PCR-based generation of shRNA libraries from cDNAs
© Du et al; licensee BioMed Central Ltd. 2006
Received: 21 October 2005
Accepted: 21 June 2006
Published: 21 June 2006
The use of small interfering RNAs (siRNAs) to silence target gene expression has greatly facilitated mammalian genetic analysis by generating loss-of-function mutants. In recent years, high-throughput, genome-wide screening of siRNA libraries has emerged as a viable approach. Two different methods have been used to generate short hairpin RNA (shRNA) libraries; one is to use chemically synthesized oligonucleotides, and the other is to convert complementary DNAs (cDNAs) into shRNA cassettes enzymatically. The high cost of chemical synthesis and the low efficiency of the enzymatic approach have hampered the widespread use of screening with shRNA libraries.
We report here an improved method for constructing genome-wide shRNA libraries enzymatically. The method includes steps of cDNA fragmentation and endonuclease MmeI digestion to generate 19-bp fragments, capping the 19-bp cDNA fragments with a hairpin oligonucleotide, and amplification of the hairpin structures by PCR. The PCR step converts hairpins into double-stranded DNAs that contain head-to-head cDNA fragments that can be cloned into a vector downstream of a Pol III promoter.
This method can readily be used to generate shRNA libraries from a small amount of mRNA and thus can be used to create cell- or tissue-specific libraries.
RNA interference (RNAi) provides a powerful tool for silencing gene expression. Large-scale phenotypic or pathway-driven screens of siRNA libraries may help to identify novel genes that may be targets for therapy in cancer and other diseases. Two different methods have been used to construct genome-wide siRNA libraries. One is to chemically synthesize oligonucleotides based on siRNA design algorithms (for reviews, see [1, 2]). Typically, the oligonucleotides are synthesized in the form of double-stranded DNA molecules containing short hairpin RNA (shRNA) templates and are cloned into a Pol III-driven expression vector. Libraries constructed with this method and targeting more than 10,000 different human genes have been successfully used for screening [3, 4]. The other method is to convert collections of cDNAs into shRNA templates. Three groups have developed protocols to produce genome-wide shRNA libraries [5–7]. These protocols share several common features, and all "measure" the appropriate length of the hairpin using the type IIS restriction endonuclease MmeI, which cuts 20/18 nt from its recognition site. The common steps, with minor variations, include (1) generating random cDNA fragments; (2) ligating the cDNA fragments with a double-stranded oligonucleotide that contains an MmeI site; (3) restriction digestion with MmeI; (4) ligating a second oligonucleotide to the digested cDNA fragments to form a double-stranded DNA with a hairpin structure; (5) using a DNA polymerase with strong strand-displacement activity to convert the hairpin DNA into double-stranded DNA; and (6) cloning the double-stranded DNA into an expression vector.
The chemical synthesis method is a very expensive and time-consuming approach that requires synthesis of thousands of oligonucleotides, followed by cloning and sequence validation. Construction of shRNA libraries from cDNAs provides an economical alternative. However, the multiple-step process in the current protocols makes the overall efficiency low and thus requires a large amount of starting mRNA. To increase the efficiency of library construction, we have developed an improved method, which includes newly designed oligonucleotides and a key PCR step to amplify and convert the hairpin structures in the abovementioned step 5 into double-stranded DNAs. The PCR amplification of the hairpin structures greatly increases the overall efficiency of the procedure and allows libraries to be constructed from small amounts mRNA.
Results and discussion
The "YIU" procedure
Generation of cDNA hairpins with noncomplementary ends. In previous reports [5–7], the first oligonucleotide used either had a CG overhang or was blunt-ended, allowing self-ligation. To avoid this problem, the "Y" oligonucleotide was designed with a single 3'-T overhang (Fig. 1), and thus cannot self-ligate (compare lanes 1 and 2, Fig. 2A). Additional features of the Y oligonucleotide include (a) an embedded MmeI site, (b) long noncomplementary arms designed for anchoring PCR primers with high melting temperatures (see below), and (c) a single basepair mismatch within the 18-bp stem region, resulting in the AflII site and MlyI site each being present on only one arm of the double-stranded PCR products, allowing the products to be directionally cloned. The unusual shape of the Y oligonucleotide causes abnormal mobility on PAGE gels (Fig. 2A). Double-stranded cDNA fragments (the "I") were generated either by restriction enzyme digestion or by partial DNase I digestion and repair with T4 polymerase. They were then treated with Taq DNA polymerase to add a single A at the 3' end ; this untemplated addition is reported to occur with an average efficiency of around 70% . This treatment prevents self-ligation of cDNA fragments but allows them to ligate to the Y oligonucleotide. Excess "Y" oligonucleotide was added to increase ligation efficiency.
The ligated "YI" molecule was digested with the restriction endonuclease MmeI, a Type IIS restriction endonuclease that cuts 20 and 18 nt away from its recognition site, yielding a 2-nt 3' overhang. A hairpin loop oligonucleotide ("U") with a random 2-nt 3' extension was ligated to the ends generated by MmeI (Fig. 1). The final product ("YIU") contains a 10-nt loop. In "YIU" molecules, the cDNA insert is 19 bp in size. Although the U molecules can ligate to themselves (compare lanes 6 and 7, Fig. 2A), the U dimers cannot be amplified by PCR.
PCR amplification of "YIU". Regular DNA hairpins (consisting only of a stem and loop) cannot be efficiently PCR-amplified because after the denaturation step, hairpins rapidly re-form upon cooling, precluding primer annealing . However, a DNA hairpin with noncomplementary arms, such as YIU molecules, can be efficiently PCR-amplified . Primers corresponding to the two noncomplementary arms were designed with high melting temperatures, allowing the annealing and extension temperature during PCR cycling to be set at 75°C to destabilize intramolecular hairpins. Vent DNA polymerase was used because of its high fidelity and its strand-displacement activity when at high temperature. Although hairpins presumably form before primer annealing, Vent polymerase can open them by strand displacement (Fig. 2D).
Cloning. The PCR product of YIU was directly digested with MlyI and AflII in the PCR buffer and separated by PAGE (Fig. 2E). The 70 bp product band was excised from gel, purified and directionally cloned into the pKSU6 expression vector.
Effect of modifications of the U6 promoter and hairpin loop on RNAi
Cloning efficiency and quality of the shRNA library
Analysis of the CCND1 shRNA library
To demonstrate the utility of this shRNA library construction method, random clones from the CCND1 shRNA library were tested for effective RNAi by cotransfection with one of two constructs expressing luciferase fused in its 3'UTR to a portion of the CCND1 gene (Fig. 4B and 4C). The shRNA constructs were also compared to three constructs targeting the luciferase portion of the fusion transcript. The two CCND1 fragments together comprise almost the entire CCND1 cDNA with the exception of a small portion of the 3' UTR containing a cluster of AUUUA motifs, which are often associated with mRNA instability . The relatively low level of expression of the "2.5 kb" construct suggests, however, that this fragment may confer instability of the fusion transcript; this may result in less apparent further destabilization by the shRNA expression constructs. A destabilizing region has been mapped within the sequences contained in the "2.5 kb" construct [16, 17]. Notably, the YIU construct targeting luciferase was at least as effective as either the "classic" construct or the construct in which the target is embedded in a microRNA structure ("miR-30"). Several of the enzymatically generated constructs effectively targeted the fusion transcript. The most effective of the tested clones, #2, is predicted to be a potentially effective shRNA by Dharmacon's program .
Construction of shRNA libraries is the first step toward the goal of performing mammalian genome-wide screens with the RNAi technology. The applications of shRNA library technology also include determining the best sequences for inhibition of infection by viruses, such as HIV [19, 20], and identifying the most effective shRNAs for a single gene.
Given the great amount of work required for a functional screen, if the construction of an shRNA library becomes much easier, tissue- or cell-specific shRNA libraries are preferable to generic synthetic shRNA libraries for the following reasons: (a) Not all genes have been identified. This is particularly likely to be the case for genes with a very limited range of tissue expression. Thus, with our current state of knowledge, any library prepared by individual chemical synthesis is necessarily incomplete. (b) Alternative splice forms of some genes may not be affected if only one or a few shRNAs are used. (c) Screening only for genes that are actually expressed in a given tissue reduces the amount of work required. Normalized, tissue-specific cDNA libraries from IMAGE collections may be the most cost-effective source of genome-wide cDNA libraries for generating shRNA libraries. However, each laboratory can pursue its specific interests using appropriate tissue-specific shRNA libraries.
A major step in constructing an shRNA library from cDNA is to normalize the cDNA. A simple cDNA normalization method has recently been reported . The key component in this method is a duplex-specific nuclease (DSN) from the Kamchakta crab. The DSN preferentially cleaves double-stranded DNA and DNA in RNA-DNA hybrid duplexes at 70°C. Using this enzyme, cDNA can be normalized after first-strand cDNA synthesis or after amplifying the cDNA. After heat-denaturation and kinetic reassociation [22, 23], abundant cDNAs reanneal more rapidly than rare cDNAs and are depleted from the mixture by DSN digestion.
The YIU method allows rapid conversion of cDNAs into shRNA templates. This method has several advantages over previous methods [5–7]. Our Y oligonucleotide was designed with a 3' T overhang to prevent self-ligation. To allow ligation with the Y oligonucleotide, a single 3' A overhang is added to cDNA fragments by incubating with Taq DNA polymerase. To create multiple fragments from each transcript, cDNAs derived from cells or tissues can be subjected to either partial DNase I digestion, sonication, or restriction digestion. A PCR step after generating the YIU molecules was introduced to amplify the desired product but not irrelevant byproducts, thereby eliminating the necessity of multiple PAGE purifications as described in previous methods [5–7] and greatly simplifying the whole process. The PCR amplification also increases the overall yield of YIU products and allows the use of small amounts of starting mRNA. This is expected to be particularly useful for small number of cells separated by FACS or microdissection.
The two-temperature PCR program used a high temperature (75°C) for a combined annealing and extension step to destabilize hairpin structures and to promote the strand-displacement activity of Vent polymerase. This approach has been used in amplifying DNA with a hairpin structure , and we showed that this method has additional applications in the siRNA field, such as amplification of shRNA expression cassettes. Conventional 3-temperature PCR cycles promote strand slippage of DNA polymerase when replicating self-annealing structures [24, 25], including the shRNA hairpin , leading to deletions. Adoption of our PCR program to the application may alleviate the problem.
Finally, the YIU method is versatile. If an initial larger loop is secondarily trimmed using a Type IIS restriction enzyme, constructs can be produced expressing an shRNA with a loop of arbitrary sequence [5–7]. In particular, with minor changes in the Y and U oligonucleotides, one potentially could prepare shRNA expression libraries in which the targeting siRNA sequences are engrafted into a microRNA stem and loop structure. The use of a microRNA stem and loop has been shown to dramatically increase the efficiency of suppression by an shRNA  and is compatible with Pol II promoters, which are more diverse and versatile than the Pol III promoters necessary for expression of "classic" shRNAs.
The "Y" oligonucleotide pair: Y-Forward: 5'-AACGACGGCCAGTGAGCGCGCGTAATACGACAACATTCTTGAGTCCAAT-3'. Y-Reverse: 5'-PO4-GTTGGACTTAAGAATGTTGTGCGCTTGGCGTAATCATGGTCATAGCTGTTTC-3'. The "U" oligonucleotide: 5'-PO4-TCGGCTCTTCCTGTCAAGCCGANN-3'.
PCR primers. Forward: 5'-CGACGCCCAGTGAGCGCGCGTAATACG-3'. Reverse: 5'-GGAAACAGCTATGACCATGATTACGCCAAGCG-3'.
Human CCND1, encoding cyclin D1, from the 4.2 kb full-length cDNA clone MGC:23386 [Genbank:BC023620] (American Type Culture Collection), used here as an example, was isolated from vector sequences with EcoRI and XhoI (yielding 2.7 and 1.5 kb fragments, due to an internal EcoRI site) and end-blunted with Klenow fragment. The gel-purified CCND1 fragments (about 1 to 1.5 μg) were digested in 1 × ThermoPol buffer (New England Biolabs, NEB) at 37°C for 1 hour individually with AluI, DpnI and HaeIII, restriction endonucleases with a 4-bp recognition sequence and blunt-end products. Concentrated dNTPs (final 200 μM each) and Taq polymerase (1 U) were added into each tube and incubated at 60°C for 3 hours. After combining the three samples, DNA was extracted with phenol/chloroform, precipitated by ethanol, and resuspended in 10 μl of H2O (about 0.3 μg/μl).
The EGFP gene was PCR-amplified from plasmid pEGFP-C2 (BD Clontech) using Taq DNA polymerase (NEB). After PCR, 1 M Tris-HCl, pH 7.0, and 0.1 M MnCl2 were added to final concentrations of 40 mM and 1 mM, respectively. Partial digestion with DNase I (0.1 U in 50 μl) was performed at room temperature for several time periods (2 to 10 minutes) to determine the optimal digestion time. The partially digested DNA samples were extracted with phenol/chloroform and precipitated with ethanol. DNA end-repair with T4 DNA polymerase (NEB) was conducted following the manufacturer's protocol. The addition of a 3' A overhang was described above.
The "YIU" procedure
(1) "YI" ligation. Three μl of digested DNA (about 0.9 μg, the "I" molecule, shown in red in Fig. 1) was mixed with 1 μl of 10 × NEB buffer 4, 1 μl of 0.1 M DTT, 1 μl of 10 mM rATP, 3 μl of 10 μM "Y" paired oligonucleotide and 1 μl T4 DNA ligase (2,000 NEB units, New England Biolabs) and placed in a thermocycler overnight, cycling between 10°C for 30 sec and 30°C for 30 sec . The T4 DNA ligase was inactivated at 65°C for 20 min.
(2) MmeI digestion. 1.5 μl of 1 mM S-adenosylmethionine (SAM),1 μl MmeI (2 U, NEB), 1 μl of 10 × NEB buffer 4, and 6.5 μl of water were added to the YI ligation mix (final 20 μl in volume), and incubated at 37°C for 1 hour.
(3) YI and "U" ligation. The MmeI-digested DNA was extracted with phenol/chloroform, precipitated with ethanol, and resuspended in 30 μl of water. Four μl of 10 × T4 DNA ligase buffer, 5 μl of "U" oligonucleotide (10 μM), and 1 μl T4 ligase (400 NEB Units) were added, and the mixture incubated in a thermocycler overnight, cycling between 10°C for 30 sec and 30°C for 30 sec.
(4) PCR amplification. To determine the optimal number of PCR cycles, four tubes were prepared, each containing 1 μl of the YIU ligation product, 1 unit of Vent DNA polymerase (NEB) and 1 × buffer, 200 μM dNTPs and 0.5 μM of primers (final conc.) in 50 μl of volume. PCR was carried out with the following cycling parameters: 95°C for 2 min, 95°C for 30 sec, 75°C for 1 min, for 15, 17, 19, or 21 cycles. After PCR amplification, the products were separated by 1.5% agarose gel electrophoresis. The band patterns from the four samples were compared, and the cycle number yielding the greatest quantity of the 160 bp product was chosen for amplifying a larger quantity for cloning. After the PCR, an equal volume of fresh PCR reaction mix (buffer, dNTPs, primers and polymerase) was added, and one additional cycle was performed.
(5) Cloning. Restriction endonucleases AflII and MlyI were added directly to the PCR product and incubated at 37°C for 2 hours. The 70 bp insert was separated on a 12% polyacrylamide-TBE gel, purified, and cloned into the expression vector pKSU6.
The shRNA expression vector pKSU6
The U6 promoter was PCR amplified from vector pAVU6+27  with primers 5'-GGAAGATCTGAGGAGGGCCTATTTCCCATG-3' (forward) and 5'-CCGGAATTCCTTAAGTTCCACAAGATATATAACTCTATC-3' (reverse). The PCR product was digested with BglII and EcoRI and inserted into the BamHI-EcoRI sites in pBluescript II KS+ (Stratagene). An XcmI site was introduced downstream of the AflII site. The Pol III transcription termination signal (T5) was embedded in the XcmI site (CCAAAAATTTTTTGG). The resulting vector, pKSU6, was cut with XcmI, blunt-ended with T4 DNA polymerase, and subsequently cut with AflII for cloning AflII-MlyI digested YIU products. The ligated products were transformed into E. coli XL-10 competent cells (Stratagene).
Control constructs targeting luciferase
The "YIU" construct targeting luciferase was generated by preparing the KSU6 vector as described above and cloning into it a double-stranded oligonucleotide prepared by annealing the oligonucleotide 5'-ttaaGTCCAACTGTGCGCTGCTGGTGCCAACTCGGCTTGACAGGAAGAGCCGAGTTGGCACCAGCAGCGCAC-3' and its complement (without ttaa). The U6 promoter of pBtU6+27  was amplified using the following primers: 5'-CCGCGGTACCCCGGGAGATCCAAGGTCGGGCAG-3' (forward) and 5'-CGCGTCTAGACCCATCGATGAGGATCCCTTTCCACAAGATATATAAAGCC-3' (reverse). The PCR product was digested with KpnI and XbaI and cloned into the corresponding sites of pBtU6+27. The resulting construct, pU6-ClaI, is similar to pKSU6, but contains BamHI and ClaI cloning sites at the 3' end of the U6 promoter. The oligonucleotide 5'-cgTGCGCTGCTGGTGCCAACTTCAAGAGAGTTGGCACCAGCAGCGCACTTTTT-3' and its complement (with 5' ctag, and lacking 3' cg) were annealed and cloned into the ClaI and XbaI sites to yield the "classic" construct targeting luciferase. The "miR-30" construct was prepared by PCR amplification using primers 5'-CAGAGGCTCGAGAAGGTATATTGCTGTTGACAGTGAGCG-3' and 5'-CTAAAGTAGCCCCTTGAATTCCGAGGCAGTAGGCA-3' and template 5'-TGCTGTTGACAGTGAGCGAGGTTGGCACCAGCAGCGCACTTAGTGAAGCCACAGATGTAAGTGCGTTGTTGGTGTCAATCCTGCCTACTGCCTCGGA-3', essentially as described . The product was digested with XhoI and EcoRI and cloned into the corresponding sites of the pSM2 vector (Open Biosystems).
Cyclin D1 (CCND1) reporter vector preparation
pGL3 Promoter construct (Invitrogen) was modified by inserting into the XbaI site a double-stranded oligonucleotide prepared by annealing the following two oligonucleotides: 5'-ctagGAATTCGATATCCCGCGGCATATGT-3' (forward) and 5'-ctagACATATGCCGCGGGATATCGAATTC-3'. A construct with the insert in the forward orientation was denoted pGL3P(2MCS), having a second multiple cloning site within the 3'UTR of luciferase. Fragments of CCND1 were inserted into this second multiple cloning site. Construct luc-CCND1(2.5 kb) was prepared in several steps, including deletion of a BstXI fragment, removing approximately 1.6 kb of 3' sequence; it includes nt 1–2173 [Genbank:BC023620] of CCND1 and additional 3' sequences. Construct luc-CCND1(1.4 kb) was prepared by cloning an EcoRI-XbaI fragment of CCND1 (nt 2750–4197) into the corresponding sites of pGL3P(2MCS). These two reporter constructs were chosen to avoid a cluster of AUUUA sequences, which in some mRNAs are associated with mRNA instability .
Cell culture and transfection
Human HEK293T or mouse NIH3T3 cells were used for RNAi assays. The cells were seeded 24 hours before transfection in DMEM plus 10% FCS in 96-well plates at 50–70% confluence. Transfection was conducted with Lipofectamine 2000 (Invitrogen) according to the manufacturer's recommendations. The amounts of DNA used per well were as follows: the reporter plasmid, pEGFP, 50 ng; Red Fluorescent Protein vector (pDsRed2, Clontech), 50 ng; and shRNA expression vector, 100 ng (~3 × molar ratio).
Northern blot hybridization
Hybridization was performed as previously described , with minor modifications. HEK293T cells were transiently transfected with either the "classic" or "YIU" shRNA construct targeting luciferase; total RNA was extracted with Trizol reagent. 40 μg of total RNA was electrophoresed on a 15% polyacrylamide gene in TBE buffer (89 mM Tris-borate, 2 mM EDTA) with 8 M urea. RNA was transferred to GeneScreen Plus membrane (PerkinElmer) with a semidry electrophoretic transfer apparatus and UV-crosslinking with 1000 μJ in a Stratagene UV Crosslinker. StarFire™ template probes (Integrated DNA Technologies, IDT) contained the following sequences: targeting luciferase, 5'-GTGCGCTGCTGGTGCCAAC-3', and 5S RNA, 5'-GACGAGATCGGGCGCGTTC-3'. Probes were labeled with [α-32P]-dATP using the Nucleic Acids Labeling System kit (IDT), following the manufacturer's protocol. The membrane was prehybridized in 0.5 M sodium phosphate buffer, pH 7.0, 1 mM EDTA, 7% SDS, 0.5% (w/v) sodium pyrophosphate at 37°C. Approximately 1.7 × 106 dpm of the luciferase probe and 4 × 105 dpm of the 5S RNA probe were added to 2 ml of hybridization buffer. After hybridization at 37°C for ~15 h, the blot was washed 3 × with 2 × SSPE (2 × buffer, 0.3 M NaCl, 20 mM sodium phosphate, pH 7.4, 2 mM EDTA), 0.1% SDS 30 min each at 37°C and then exposed overnight to X-ray film.
HEK293 cells in 24-well plates were cotransfected with 300 ng Luc-CCND1 reporter plasmid, 900 ng shRNA expression plasmid, and 20 ng pRL-TK (Renilla luciferase control). Samples were extracted 48 h after transfection and analyzed using the Dual-Luciferase Reporter Assay System (Promega), according to the manufacturer's protocol.
We wish to thank Dr. David R. Engelke for providing plasmids, Xiaoyan Feng for technical assistance, Dr. Yulei Shen and Radha M. Golla for preparation of constructs, and members of the Chan and McKeithan laboratories for helpful comments. We also thank Linda M. Wilkie of the University of Nebraska Medical Center Cell Analysis Facility for flow cytometry. This work was partially supported by a Lymphoma Research Foundation grant to TWM and NCI grant U01 CA84967 to WCC.
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