduction of the CSPG4 gene into the pgRNA virus on the efficiency of deletion of large fragments. pgRNA (2+ 2' in c, designed to produce a 4.0kb deletion) was delivered to Huh7.5 by lentivirus infection_OCIn the cells, genomic DNA was extracted at given different time points. Quantification was performed using the primers L2/R2 corresponding to the sequences flanking the target site of the pgRNA, and normalization was performed using the primers L3/R3 corresponding to the sequences distant from the target site. All primer sequences are listed in table 12. Images were analyzed by ImageJ software and data were expressed as mean ± standard deviation (n ═ 3). (e) DNA sequencing analysis of large fragment deletions induced by two CSPG 4-targeting pgRNAs (p 2and p4, representing 2+2 'and 4+ 4', respectively) in FIG. 1d and two MALAT 1-targeting pgRNAs (p 2and p3, representing 2+2 'and 3+ 3', respectively) in FIG. 2c in mixed cells 3 weeks after infection. The partial sequence of the target gene containing the target regions of the two grnas in the genome is underlined, and the shaded nucleotides represent the PAM sequence. The dash represents a deletion.

FIG. 3 is the design, cloning and screening of a pgRNA library. (a) Construction of a pgRNA plasmid library. Each synthetic 137-nt DNA oligonucleotide contains two gRNA pairing sequence coding sequences. Oligos were amplified to generate dsDNA molecules and cloned into the lentiviral backbone using the Gibson reaction. After insertion of the linker segments by BsmBI digestion and ligation, the final construct (hereinafter "materials and methods" section) was obtained. (b) Delivery of pgRNA libraries to Huh7.5 by lentivirus infection_OCIn cells, the MOI is about 0.3. Infected cells were harvested by FACS with green fluorescence 3 days after infection. For screening, library cells were cultured for 30 days before genomic DNA extraction and high throughput sequencing analysis.

Fig. 4 is a DNA sequence of the designed oligonucleotide and the linker between the two grnas of each pair, and PCR amplification was performed for deep sequencing analysis, as shown in the electropherogram. (a) The characteristics and sequence of each oligonucleotide. The left and right arms were used for primer targeting during amplification. (b) The characteristics of the linker constructed from the pgRNA plasmid, as well as its unique sequence between the end of the first gRNA backbone and the beginning of the second U6 promoter. (c) From Huh7.5_OCIsolated genomes of libraries and wild-type cellsThe DNA was used as a template for PCR amplification.

FIG. 5 shows the identification of lncRNA for negative (negative) and positive (positive) screens. (a) Distribution of changes in abundance (log fold) of pgrnas targeting negative control, positive control, and lncrnas. P < 0.05; p < 0.01; wilcox rank sum test. The middle line represents the median value; boxes represent quartering bits; each whisker (whiskers) extends to 1.5 times the interquartile; the dots represent outliers. (b) Summary of log fold (log fold) and genomic position of all pgrnas targeting one positive control gene EZH 2. Most of the pgRNA was reduced (i.e., the cells transformed with the pgRNA were reduced), including pgRNA targeting the promoter, promoter + exon and intron of EZH2 (log FC < -1). (c, d) top ranking negative screening lncRNA from MAGECK calculation (c) and positive screening lncRNA (d) Rank Aggregation (RRA) scores. Some positive control genes that were negatively screened are also shown in black triangles. A smaller RRA score indicates a stronger selection of the corresponding lncRNA.

FIG. 6 is the correlation and read distribution of independent experimental replicates. (a) Correlation between replicate groups of control samples. (b) Correlation between replicate groups of 30-day enriched samples. The Pearson Correlation Coefficient (PCC) between the repetitions is also given.

FIG. 7 shows the mean read counts for pgRNAs for negative screening lncRNA (a) and positive screening lncRNA (b) in the top ranking. (c) Mean read counts of pgrnas targeting AAVS1 (left) and non-targeting control (right).

FIG. 8 is pgRNAread counts of lncRNA selected for negative (a) and positive (b) screens for validation.

FIG. 9 is a validation of candidate lncRNA. (a-c) Huh7.5_OCEffect of large fragment deletion of RPL18A (a), negatively selected lncrna (b), and positively selected lncrna (c) in cells on cell proliferation. Each lncRNA selection 3-5 was validated against promoter or promoter + exon-targeted pgRNA. pgRNA was delivered into cells by lentiviral infection and EGFP was targeted by FACS⁺The cells of (3) are quantified. The first quantification was started three days after viral infection, in this figure and aboveThe latter panels are marked as day 0. EGFP for a given time point with control (day 0)⁺The percentages are normalized to determine the cell proliferation rate. The newly designed pgrnas, which differ from those used in the original library, are marked with an asterisk. The arrow points to the transcription start site. Open and shaded boxes refer to exons of non-coding and coding genes, respectively. (d) Effect of transcriptional inhibition of negatively screened lncrnas on cell proliferation. mRNA levels (normalized to GAPDH) were quantified for RPL18A, AC004463.6, RP11-439K3.1, and AC 095067.1. All primers used for quantitative PCR are listed in table 13. (e) Huh7.5_OCEffect of sgRNA and pgRNA targeting lncRNA screened negatively in cells on cell proliferation. Data are presented as mean ± standard deviation (n ═ 3). P values were calculated by Student's t test and corrected for multiple comparisons using the Benjamini Hochberg program<0.05；**P<0.01；***P<0.001; and NS: has no significance.

FIG. 10 is a genomic validation of candidate lncRNA in Huh7.5 cells and genomic deletion induced by RP11-439K3.1 targeting pgRNA. (a) Huh7.5_OCEffect of deletion of large fragments of negatively selected lncRNA in cells on cell proliferation. (b) Huh7.5_OCEffect of deletion of large fragments of forward-selected lncrnas in cells on cell proliferation. (c) Huh7.5 containing LINC 00882-targeting pgRNA_OCLINC00882mRNA levels in cells, and Huh7.5 after complementation of LINC00882 cDNA_OCLINC00882mRNA levels (normalized to GAPDH) in cells were quantified. All primers used for quantitative PCR are listed in table 13. (d) cDNA complementation of LINC 00882-Targeted pgRNA and LINC00882 for Huh7.5_OCThe effect of cell proliferation. Data are presented as mean ± standard deviation (n ═ 3). P values were calculated using the Student's t test and corrected for multiple comparisons using the Benjamini Hochberg program<0.01. (e) RP11-439K3.1_ p3 and RP11-439K3.1_ p4 were delivered to Huh7.5 by lentiviruses_OCIn cells, deletions were detected by genomic PCR. (f) Huh7.5_OCEffect of sgRNA (transcriptional repression) and pgRNA targeting negatively selected lncrnas in cells on cell proliferation. The cell proliferation assay was the same as described in figure 9. In this and the remaining figures, the new designDifferent gRNA pairs than used in the original library were marked with an asterisk (#). The arrow indicates the transcription start site. Open and shaded boxes refer to exons of non-coding and coding genes, respectively. Data are presented as mean ± standard deviation (n ═ 3). P values were calculated using the Student's t test and corrected for multiple comparisons using the Benjamini Hochberg program<0.05；**P<0.01；***P<0.001; and NS: not significant.

FIG. 11 is a graph showing the effect of pgRNA and transcription inhibited sgRNA presented on Huh7.5 cell viability. All pgRNAs and sgRNAs were delivered to Huh7.5 by lentiviral infection_OCIn the cell. FACS enrichment of cells was performed 72h post infection and LDH lethality was detected 1-3 days post FACS. Data are presented as mean ± standard deviation (n ═ 3).

FIG. 12 shows the effect of forward screening for the transcriptional activation of IncRNA on cell viability. (a) Levels of LINC01087 and LINC00882mRNA (normalized to GAPDH) were quantified. All primers used for quantitative PCR are listed in Table 13. (b) All sgrnas were delivered into huh7.5 cells by transient transfection. FACS enrichment of cells was performed 72h post-infection and LDH lethality was detected 1-3 days after FACS. Data are presented as mean ± standard deviation (n ═ 3).

Fig. 13 is the complete sequence of the paired sgrnas (pgrnas).

Detailed Description

The invention constructs a pgRNA expression plasmid library of multiple nucleic acid sequences, such as non-coding elements (such as lncRNA genes) or paired genes, in targeted cells, realizes the knockout of the target nucleic acid sequence in the cells by using a Cas9/CRISPR (Clustered Regularly interspaced short Palindromic Repeats) system and the pgRNA library, and screens functional nucleic acid sequences, such as functional non-coding target genes (such as functional lncRNA genes) or functional paired genes, through phenotypic characteristics.

The term "nucleic acid sequence" or "target nucleic acid sequence" as used herein may refer to any nucleic acid sequence having a known or unknown function present in a genome, such as, but not limited to, noncoding elements, paired genes, nucleic acid sequences of unknown function, and the like, unless the context makes clear that it has another meaning. As used herein, "a nucleic acid sequence" refers to the entire nucleic acid sequence having a function or possibly a function. The "different nucleic acid sequences" as used herein refers to nucleic acid sequences having different functions, respectively.

The term "non-coding element" as used herein, which may also be referred to as a "non-coding sequence," refers to a nucleotide sequence present in the genome of a cell that does not encode an amino acid, which may be a non-coding gene, or may be other non-coding elements that are non-genic, including but not limited to lncRNA genes, microRNAs, cis-regulatory elements, and other unannotated elements.

The term "lncRNA gene" used herein may be abbreviated as "lncRNA" and means a long non-coding RNA (long non-coding RNA) having a length of more than 200 nucleotides. The term "non-coding RNA" refers to a functional RNA molecule that cannot be translated into a protein, including but not limited to small interfering RNA, long non-coding RNA, and the like. Research shows that lncRNA plays an important role in a plurality of life activities such as dose compensation effect (Dosage compensation effect), epigenetic regulation, cell cycle regulation, cell differentiation regulation and the like, and is a hotspot of genetic research. The term "functional incrna gene" as used herein refers to a functional incrna gene that functions in a cell.

The term "paired genes" or "functional pair of genes" as used herein refers to a pair of genes that exhibit a phenotype associated with function only when both genes are knocked out, and that do not exhibit a phenotype associated with function when only one gene is knocked out. The paired genes may be genes having the same or similar functions, and when only one gene is knocked out, a phenotype associated with the function cannot be exhibited due to the compensatory effect of the other gene. In some embodiments, the paired genes are paired encoding genes.

The Cas9/CRISPR system utilizes RNA-guided DNA binding to sequence-specific cleave target DNA, from crRNA (CRISPR-derived RNA) by base pairing to tracrRNA (trans-activating RNA) to form a tracrRNA/crRNA complex that directs the nuclease Cas9 protein to cleave double-stranded DNA at a specific position on the target sequence that is paired with the crRNA. The target sequence that pairs with the crRNA is typically a sequence of about 20 nucleotides located upstream of the genomic PAM (protospacer adjacent motif) site (NNG).

Cleavage of the target site by Cas9 protein requires the aid of a guide RNA. The term "guide RNA" is also known as gRNA (guide RNA), which typically includes nucleotides on the crRNA that are complementary to the target sequence and an RNA backbone (Scaffold) formed by base pairing of the crRNA to the tracrRNA, and is capable of recognizing the target sequence to which the crRNA is paired. The gRNA can form a complex with Cas9 protein and bring Cas9 protein to the target sequence and cleave the target site therein.

Traditionally, grnas are usually in the form of sgrnas (single guide rnas). sgRNA, also called "single-stranded guide RNA," is an RNA strand formed by fusion of crRNA and trancrna. Typically, as is well known to those skilled in the art, sgrnas comprise sequences that pair with a target sequence (also referred to as gRNA pairing sequences or sgRNA pairing sequences), a backbone (Scaffold) sequence (also referred to as a gRNA backbone sequence), and a transcription terminator (e.g., polyT). In the present invention, unless otherwise specified, grnas and sgrnas may be used interchangeably.

The term "gRNA framework sequence" or "framework sequence" as used herein refers to a sequence in the sgRNA between the gRNA pairing sequence and the transcription terminator.

Methods and tools for designing gRNAs (sgRNAs) are well known in the art and may be obtained in a variety of ways, such as, but not limited to, the gUIDEboot online platform available from the Horizon Discovery company.

The term "pgRNA" as used herein refers to a paired gRNA (paired rna) or paired sgRNA, and refers to a pair of grnas (or sgrnas). In some embodiments, a pgRNA described herein is a pair of grnas (or sgrnas) that target different target sites on the same nucleic acid fragment or target different nucleic acid fragments, respectively. In some embodiments, a pgRNA described herein is a pair of grnas (or sgrnas) that target different target sites on the same non-coding element. Preferably, the target sites of the two gRNAs in a pgRNA pair are separated by 200bp-10 kb. In other embodiments, a pgRNA described herein is a pair of grnas (or sgrnas) that target two genes in a paired gene, respectively.

The invention can simultaneously cut two target sites aimed at by pgRNA by applying a Cas9/CRISPR system, thereby achieving the purpose of gene knockout. For example, the invention can use Cas9/CRISPR system to cut the fragment between two target sites targeted by pgRNA, thereby deleting the sequence between the two target sites, and achieving the purpose of gene knockout (for example, the target sites are on non-coding elements). For another example, the Cas9/CRISPR system can be used to cleave two target genes targeted by pgRNA, so as to achieve the knockout of the two target genes (for example, the two target genes are paired genes). The pgRNA expression plasmid library constructed by the invention can realize high-throughput knockout of a plurality of nucleic acid sequences, such as non-coding elements (such as lncRNA genes) or a plurality of pairs of paired genes, thereby realizing high-throughput screening of functional nucleic acid sequences, such as functional non-coding elements or functional paired genes.

In some embodiments, the present invention provides methods for constructing a library of pgRNA expression plasmids comprising each pgRNA expression plasmid having two gRNA sequences operably linked to respective U6 promoters in sequential linkage, the two grnas constituting a pgRNA, targeting a nucleic acid sequence or targeting different nucleic acid sequences, e.g., targeting a non-coding element (e.g., an incrna gene) or targeting a pair of genes (wherein the two grnas target two of the pair of genes, respectively). The library construction method is realized by two-step ligation reaction, firstly providing a plasmid (namely an initial plasmid) containing a first U6 promoter, a second gRNA framework sequence coding sequence and a transcription terminator which are sequentially connected, then respectively inserting each of a plurality of first gRNA pairing sequence coding sequence-spacer sequence-second gRNA pairing sequence coding sequence between a first U6 promoter and a second gRNA framework sequence coding sequence on the plasmid through the first-step ligation reaction, then transforming competent cells to obtain a second plasmid mixture, then inserting the first gRNA framework sequence, the transcription terminator and a second U6 promoter which are sequentially connected between two gRNA plasmid sequences in a second plasmid through the second-step ligation reaction, and then transforming the competent cells to obtain an RNA expression plasmid library, wherein each pgRNA expression comprises a first U6 promoter, a first gRNA pairing sequence and a first gRNA framework sequence which are sequentially connected -a transcription terminator-a second U6 promoter-a second gRNA pairing sequence-a second gRNA backbone sequence-a transcription terminator ". The first U6 promoter and the second U6 promoter may be the same or different in the present invention. The first gRNA framework sequence and the second gRNA framework sequence can be the same or different in the present invention.

The term "plasmid" as used herein may also be referred to as an expression vector, which when introduced into a cell, expresses the coding sequence contained therein in association with a promoter. Plasmids usually contain elements necessary for gene expression. In the present invention, the plasmid used may be a lentiviral vector plasmid, or any other plasmid capable of expressing the gene encoded therein, preferably a lentiviral vector plasmid.

The term "coding sequence" of an RNA (e.g. a pgRNA or gRNA backbone sequence or a gRNA pairing sequence) as used herein refers to a DNA sequence encoding the RNA.

The term "transcription terminator" as used herein refers to a DNA sequence that gives a signal for transcription termination by RNA polymerase. Useful transcription terminators are well known to those skilled in the art. Any suitable transcription terminator may be used in the present invention. The transcription terminator used in the present invention is preferably polyT.

In some embodiments, the pgRNA targets a non-coding element. In some embodiments, the pgRNA expression plasmid library targets more than 50 non-coding elements, preferably more than 100 non-coding elements, more than 200 non-coding elements, more than 500 non-coding elements, even more than 1000 non-coding elements.

In some embodiments, the paired genes are preferably paired encoding genes.

In some embodiments, a suicide gene having restriction endonuclease cut sites at both ends thereof may be contained between the first U6 promoter and the second gRNA backbone sequence coding sequence in the initial plasmid. The restriction endonuclease is preferably a type II restriction endonuclease, more preferably BsmBI.

The term "suicide gene" as used herein refers to a gene that, when expressed in a host cell, has a lethal effect on the host cell. Suicide genes can be constructed into expression vectors, and as a selectable marker during transformation, the expression product of the suicide gene can inhibit host cell growth, which would not occur if the clone was transformed with a plasmid that was not cut or circularized. The suicide gene preferably used is the ccdB gene, a toxin protein gene, for example, as introduced by InvitrogenA vector transformation system.

In some embodiments, the first ligation reaction is a ligation reaction of an initial plasmid cleaved at the 3 'end of the first U6 promoter and the 5' end of the second gRNA backbone sequence encoding sequence with a mixture of a plurality of DNA oligonucleotide sequences. In a preferred embodiment, the starting plasmid is cleaved by a restriction endonuclease. The initial plasmid is first cleaved by a restriction endonuclease, preferably a Type II (Type IIs) restriction endonuclease, more preferably BsmBI, such that the initial plasmid is cleaved at the 3 'end of the first U6 promoter and the 5' end of the second gRNA backbone sequence coding sequence prior to ligation with a mixture of multiple DNA oligonucleotide sequences. Each of the plurality of DNA oligonucleotide sequences comprises two gRNA pairing sequences of one pgRNA, i.e., a first gRNA pairing sequence, a second gRNA pairing sequence, and comprises a sequence of "first gRNA pairing sequence coding sequence-spacer sequence-second gRNA pairing sequence coding sequence" that are sequentially linked. The spacer sequence of the DNA oligonucleotide sequence includes a cleavage site (e.g., a restriction endonuclease cleavage site, preferably a type II (TypeIIs) restriction endonuclease cleavage site, more preferably a BsmBI cleavage site) in the spacer sequence to facilitate cleavage between the first gRNA mate sequence coding sequence and the second gRNA mate sequence coding sequence for the second ligation reaction. The DNA oligonucleotide sequence may also have sequences at both ends that pair with primers to facilitate amplification of the DNA oligonucleotide sequence by a primer-pairing mediated amplification reaction. Preferably, the DNA oligonucleotide sequence mixture is amplified prior to performing the first ligation reaction.

In some embodiments, the first ligation reaction is performed by the Gibson assembly method. In this case, the primers used to amplify the DNA oligonucleotide sequence mixture have sequences homologous to the original plasmid cleaved by the restriction endonuclease required for Gibson assembly at both ends.

The term "Gibson assembly" as used in the present invention is also known as GibsonDoctor Daniel Gibson, institute j.craig Venter, was filed in 2009. The Gibson assembly method is suitable for splicing multiple linear DNA fragments and also for inserting the DNA of interest into a vector. For Gibson assembly, homologous fragments must first be added to the ends of the DNA fragmentsThen mixing and incubating the DNA fragments and master mix for one hour, wherein the master mix consists of three enzymes, DNA exonuclease firstly degrades nucleotides from the 5' end to generate a cohesive end, then overlapping sequences between adjacent fragments are annealed, and finally DNA polymerase and DNA ligase fill the sequences to form a complete double-stranded DNA molecule so as to realize traceless splicing. The master mix can be purchased from NEB or SGI-DNA companies, or formulated by itself (see Miller Lab Protocol).

In some embodiments, the reaction mixture of the first ligation reaction transforms the competent cells to obtain a second plasmid mixture. Each second plasmid contained in the second plasmid mixture comprises a "first U6 promoter-first gRNA mate sequence coding sequence-spacer sequence-second gRNA mate sequence coding sequence-second gRNA backbone sequence coding sequence-transcription terminator" sequence that are sequentially linked. In some embodiments, there is a close proximity linkage in each second plasmid between the first U6 promoter and the first gRNA pairing sequence coding sequence, and between the second gRNA pairing sequence coding sequence and the second gRNA backbone sequence coding sequence.

In some embodiments, the second ligation reaction is a ligation reaction of a second plasmid mixture that is cleaved 3 'of the first gRNA pairing sequence coding sequence and 5' of the second gRNA pairing sequence coding sequence with a DNA fragment comprising a "first gRNA backbone sequence coding sequence-transcription terminator-second U6 promoter" sequence. In a preferred embodiment, the second plasmid is cleaved by a restriction endonuclease, preferably a Type II (Type IIs) restriction endonuclease, more preferably BsmBI. The second plasmid mixture is first cleaved by a restriction endonuclease, preferably a Type II (Type IIs) restriction endonuclease, more preferably BsmBI, such that the second plasmid is cleaved 3 'of the first gRNA mate sequence coding sequence and 5' of the second gRNA mate sequence coding sequence prior to ligation with a DNA fragment comprising the sequence "first gRNA backbone sequence coding sequence-transcription terminator-second U6 promoter". The DNA fragment comprising the "first gRNA backbone sequence coding sequence-transcription terminator-second U6 promoter" sequence further comprises an adapter sequence, preferably between the transcription terminator and the second U6 promoter, which may comprise a primer binding region for amplifying a fragment containing the first gRNA pairing sequence coding sequence and/or the second gRNA pairing sequence coding sequence.

In some embodiments, the reaction mixture from the second ligation step transforms competent cells to obtain a pgRNA expression plasmid library. Each pgRNA expression plasmid in the pgRNA expression plasmid library comprises the sequence of "first U6 promoter-first gRNA mate sequence coding sequence-first gRNA backbone sequence coding sequence-transcriptional terminator-second U6 promoter-second gRNA mate sequence coding sequence-second gRNA backbone sequence coding sequence-transcriptional terminator" in sequential linkage. Wherein the "first gRNA pairing sequence-first gRNA framework sequence-transcription terminator" constitutes a first gRNA (or sgRNA) sequence, and the "second gRNA pairing sequence-second gRNA framework sequence-transcription terminator" constitutes a second gRNA (or sgRNA) sequence. In some embodiments, there is a close linkage between the first U6 promoter and the first gRNA mate sequence coding sequence, and between the first gRNA mate sequence and the first gRNA backbone sequence coding sequence in each pgRNA expression plasmid. In some embodiments, there is a close linkage between the second U6 promoter and the second gRNA mate sequence coding sequence, and between the second gRNA mate sequence and the second gRNA backbone sequence coding sequence in each pgRNA expression plasmid.

In the present invention, the expression "first sequence-second sequence-third sequence" and the similar expressions mean that the sequences contained therein are connected in the order of the words. In some embodiments, there may or may not be other nucleotides or nucleotide sequences between the individual sequences.

Preferably, in the present invention, the adjacent ligation between the gRNA mate sequence coding sequence and the gRNA backbone sequence coding sequence, and between the gRNA mate sequence coding sequence and the transcription promoter, is in close proximity both in the plasmids and DNA fragments used and generated in the library construction method, and in the final prepared pgRNA expression plasmid library. The term "immediately adjacent to" or "immediately adjacent to" as used herein means that there is no other nucleotide present between two elements (e.g., DNA sequences) that are sequentially linked.

The term "sequentially linked" as used herein means that two or more elements (e.g., DNA sequences) are linked in the order indicated by the text. In some embodiments, two or more elements (e.g., DNA sequences) that are connected sequentially may or may not have other nucleotides or nucleotide sequences.

The starting plasmid used in the present invention may also comprise a marker gene operably linked to a promoter for use in screening cells containing a pgRNA expression plasmid.

The term "marker gene" as used herein refers to any marker gene whose expression can be selected or enriched, i.e., when the marker gene is expressed in a cell, the cell expressing the marker gene can be selected and enriched in a manner. Marker genes that can be used in the present invention include, but are not limited to, fluorescent protein genes that can be sorted by FACS after expression, or resistance genes that can be selected using antibiotics, or protein genes that can be recognized by corresponding antibodies after expression and selected by immunostaining or magnetic bead adsorption. Resistance genes that may be used in the present invention include, but are not limited to, resistance genes for Blasticidin (Blasticidin), Geneticin (Geneticin, G-418), Hygromycin (Hygromycin B), Mycophenolic Acid (Mycophenoic Acid), Puromycin (Puromycin), bleomycin (Zeocin), or Neomycin (Neomycin). Fluorescent Protein genes that can be used in the present invention include, but are not limited to, genes of blue Fluorescent Protein (cyanfluorescent Protein), Green Fluorescent Protein (Green Fluorescent Protein), enhanced Green Fluorescent Protein (enhanced Green Fluorescent Protein), Yellow Fluorescent Protein (Yellow Fluorescent Protein), Orange Fluorescent Protein (Orange Fluorescent Protein), Red Fluorescent Protein (Red Fluorescent Protein), Far-Red Fluorescent Protein (Far-Red Fluorescent Protein), or Switchable Fluorescent Protein (Switchable Fluorescent proteins), preferably Enhanced Green Fluorescent Protein (EGFP).

The promoter operably linked to the marker gene may be any promoter commonly used in the art for marker gene expression, including but not limited to the CMV promoter.

In a preferred embodiment, the initial plasmid is a lentiviral vector comprising, in order from 5 'to 3', a first U6 promoter, a ccdB gene with a BsmBI cleavage site at both ends, a second gRNA backbone sequence coding sequence, a polyT, a CMV promoter, an EGFP gene. The DNA oligonucleotide sequence contains two gRNA pairing sequence coding sequences of a pair of pgRNAs, and both ends of a spacer sequence between the two gRNA pairing sequence coding sequences are provided with BsmBI enzyme cutting sites. The DNA oligonucleotide sequence has primer pair sequences at both ends. A DNA fragment comprising a "first gRNA backbone sequence coding sequence-transcription terminator-second U6 promoter" sequence has a BsmBI cleavage site 5 'of the first gRNA backbone sequence coding sequence and 3' of the second U6 promoter, and a linker sequence between the transcription terminator and the second U6 promoter. Amplification was performed with primers targeting sequences flanking the DNA oligonucleotide sequences, with sequences homologous to the 3 'region of the first U6 promoter and the 5' region of the second gRNA backbone sequence coding sequence on both primers, respectively, to generate 60bp homologous sequences homologous to the BsmBI digested starting plasmid. The amplified mixture of DNA oligonucleotide sequences was ligated to the original plasmid by Gibson assembly and transformed into Trans1-T1 competent cells to obtain a second plasmid, which was then digested with BsmBI and ligated to a DNA fragment containing the sequence "first gRNA backbone sequence coding sequence-transcription terminator-second U6 promoter" digested with BsmBI, and the ligation mixture was transformed into Trans1-T1 competent cells to obtain the final library plasmid.

It is also within the scope of the present invention to construct a library of pgRNA expression plasmids obtained by the above-described method.

In some embodiments, the present invention provides methods of constructing a nucleic acid sequence, such as a non-coding element (e.g., lncRNA gene) or a paired knock-out cell library, comprising delivering a pgRNA expression plasmid library of the present invention into a target cell, introducing Cas9 nuclease into the target cell, culturing the cell, and screening for cells successfully transformed with the pgRNA expression plasmid library to obtain a nucleic acid sequence, such as a non-coding element (e.g., lncRNA gene) or a paired knock-out cell library.

Methods for delivering plasmid libraries into target cells are well known in the art, such as, but not limited to, by electroporation, microinjection, gene gun, calcium phosphate co-precipitation, lipofection, virus-mediated transfection techniques. Virus-mediated transfection techniques may be by retroviral, adenoviral, or lentiviral transfection. Lentivirus transfection requires the preparation of a plasmid library using a lentivirus vector, then co-transfecting host cells with the plasmid library and a lentivirus packaging plasmid to generate lentiviruses of the plasmid library, then transducing target cells with the lentiviruses, and after infection, screening the successfully infected target cells. The host cell used may be any cell that can be used to co-transfect lentivirus in vivo cells with a lentivirus packaging plasmid to produce lentivirus, including but not limited to HEK293T cells.

In some embodiments of the invention, the pgRNA expression plasmid library is delivered to the target cell by lentiviral infection. In some embodiments, the pgRNA expression plasmid library is co-transfected with a viral packaging plasmid into a host cell to produce a lentivirus containing the pgRNA expression plasmid library, and the target cell is infected with the lentivirus, thereby delivering the pgRNA expression plasmid library to the target cell.

In some embodiments, the MOI of the lentivirus-infected target cells is 0.3 or less, such that one lentivirus infects one target cell.

In some embodiments, the culture time between infection of cells and collection of infected cells is about 48h to about 72 h.

In some embodiments, infected cells are harvested by drug resistance or FACS methods.

It is also within the scope of the invention to construct a library of nucleic acid sequence knock-out cells, e.g., a library of noncoding elements or paired gene knock-out cells, by the methods described above.

In the present invention, Cas9 nuclease can be introduced into cells in the form of a protein or in the form of its encoding nucleic acid sequence (e.g., mRNA or cDNA). The nucleic acid encoding Cas9 can be introduced into a cell, e.g., by transfection, contained in a plasmid or viral vector (e.g., a lentiviral vector). The nucleic acid encoding Cas9 can also be delivered directly into cells by electroporation, liposomes, microinjection, and the like.

In the present invention, Cas9 and the pgRNA expression plasmid library can be introduced into the cell simultaneously, or, for example, Cas9 can be introduced into the cell first, and then the pgRNA expression plasmid library can be introduced into the cell. In some embodiments, the cells are co-transfected with a vector comprising Cas9 and a pgRNA expression plasmid library. In other embodiments, the Cas9 and pgRNA expression plasmid libraries are assembled into complexes in vitro, and the cells are transfected. In other embodiments, Cas9 is stably expressed in the cells prior to transfecting the cells with the pgRNA expression plasmid library.

The cell of the present invention may be any eukaryotic cell, such as an isolated animal cell, e.g., a totipotent cell, a pluripotent cell, an adult stem cell, a fertilized egg, or a somatic cell, etc. In some embodiments, the cell is a vertebrate cell. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a human cell, e.g., a HEK293T cell, a HeLa cell, huh7.5 cell_OCCells, and the like. In some embodiments, the cell is a bovine, goat, sheep, cat, dog, horse, rodent, fish, primate cell. In some embodiments, the rodent comprises a mouse, a rat, a rabbit.

In some embodiments, the invention also provides a method of screening for a functional nucleic acid sequence, e.g., a functional non-coding element (e.g., a incrna gene) or a functional pair of genes, comprising: culturing the cell library or culturing the cell library under specific screening conditions, extracting genomic DNA from the cell library mixture, amplifying DNA fragments comprising the sequences encoding either or both of the gRNA pairing sequences, sequencing the amplified products using deep sequencing techniques, and analyzing the sequencing results to determine the function of the target nucleic acid sequence of the pgRNA, e.g., the target noncoding element (e.g., lncRNA gene) or the target pairing gene.

In some embodiments, the purpose of amplifying DNA fragments comprising either or both of the gRNA mate sequence coding sequences in the pgRNA is to deep sequence these DNA fragments, thereby identifying which cells comprising which pgRNA are present in the cultured cell library and the proportion thereof. Amplification can be performed with the sequence preceding the first U6 promoter, the linker sequence between the transcription terminator and the second U6 promoter, and/or the sequence following the sequence encoding the second gRNA backbone sequence in the pgRNA expression plasmid as the target sequence for the primers.

The term "deep sequencing" as used herein, which may also be referred to as "high-throughput sequencing" or "next generation sequencing," can be performed on hundreds of thousands to millions of DNA molecules in parallel at a time. The sequencing result of deep sequencing may be the number of reads for each different sequenced DNA fragment. Deep sequencing technology is a mature technology in the art, and there are a number of manufacturers that provide deep sequencing services, or provide kits, instruments, and instructions for performing deep sequencing. Deep sequencing techniques that may be used include, but are not limited to, deep sequencing on Ion Torrent or Illumina sequencing platforms, e.g., using Illumina hiseq 2500.

All pgRNA-related sequences contained in the cell library obtained by amplification (i.e. DNA fragments containing the sequences encoding either or both gRNA-mate sequences in the pgRNA) can be sequenced by deep sequencing. The sequencing result of deep sequencing can be the number of reads of each different sequenced DNA fragment or the proportion of each different sequenced DNA fragment. The "occupancy ratio" of each different sequenced DNA fragment (e.g., each different pgRNA-related sequence) may refer to the ratio of the reads number of each different sequenced DNA fragment (e.g., each different pgRNA-related sequence) to the reads number of all sequenced DNA fragments (i.e., all pgRNA-related sequences obtained by amplification). According to the deep sequencing results, if certain pgRNAs have an increased proportion compared to the control, it is indicated that the cells containing these pgRNAs are enriched during the culture and that the knockout of their target nucleic acid sequence favours the proliferation of the cells; if certain pgRNAs are present in a reduced proportion compared to the control, it is indicated that the cells containing these pgRNAs are gradually depleted during the culture, and the knock-out of their target nucleic acid sequences is detrimental to cell proliferation.

The cell libraries of the invention can be used to screen for functional nucleic acid sequences, such as non-coding elements (e.g., lncRNA genes) or paired genes, for example, to screen for functional nucleic acid sequences that modulate cell proliferation, such as non-coding elements (e.g., lncRNA genes) or paired genes. Such functions include, but are not limited to, for example, a knockout of the nucleic acid sequence promoting or inhibiting cell growth, or a knockout of the nucleic acid sequence resulting in cell growth being inhibited or promoted by a particular drug. The "specific screening conditions" include, but are not limited to, the presence of a drug, the presence of a protein, and the like.

In one embodiment, for example, a nucleic acid sequence knockout cell library of the invention is cultured, genomic DNA of the cell library mixture is then extracted, DNA fragments comprising either or both of the gRNA paired sequence coding sequences in the pgRNA pair are amplified, the amplified product is sequenced using deep sequencing techniques, the sequencing results are analyzed, the cell library sequencing results at the beginning of the culture are used as controls, a pgRNA whose percentage in the sequencing results is increased compared to the controls indicates that knockout of its target nucleic acid sequence favors cell proliferation, and a pgRNA whose percentage in the sequencing results is decreased compared to the controls indicates that knockout of its target nucleic acid sequence does not favor cell proliferation.

In another embodiment, for example, a nucleic acid sequence knockout cell library of the invention is cultured under specific screening conditions, genomic DNA of the cell library mixture is then extracted, DNA fragments comprising either or both of the gRNA mate sequence coding sequences in the pgRNA pair are amplified, the amplification product is sequenced using deep sequencing techniques, the sequencing results are analyzed, with the sequencing results of a cell library not cultured under the specific screening conditions being used as a control, a pgRNA in the sequencing results having an increased proportion compared to the control indicating that knockout of its target nucleic acid sequence is favorable for cell proliferation under the screening conditions, and a pgRNA in the sequencing results having a decreased proportion compared to the control indicating that knockout of its target nucleic acid sequence is unfavorable for cell proliferation under the screening conditions.

In another specific embodiment, for example, a nucleic acid sequence knockout cell library of the invention is cultured in the presence of a particular drug, genomic DNA of the cell library mixture is then extracted, DNA fragments comprising the sequences encoding either or both of the gRNA mate sequences in the pgRNA pair are amplified, the amplified product is sequenced using deep sequencing techniques, the sequencing results are analyzed, and with the cell library not cultured under the particular screening condition as a control, the pgRNA whose percentage in the sequencing results is increased compared to the control indicates that knockout of its target nucleic acid sequence facilitates proliferation of the cell under the screening condition, i.e., knockout of its target nucleic acid sequence increases tolerance of the cell to the particular drug; the pgRNA with a reduced proportion compared with the control in the sequencing result indicates that the knockout of the target nucleic acid sequence is not beneficial to the proliferation of the cell under the screening condition, i.e., the knockout of the target nucleic acid sequence can reduce the tolerance of the cell to the specific drug.

The term "control" in the context of comparison of sequencing results refers to a control that reflects the function of the nucleic acid sequence under study, and such control is chosen in accordance with routine skill in the art. For example, when the effect of nucleic acid sequence knock-out on cell proliferation is examined, the results of cell library sequencing at the start of culture are used as controls. For another example, when the effect of nucleic acid sequence knock-out on cell proliferation under a particular screening condition is examined, the sequencing results of a library of cells not cultured under the particular screening condition are used as controls.

The term "forward screening" as used herein (e.g., in the examples) refers to the knockout of a target nucleic acid sequence, e.g., a non-coding element (e.g., lncRNA gene), resulting in the promotion of cell growth.

The term "negative screening" as used in the present invention (e.g., examples) means that knockout of a target nucleic acid sequence, e.g., a non-coding element (e.g., lncRNA gene), results in inhibition of cell growth.

The term "comprising" or "comprises" as used herein means "including but not limited to," consisting essentially of … …, "or" consisting of … ….

The invention is further illustrated by the following examples and figures, which are given by way of illustration only and are not intended to limit the scope of the invention. The examples are carried out according to conventional experimental conditions, if not indicated otherwise, such as the Molecular cloning handbook, Sambrook et al (Sambrook J & Russell DW, Molecular cloning: a laboratory Manual,2001), or according to the instructions provided by the manufacturer.

Example 1 delivery of paired guide RNAs (pgRNAs) by lentivirus

1. Construction of a CRISPR pgRNA library such that the genomic sequence between the two gRNA target sites is deleted simultaneously

Two methods were tested to express pgRNA in a lentiviral backbone, one using two U6 promoters driving two gRNAs separately (U6)₂) The other is to drive two sequentially linked gRNAs with a single U6 promoter (U6)₁) (FIG. 1 a). To compare these two approaches, six pairs of grnas targeting the CSPG4 gene (see table 1 and table 2 below, fig. 1b) were used to induce large fragment deletions, which were predicted to delete 2kb-4.5kb of the human CSPG4 locus (gene CSPG4 encodes complete membrane chondroitin sulfate proteoglycan) (fig. 1b and c).

TABLE 1 design of CSPG 4-targeted paired gRNAs (pgRNAs) for DNA fragment deletion

TABLE 2 sequences of paired gRNAs (pgRNAs) targeting CSPG4 for DNA fragment deletion

sgRNA^CSPG4	Sequence of
		sgRNA1	5’-AGGAGACTGGAGGTAAGACA
sgRNA1’	5’-TCACTCCTGTGCACAGCAGC
		sgRNA2	5’-AGAAGAGCTGGCCCAGCAGC
sgRNA2'	5’-CCACCACATACACACCTATG
		sgRNA3	5’-AGTCTAGTGAGACGGAGGCG
sgRNA3’	5’-TGCTGGGAGGAGGTTTGAGA
		sgRNA4	5’-TCAGTCTCGGGATCTCTGAT
sgRNA4’	5’-TGGCCAGTGATGAGCCTTCT
		sgRNA5	5’-GTGCTGGGACTTGCTGTGGT
sgRNA5’	5’-CAGAAAGGCAACTAAACAGA
		sgRNA6	5’-ACACCTCTTGCCAGTCTGCT
sgRNA6’	5’-GTTGTAAGCTCCATGGGATT
		sgRNA^AAVS1	5’-CGGAACCTGAAGGAGGCGGC

Hepatoma cell line Huh7.5 stably expressing Cas9 and OCT1 genes (OCT1 gene encoding transcription factors)_OC[8,16]In U6₂All six pgRNAs in the vector produced the correct size genome deletion, but U6₁Only two pgRNAs in the vector produced the correct deletion, which was much less efficient (FIG. 1c), indicating the use of U6₂More preferably, U6 is used₂Subsequent experiments were performed.

U6 was found₂Five pgrnas targeting lncRNA MALAT1 (see tables 3 and 4) also produced genome deletions of the correct size with high efficiency (fig. 2a and b).

TABLE 3 design of paired gRNAs (pgRNAs) targeting MALAT1 for DNA fragment deletion

TABLE 4 sequences of paired gRNAs (pgRNAs) targeting MALAT1 for DNA fragment deletion

sgRNA^MALAT1	Sequence of
		sgRNA1	5’-CCGCAGATCAGAGTGGGCCAC
sgRNA1’	5’-GGATAGTACACTTCACTCAG
		sgRNA2	5’-ACACAAGAAGTGCTTTAAG
sgRNA2'	5’-GGGATCAAGTGGATTGAGG
		sgRNA3	5’-CCCGAATTAATACCAATAGA
sgRNA3’	5’-CTTGAATGTCTCTTAGAGGG
		sgRNA4	5’-CCCATCAATTTAATTTCTGG
sgRNA4’	5’-CCAGTTTGAATTGGGAAGCT
		sgRNA5	5’-GAGCCAGTGCGATTTGGTGA
sgRNA5’	5’-GGTCTTAACAGGGAAGAGAG

Next it was investigated whether the post-transduction culture time of lentiviral delivered pgRNA affected the efficiency of gene deletion and genome deletions were observed to continue over time and reach a plateau approximately 15 days after transduction (fig. 1 d). Similar results were obtained when genome deletions were induced with another pgRNA targeting CSPG4 (2+2 ', fig. 1b) and another pgRNA targeting MALAT1 (2+ 2', fig. 2a, c and d). Thus, it is desirable to culture the library cells for at least 2 weeks after transduction, so that there is sufficient time to produce a genomic deletion in the mammalian cells, at a level suitable for screening. Total genomic sequencing of five pgRNA-targeted regions (3 of which target CSPG4 and 2 of which target MALAT1) revealed almost 80% of deletions at each site to be the exact junction of the two Cas9 cleavage sites 3nt upstream of the promiscuous sequence adjacent motif (PAM) (FIGS. 1e and 2e), consistent with previous findings [17 ]. Together, these results indicate that lentiviral delivered pgRNA is capable of efficiently producing large fragment genomic deletions in mammals.

Example 2 construction of pgRNA library and genome-wide IncRNA deletion screening

A pgRNA library was designed that targets approximately 700 incrna genes (table 5), which have known or predicted roles in cancer or other diseases [18 ].

TABLE 5 summary of pgRNA libraries for functional screening

We developed a rapid and precise method for cloning pgRNA into lentiviral expression vectors (FIG. 3a, FIGS. 4a and 4 b).

Since both grnas in each gRNA pair are driven by the same type of U6 promoter and contain the same 3' backbone sequence, recombination can occur, which can lead to erroneous pgRNA assembly. The recombination rate in the plasmid constructs of the two pgRNA libraries and the integration of the chromosomes in the cells after transduction were thus tested and found to occur after viral transduction in the cells, with a recombination rate of about 7.5%, which is comparable to the error rate in oligonucleotide (oligo) synthesis (Table 6). This indicates that recombination has negligible effect on the pgRNA library screening.

TABLE 6 qualitative summary of pgRNA libraries

Categories	PlasmidsLibraries	Cell library
			Success rate	92.5％	84.2％
Mutation rate	7.5％	8.3％
			Rate of recombination	0％	7.5％

Note: sequences of 80 and 120 grnas randomly selected from plasmid and cell libraries, respectively, were verified.

At U6₂The pgRNA library was constructed and transferred to Huh7.5 at a low multiplicity of infection (MOI ═ 0.3) (low multiplicity of infection so that only one cell was transferred to one pair of grnas)_OCMiddle, Huh7.5_OCHave previously been used to functionally screen coding genes [16]. The cultures were incubated for 30 days after transduction in an attempt to identify lncrnas that had a positive or negative impact on cell growth and viability and to maximize the identification of these lncrnas. PCR amplification of gRNA coding regions from extracted cellular genomic DNA was performed before or after CRISPR screening for deep sequencing analysis (fig. 3b and 4 c). Overall, the read distribution of 3 independent experimental replicates under each condition showed a high level of correlation (fig. 6). After 30 days of culture, pgRNA targeting the positive control gene (mostly ribosomal gene) or lncRNA was depleted (i.e. the cells transferred into the pgRNA decreased) compared to the negative control pgRNA (non-targeting pgRNA or pgRNA targeting the non-functional AAVS1 locus) (fig. 5a and 5b), indicating their effect on cell survival or proliferation.

The top selected incrna genes (the top hits) were identified by comparing the day 30 samples to day 0 controls using the MAGeCK algorithm [21 ]. MAGeCK uses a Negative Binomial (NB) model to assess the statistical significance of individual pgRNA abundance changes and compares the ranking (ranks) of pgrnas targeting each incrna using a uniformly distributed zero model (see materials and methods section below). The output of MAGeCK is a set of lncrnas that are negatively (or positively) selected, or whose knock-out destroys (or stimulates) lncrnas that proliferate in cells. MAGeCK identified 43 negatively screened and 8 positively screened lncrnas with statistical significance (false discovery rate < 0.25).

Gene Set Enrichment Analysis (Gene Set Enrichment Analysis, GSEA) showed that positive control pgRNAs were significantly enriched in the ranked list of negative screening pgRNAs, showing the essential function of their target genes as expected [22 ]. The top negative screening genes included two positive control genes: RPL18A, an essential gene, and EZH2, a gene encoding a polycomb family member, which plays an important role in the proliferation of hepatoma cells [23 ]. The promoters and exons of RPL18A and EZH2 were depleted together (fig. 5 b). Similarly, 89% of pgrnas targeting top-ranked negatively-selected lncrnas were depleted when 76% of lncrnas targeting the positive-selection were enriched (fig. 5c and 5d, fig. 7a and 7 b). In contrast, abundance of pgRNA of non-targeted control and pgRNA of targeted AAVS1 locus were similar under control and treatment conditions (fig. 7c, table 7).

TABLE 7 screening results for negative control pgRNA

Interestingly, targeting 266 pgrnas in the intron region of the essential gene reduced cell viability (fig. 5b), possibly due to deletion of regulatory elements or regulation of alternative splicing of the target gene [24,25 ].

Example 3 validation of selected lncRNA candidates

From the statistically significant incrnas for positive or negative screens, selected incrna genes (hits) were obtained that ranked top, and their corresponding pgrnas were consistently depleted (for negative screens) or enriched (for positive screens) in 3 independent experimental replicates, respectively (fig. 5c and 5d and fig. 8, table 8 and table 9).

TABLE 8 negative selection of lncRNA genes

TABLE 9 forward screening of lncRNA genes

To verify the function of some of these lncrnas, two pairs of grnas originally present in the library were selected and up to 3 additional novel pgrnas were designed for each gene. In addition, 3 pairs of grnas were designed to target the AAVS1 locus as a negative control (table 10).

TABLE 10 functional verification of selected lncRNA pgRNA design

Retransduced all pgRNAs to Huh7.5 with a CMV-EGFP-carrying lentiviral backbone_OCIn the cell. Cell proliferation was quantified as a percentage change in EGFP-positive cells. Deletion of the promoter of RPL18A, the first ribosomal gene from the negative screen list, significantly reduced cell proliferation, and deletion of the AAVS1 locus had negligible effect on cell growth (fig. 9 a).

Using the same method, selection from the pgRNA library screening did not show any weight in comparison with the encoding geneValidation of the stacked lncRNA. 5 lncRNAs were selected, and their deletion appeared to inhibit cell proliferation in the primary screen: AC004463.6, AC095067.1, HM13-AS1, RP11-128M1.1 and RP11-439K 3.1. 4 lncrnas were also selected, and their deletion appeared to positively regulate cell growth: LINC00176, LINC01087, LINC00882and LINC 00883. pgRNA is designed to target the promoter or exon of lncRNA. For a pair of lncrnas transcribed separately but sharing the same promoter: LINC00882and LINC00883, another 3 pgRNA targeting exons were designed. Based on the results of the single deletion, all 5 incrnas from the negative screen were found to be essential for cell proliferation, and all 4 incrnas from the positive screen were shown to negatively regulate cell proliferation (fig. 9b and 9c, fig. 10a and 10 b). Introduction of cDNA clone of LINC00882 into two sets of Huh7.5 containing pgRNA targeting LINC00882and deleted LINC00882_OCIn cells, and exogenous anaplerotic expression of LINC00882 was shown to negatively regulate cell proliferation (fig. 10c and 10 d). Some pgRNAs did not produce phenotypes, such as RP11-439K3.1_ p3 and RP11-439K3.1_ p4 (FIG. 9b), and it has been demonstrated that this was because these pgRNAs were not capable of producing genomic deletions (FIG. 10 e). To further validate candidate genes, the CRISPR inhibitor (CRISPRi) method was used [26]This method can reduce transcription of the target region. Suppression of three negatively screened lncrnas (AC004463.6, RP11-439K3.1 and AC095067.1) using CRISPRi was found to significantly reduce cell proliferation (fig. 9 d). Five cell lines that knocked out negatively screened lncRNA and CRISPRi cell lines (transcription of lncRNA is repressed) were also examined for cell death signals, and all 5 lncrnas were found to be essential for cell survival (figure 11 and table 11).

Table 11 sgRNA design for CRISPR inhibition and CRISPR upregulation for functional validation of selected lncrnas

For forward screened gene candidates, we used CRISPR up-regulation (CRISPRa) method [27] to up-regulate transcription of LINC01087 and LINC00882, and found that overexpression of both lncrnas was lethal (fig. 12 and table 11). Therefore, the CRISPR/Cas9 screening strategy for genome deletion is effective for negative and positive screening of lncrnas, with high efficiency and reliability.

For library screening and candidate validation, paired grnas were introduced into cells. It is likely that the observed phenotypic change is due to the effect of a gRNA-mediated Double Strand Break (DSB), rather than the effect of pgRNA-mediated genome deletion. To rule out this possibility, we compared the effect of pgrnas targeting AC004463.6 and AC095067.1 with the effect of introducing only one of their corresponding grnas. In both cases, only pgRNA significantly affected cell proliferation, while single grnas targeting either introns or exons did not alter cell survival (fig. 9e and fig. 10 f). This suggests that pgRNA-mediated genome deletion is essential for generating a functional knockout of lncRNA, at least for these two loci, and indels generated by a single gRNA are unlikely to achieve such an effect.

Using this method, approximately 700 human lncRNA were screened and lncRNA having carcinogenic or tumor suppressive effects in cancer cells could be identified. By applying the screening method, 51 lncRNAs positively or negatively regulating the growth of human cancer cells are identified. 9 of the 9 lncrnas were each validated by CRISPR/Cas 9-mediated genome deletion and functional complementation (functional restore), CRISPR upregulation or inhibition, and gene expression profiling alone.

The method of the present invention does not distinguish the mechanism of action of lncRNA [30], and detailed studies are required to further investigate the function of the identified lncRNA. More than 30% of lncRNA we identified are located in introns of other coding genes with diverse biological functions [33 ]. Further characterization of these lncrnas is challenging, as the disrupted intron can have a deleterious effect on cell proliferation (e.g., intron-targeted pgRNA in fig. 5 b).

Although the CRISPR pgRNA library may cause incorrect pgRNA assembly due to recombination of paired grnas during lentiviral packaging and integration steps, due to sequence similarity between the two U6 promoters and the two gRNA backbone sequences, the screening method was not affected due to limited recombination rates. However, this approach can be optimized by using different types of U6 promoters (of human and murine origin, respectively) [34] and alternative sgRNA backbone sequences to further reduce the possible rate of lentiviral recombination. The method can also be extended to study other phenotypic changes of interest besides simple growth by introducing a reporter system. The paired guide RNA screening strategy of the present invention can be more broadly applied to the study of other non-coding sequences, including microRNAs, cis-elements and other unannotated elements.

Materials and methods

Cells and reagents

Huh7.5 cells were obtained from Stanley Cohen's laboratory (Stanford university of medicine school) and cultured in Dulbecco's modified Eagle's Medium (DMEM, Gibco) supplemented with non-essential amino acids (NEAA, Gibco). 22RV1 cells were obtained from Myles Brown's laboratory and cultured in RPMI1640 medium (Gibco). HeLa cells were obtained from Jiangyuan laboratories (Beijing university) and maintained in Dulbecco's modified Eagle medium (DMEM, Gibco). All media were supplemented with 10% fetal bovine serum (FBS, CellMax) at 5% CO₂And culturing at 37 ℃ in an incubator. All cells were checked to ensure that they were free of mycoplasma contamination.

Plasmid construction

The human U6 promoter, ccdB gene and gRNA backbone were cloned into pLL3.7(Addgene, Inc.) replacing its original U6 promoter, creating a lentiviral pgRNA-expression vector [8 ]. The backbone-linker-U6 fragment was cloned into pEASY-Blunt plasmid (TransGen Biotech).

Positive and negative controls

Positive controlThe target genes for the positive control consisted of 20 genes, including 17 ribosomal genes and 3 cancer-associated genes, FOXA1, HOXB13 and EZH 2. We designed 100 pairs of grnas for each positive control gene, including 20 pairs of targeted promotersGrnas (distance between two grnas in each pair is between 200bp-5 kb), 80 grnas targeting promoter plus exon.

Negative controlThree different types of negative controls of 500 pgRNAs were designed. The first type of negative control (100 pairs of grnas) consists of pgRNA that does not target any site in the human genome. These pgRNAs were generated directly from the GeCKO v2 library [38]The existing non-targeted control gRNA. The second type of control (100 versus gRNA) consists of a pgRNA targeting the AAVS1 region, a non-essential region in the genome, the AAVS1 region, often used in CRISPR studies for performing efficiency tests. A third type of negative control (300 pairs of grnas) consists of pgRNA targeting the intron of the positive control gene.

Construction of CRISPR/Cas9 pgRNA library

A library was constructed with 12472 on grnas, targeting 671 lncrnas. Referring to FIG. 3a, a 137-nt oligonucleotide (CustomAlray, Inc.) containing the sequence encoding the gRNA pairing sequence for each pair of pgRNAs was designed and synthesized. Amplification was performed with primers targeting these oligonucleotide flanking sequences to generate 60bp homologous sequences homologous to the BsmBI digested backbone of the expressed pgRNA. Amplified DNA products were assembled by Gibson's method [39 ]]Was ligated into a lentiviral vector and transformed into Transs 1-T1 competent cells (Transgen, Biotech) to obtain a plasmid. The plasmid obtained in the previous step was then digested with BsmBI and ligated to BsmBI-digested backbone-Linker-U6 fragment (FIG. 4), and the ligation mixture was transformed into Transs 1-T1 competent cells (Transgen, Biotech) to obtain the final library plasmid (FIG. 13). The library plasmids were co-transfected into HEK293T cells with two viral packaging plasmids, pvsg and pr8.74(Addgene, Inc.) using X-tremagene HP DNA transfection reagent (Roche), generating lentiviruses of the pgRNA library. Construction of Huh7.5 by transduction of Low MOI (. about.0.3) Virus_OCCell library, 72h post infection, FACS screening was performed to infect successful cells.

Calculation of recombination Rate

The recombination rate for chromosomal integration in the plasmid construct and in the cells after transduction was calculated. For plasmids, the entire pgRNA sequence was amplified from the library plasmids as a template. For chromosome integration in cells, pgRNA sequences are amplified from the library genome as templates. The PCR product was then cloned into a vector for sequencing analysis. 80 and 120 clones were randomly selected from plasmid and cell libraries, respectively, for sequencing.

CRISPR/Cas9 pgRNA library screening

Will be 1.2X 10 in total⁷One pgRNA library cell was seeded onto a 150mm Petri plate, and three replicate groups were set up. The library cells of the control group were collected, and the library cells of the experimental group were cultured for one month, and the cells of the experimental group were collected. From 4X 10 using DNeasy blood and Tissue kit (Qiagen)⁶Genomic DNA was isolated from each replicate in individual cells. PCR amplification (TransTaq DNA Polymerase High Fidelity, TransGen) was then integrated into the gRNA-coding region in the chromosome by 28 cycles of reaction with primers targeting the U6 promoter and the junction between the two grnas of each pair (fig. 4 and table 12). In each tube, using 0.6. mu.g of genomic DNA as template, 20 PCR reactions were performed for each replicate. Mixing the PCR products of each repeat and using DNA Clean&Concentrator-25(Zymo Research Corporation) was purified and then subjected to deep sequencing analysis (Illumina HiSeq 2500).

Computational analysis of screens

Data analysis after library screening was performed using the latest version of MAGeCK (0.5.3) [21 ]. The MAGECK algorithm consists of 4 steps: normalization, pgRNA mean variance modeling, pgRNA ranking and lncRNA ranking. And obtaining genes which are remarkably enriched in the negative screening and the positive screening by comprehensively analyzing the enrichment degree of different pgRNAs targeting each lncRNA, the parallelism of different repeat groups and the quantity of pgRNAs with remarkably changed each lncRNA to give a comprehensive score, and carrying out subsequent genetics and functional verification.

Cell proliferation assay

All pgrnas targeted to the positive control gene and lncRNA to be verified were cloned into a lentiviral expression backbone carrying EGFP driven by the CMV promoter and delivered into the cells by viral infection. EGFP-FACS pair⁺The percentage of cells was quantified. First of allThe sub-quantification starts three days after the viral infection and is marked as day 0, as a standardized control. EGFP of given time point⁺The percentages were normalized to day 0 controls to determine the cell proliferation rate.

Cell death signal detection

All pgRNAs targeting negative selection of lncRNA were delivered to Huh7.5 by lentiviral infection_OCIn cells, all sgrnas designed to block or up-regulate the transcriptional level of lncrnas were delivered into huh7.5 cells by transient transfection. Cells were FACS enriched 72 hours post infection or transfection and LDH death signal detection was performed one to three days post FACS. LDH staining and detection was performed as described in the product literature (CytTox96, Promega). The death signal represented by the amount of LDH released was normalized to the well for maximal LDH activity of all lysed cells.

CRISPR-inhibited transcription and CRISPR up-regulation transcription

For CRISPRi, the KRAB-dCas9-P2A-mCherry (Addgene #60954) plasmid was delivered into huh7.5 cells by lentiviral infection. mCherry-positive cells were enriched by FACS 3 days after infection. Sgrnas targeting negatively selected lncrnas were then delivered by lentiviral transfection into cells stably expressing dCas9-KRAB, followed by cell proliferation assays and cell lethality assays. For CRISPRa, three plasmids dCAS-VP64_ Blast (addge #61425), MS2-P65-HSF1_ Hygro (addge #61426) and sgrnas carrying EGFP for each forward screening lncRNA were delivered into cells by transient transfection. EGFP positive cells were enriched by FACS 3 days after transfection and then tested for cell lethality.

Real-time PCR

RNA from cultured cells was extracted using RNAprep Pure Micro kit (TIANGEN, DP420), and cDNA was synthesized using QuantScript RT kit (TIANGEN, KR 103-03). Real-time PCR was performed on a LightCycler96qPCR system using SYBR Premix Ex Taq II (TaKaRa, RR 820A). GAPDH transcript levels were measured as a normalized control.

Primers for PCR amplification of genomic DNA and library construction

TABLE 12 primers used for PCR amplification of genomic DNA and library construction

Primer for quantitative PCR

TABLE 13 primers for quantitative PCR

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Claims

1. A method of constructing a library of pgRNA expression plasmids comprising:

(3) inserting a first gRNA framework sequence, a transcription terminator and a second U6 promoter which are sequentially connected into a second plasmid between two gRNA pairing sequences through a second-step connection reaction, and then transforming competent cells to obtain a pgRNA expression plasmid library;

wherein the interval between the target sites of the first gRNA and the second gRNA is 200bp-10 kb.

2. The method according to claim 1, wherein the first ligation reaction is a ligation reaction of an initial plasmid cleaved at the 3 'end of the first U6 promoter and the 5' end of the second gRNA backbone sequence coding sequence with a mixture of a plurality of DNA oligonucleotide sequences each comprising a sequence of "first gRNA mate sequence coding sequence-spacer sequence-second gRNA mate sequence coding sequence" joined in sequence, wherein the first gRNA mate sequence and the second gRNA mate sequence are two gRNA mate sequences of one pgRNA.

3. The method according to claim 2, wherein the DNA oligonucleotide sequence includes a cleavage site in the spacer sequence to facilitate cleavage between the first gRNA mate sequence encoding sequence and the second gRNA mate sequence encoding sequence for the second ligation reaction.

4. The method according to claim 3, wherein the cleavage site is a cleavage site for a restriction endonuclease by which the second plasmid mixture is cleaved.

5. The method according to claim 4, wherein the restriction endonuclease is a Type II (Type IIs) restriction endonuclease.

6. The method according to claim 5, wherein the Type II (Type IIs) restriction endonuclease is BsmBI.

7. A method according to claim 2, wherein the initial plasmid is cleaved by a restriction endonuclease.

8. The method according to claim 7, wherein the restriction endonuclease is a Type II (Type IIs) restriction endonuclease.

9. The method according to claim 8, wherein the Type II (Type IIs) restriction endonuclease is BsmBI.

10. The method according to any one of claims 1 to 3, wherein the DNA oligonucleotide sequence mixture is amplified before the first ligation reaction is performed.

11. The method according to claim 10, wherein the first ligation reaction is performed by the Gibson assembly method.

12. The method according to any one of claims 1-3, wherein there is a close linkage between the first U6 promoter and the first gRNA pairing sequence coding sequence, and between the second gRNA pairing sequence coding sequence and the second gRNA backbone sequence coding sequence in each second plasmid.

13. The method according to any one of claims 1-3, wherein the second ligation reaction is a ligation reaction of a second plasmid mixture that is cleaved 3 'of the first gRNA pairing sequence coding sequence and 5' of the second gRNA pairing sequence coding sequence with a DNA fragment comprising a "first gRNA backbone sequence coding sequence-transcription terminator-second U6 promoter" sequence.

14. The method according to claim 13, wherein the DNA fragment comprising the "first gRNA backbone sequence coding sequence-transcription terminator-second U6 promoter" sequence further comprises a linker sequence between the transcription terminator and the second U6 promoter.

15. The method according to claim 14, wherein the adapter sequence serves as a primer pairing sequence for amplifying a fragment containing the first gRNA pairing sequence coding sequence and/or the second gRNA pairing sequence coding sequence.

16. A method according to any one of claims 1-3, wherein: each pgRNA expression plasmid has a contiguous linkage between the first U6 promoter and the first gRNA mate sequence coding sequence, and between the first gRNA mate sequence and the first gRNA backbone sequence coding sequence; and/or in each pgRNA expression plasmid between the second U6 promoter and the second gRNA mate sequence coding sequence, and between the second gRNA mate sequence and the second gRNA backbone sequence coding sequence.

17. A method according to any one of claims 1-3, wherein: the first U6 promoter and the second U6 promoter are the same or different, and/or the first gRNA framework sequence and the second gRNA framework sequence are the same or different.

18. A method according to any one of claims 1 to 3, wherein the initial plasmid is a lentiviral vector plasmid.

19. The method according to claim 18, wherein the initial plasmid further comprises a marker gene operably linked to the promoter.

20. The method according to claim 19, wherein the marker gene is an antibiotic resistance gene or a fluorescent protein gene.

21. The method according to any one of claims 1-3, wherein said pgRNA targets a non-coding element or a paired gene.

22. The method of claim 21, wherein said non-coding element is a lncRNA gene, microRNA, cis-regulatory element, or other element of unknown function.

23. A method of constructing a library of nucleic acid sequence knock-out cells comprising: constructing a pgRNA expression plasmid library using the method of any one of claims 1-22, delivering the pgRNA expression plasmid library into a target cell, introducing Cas9 nuclease into the target cell, culturing the cells, and screening the cells successfully transformed into the pgRNA expression plasmid library to obtain a nucleic acid sequence knock-out cell library; wherein a region between the two gRNA target sites on the nucleic acid sequence is deleted.

24. The method of claim 23, delivering said library of pgRNA expression plasmids into target cells by lentiviral infection.

25. The method of claim 24, wherein the lentivirus infection has an MOI of 0.3 or less.

26. The method according to any one of claims 23-25, wherein said nucleic acid sequence is a non-coding element or a paired gene.

27. The method of claim 26, wherein said non-coding element is a lncRNA gene, microRNA, cis-regulatory element, or other element of unknown function.

28. A library of nucleic acid sequence knockout cells obtained by the method of any one of claims 23-25 and 27.

29. A method of screening for a functional nucleic acid sequence comprising: culturing the library of cells of claim 28 or culturing the library of cells of claim 28 under specific screening conditions, followed by extracting genomic DNA from the cell library mixture, amplifying DNA fragments comprising the sequences encoding either or both of the gRNA mate sequences in the pgRNA pair, sequencing the amplified products using deep sequencing techniques, and analyzing the sequencing results to determine the function of the target nucleic acid sequence of the pgRNA; wherein the specific screening condition is in the presence of a drug or in the presence of a protein.

30. The method according to claim 29, wherein said nucleic acid sequence is a non-coding element or a paired gene.

31. The method of claim 30, wherein said non-coding element is a lncRNA gene, microRNA, cis-regulatory element, or other element of unknown function.

32. The method according to any one of claims 29-31, wherein a pgRNA with an increased proportion in the sequencing result compared to the control indicates that the knockout of its target nucleic acid sequence is favorable for cell proliferation, and a pgRNA with a decreased proportion in the sequencing result compared to the control indicates that the knockout of its target nucleic acid sequence is unfavorable for cell proliferation.

33. A library of nucleic acid sequence knockout cells obtained by the method of claim 26.