CRISPR: Evolution, Mechanisms and Infection
Programmable DNA cleavage in vitro by Cas9 Tautvydas Karvelis*1 , Giedrius Gasiunas*1 and Virginijus Siksnys*2 *Institute of Biotechnology, Vilnius University, Graiciuno 8, LT-02241, Vilnius, Lithuania
Biochemical Society Transactions
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Abstract The ternary Cas9–crRNA–tracrRNA complex (Cas9t) of the Type II CRISPR (clustered regularly interspaced short palindromic repeats)–Cas (CRISPR-associated) system functions as an Mg2 + -dependent RNA-directed DNA endonuclease that locates its DNA target guided by the crRNA (CRISPR RNA) in the tracrRNA–crRNA structure and introduces a double-strand break at a specific site in DNA. The simple modular organization of Cas9t, where specificity for the DNA target is encoded by a small crRNA and the cleavage reaction is executed by the Cas9 endonuclease, provides a versatile platform for the engineering of universal RNA-directed DNA endonucleases. By altering the crRNA sequence within the Cas9t complex, programmable endonucleases can be designed for both in vitro and in vivo applications. Cas9t has been recently employed as a geneediting tool in various eukaryotic cell types. Using Streptococcus thermophilus Cas9t as a model system, we demonstrate the feasibility of Cas9t as a programmable molecular tool for in vitro DNA manipulations.
Introduction CRISPR (clustered regularly interspaced short palindromic repeats) is a microbial adaptive immune system which provides acquired resistance against viruses and plasmids [1]. The CRISPR array consists of short conserved repeat sequences interspaced by unique spacer sequences, which often originate from phage or plasmid DNA. The cas (CRISPR-associated) genes are located adjacent to the CRISPR array. CRISPR–Cas systems hijack fragments of the invading nucleic acid to incorporate them as spacers into a host genome and later use them as templates to generate small RNA molecules [crRNA (CRISPR RNA)] which together with Cas proteins are assembled into an effector complex which silences foreign nucleic acids in the subsequent rounds of infection [2–7]. CRISPR–Cas systems have been categorized into three main types, on the basis of core element content and sequences [8]. The composition and stoichiometry of effector complexes differ strikingly between different CRISPR subtypes [9,10]. In the Type I and Type III systems, effector complexes are arranged as large multisubunit ribonucleoprotein complexes [11–16]. In Type II CRISPR–Cas systems, the effector complex required for DNA interference comprises a sole Cas9 protein bound to a dual RNA molecule [17–20]. Cas9 is a large multidomain protein which contains two nuclease domains, an RuvC-like nuclease domain near the N-terminus and a HNH (His-AsnHis)-like nuclease domain in the middle of the protein [17– 19,21]. Cas9 forms a ternary complex (Cas9t) with two RNA
Key words: cloning, clustered regularly interspaced short palindromic repeats (CRISPR), CRISPR RNA (crRNA), RNA-guided endonuclease, site-specific cleavage, targeted genome modification. Abbreviations used: CRISPR, clustered regularly interspaced short palindromic repeats; crRNA, CRISPR RNA; PAM, protospacer-adjacent motif; qPCR, quantitative real-time PCR; St-Cas9t, Cas9– crRNA–tracrRNA ternary complex of the Streptococcus thermophilus; tracrRNA, transactivating crRNA. 1 2
These authors contributed equally to this work. To whom correspondence should be addressed (email [email protected]).
Biochem. Soc. Trans. (2013) 41, 1401–1406; doi:10.1042/BST20130164
molecules: crRNA and tracrRNA (transactivating crRNA) [18–20]. The 42 nt mature crRNA consists of the 22-nt 3 -handle originating from the repeat sequence and 20-nt unique spacer fragment, which guides Cas9t to the matching DNA target [22]. tracrRNA is partially complementary to crRNA and is essential for the crRNA maturation [20,22] and DNA cleavage by Cas9t [18–20]. Cas9t binding to the target site (a protospacer) complementary to the crRNA occurs only if a short nucleotide sequence, called a PAM (protospacer-adjacent motif), is located in the vicinity of a protospacer [18,19]. If both the protospacer and PAM are present, Cas9t binds to the target site generating the Rloop, where one DNA strand is displaced while another is engaged into a heteroduplex with crRNA. DNA cleavage is mediated by a pair of Cas9 active sites that act on opposite DNA strands: RuvC cuts the displaced DNA strand, whereas HNH cuts DNA strand paired with RNA [18,19]. Doublestrand breaks introduced by the Cas9 protein of Streptococcus thermophilus occurs within a protospacer, 3 nt away from the PAM sequence, to produce blunt-end DNA products [18,19,23,24]. Thus the Cas9t complex functions as an Mg2 + dependent RNA-directed DNA endonuclease that requires five obligatory components for a site-specific DNA cleavage: the Cas9 protein, crRNA, tracrRNA, a protospacer sequence complementary to the crRNA and a PAM. The simple modular organization of the Cas9t complex, where specificity for DNA targets is encoded by a small crRNA and the cleavage machinery consists of a single multidomain Cas protein, provides a versatile platform for the engineering of universal RNA-guided DNA endonucleases. Indeed, by altering the RNA sequence within the Cas9t complex, programmable endonucleases can be designed for both in vitro and in vivo applications. Cas9t has been recently employed as a gene-editing tool in various eukaryotic cells, including human, mouse, zebrafish, yeast and Drosophila [25–36]. In the present article, we show that C The
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Cas9t provides a versatile programmable molecular tool for in vitro applications.
In vitro assembly of a programmable Cas9t complex We have shown previously that the Cas9–crRNA–tracrRNA ternary complex of the S. thermophilus DGCC7710 CRISPR3–Cas system (St-Cas9t) (Figure 1A) isolated from the recombinant host cleaves both a synthetic oligodeoxynucleotide duplex and a plasmid DNA bearing a nucleotide sequence complementary to the crRNA, in a PAM-dependent manner [18,20]. However, the low yield of the St-Cas9t complex isolated from the recombinant Escherichia coli host and traces of nucleases contaminating the St-Cas9t preparation hindered further applications. We therefore have developed a method to obtain in vitro an active St-Cas9t from individual components [20] (see the Supplementary Online Data at http://www. biochemsoctrans.org/bst/041/bst0411401add.htm). Two different experimental procedures have been used for the StCas9t complex assembly: (i) mixing of the Cas9 apoprotein with ‘mature’ forms of tracrRNA (78 nt) and crRNA (42 nt), and (ii) mixing of the Cas9 apoprotein with primary transcripts of tracrRNA (105 nt) and pre-crRNA (150 nt) followed by a subsequent incubation with RNase III [20]. RNase III together with tracrRNA are required for the long pre-crRNA processing in the Type II systems, including Streptococcus pyogenes and S. thermophilus [20,22]; however, RNase III turned out to be dispensable if pre-crRNA is transcribed from a minimal R-SP-R (where R is a repeat unit and SP is a spacer) CRISPR region [20]. In the latter case, the unprocessed crRNA carrying flanking repeat sequences together with tracrRNA and Cas9 protein assemble into a functionally active Cas9t complex. Subsequent RNase III treatment results in a ∼2-fold increase in Cas9t DNA cleavage activity. Therefore, in our study of Cas9t complex assembly, we first incubated the 105 nt tracrRNA transcript (101nt-long putative tracrRNA transcript + 4 nt for in vitro transcription using T7 RNA polymerase) with 150-nt-long pre-crRNA (20 nt leader, R-SP-R unit and downstream flanking sequence) in the presence of St-Cas9 protein to promote tracrRNA–pre-crRNA duplex formation and then added RNase III for the crRNA end-processing to generate a functional complex (Figure 1B). The finding that an active Cas9t can be assembled using crRNA and tracrRNA obtained by in vitro transcription prompted us to engineer a recombinant plasmid, pcas-2R, by inserting a cassette encoding a spacer sequence flanked by two repeat units between SapI and Eco31I restriction sites (Figure 1C). This plasmid allows an easy reprogramming of the Cas9t complex by ligating a synthetic oligoduplex carrying a new spacer sequence into the SapI/Eco31I precleaved plasmid followed by in vitro transcription of the PCR product carrying the R-SP-R unit to generate a desired precrRNA. C The
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DNA cleavage by the in vitro assembled St-Cas9t complex First, the DNA cleavage activity of the in vitro assembled St-Cas9t complex was assayed on oligodeoxynucleotide and plasmid substrates containing protospacer and PAM sequences. Cas9t loaded by the dual tracrRNA–crRNA matching the protospacer SP1 in the 55 bp oligoduplex cleaved both strands at a fixed position, 3 nt upstream of the PAM (5 -NGGNG-3 ) sequence, leaving blunt ends (Figure 1D), identically with Cas9t complex purified from E. coli [18]. As expected, an oligoduplex lacking a protospacer SP1 was resistant to the Cas9t cleavage. Furthermore, upon Cas9t treatment the pSP1 plasmid containing a protospacer SP1 and PAM sequence was converted into a linear form (Figure 1E), indicating that both DNA strands were cleaved specifically within the protospacer. Re-ligation of the linear pSP1 form by DNA ligase followed by a transformation of E. coli cells yielded multiple ampicillin-resistant clones. Sequencing of DNA purified from selected clones revealed no changes in the protospacer SP1 sequence. In a control experiment, E. coli transformation by the ligation mixture in the absence of T4 DNA ligase produced no transformants, indicating that a supercoiled plasmid does not co-purify with a linear reaction product. Taken together, these results demonstrate that (i) no mutations or indels are introduced upon Cas9t cleavage of plasmid DNA, and (ii) DNA ends generated by Cas9t cleavage are substrates for T4 DNA ligase, and therefore must contain a phosphate at the 5 terminus and a free hydroxy group at the 3 terminus [37]. In addition, using the procedure described above for the Cas9 CRISPR3–Cas system, we were able to assemble in vitro an active ternary Cas9 complex of the S. thermophilus CRISPR1–Cas [1,23,38] system (results not shown). Unlike the Cas9 from CRISPR3–Cas which depends on the 5 -NGGNG-3 PAM sequence, Cas9t of CRISPR1–Cas requires a 5 -AGAAW-3 PAM located in the vicinity of the target complimentary to the crRNA [38,39]. The availability of functional Cas9t variants with different PAM requirements expands the range of targets accessible for the Cas9t cleavage.
St-Cas9t complex as a tool for DNA manipulation Restriction endonucleases recognize short nucleotide sequences usually 4–8 bp in length and, in the presence of Mg2 + , ions cut both DNA strands within or close to the target site. Owing to their unique specificity, restriction enzymes became indispensable tools for DNA cloning [40]. However, the strict specificity of restriction enzymes limits flexibility when inserting gene fragments into a plasmid vector. To demonstrate that Cas9t could be used as a universal RNA-guided DNA endonuclease programmed by the crRNA to target any DNA site, we adapted it for a cloning procedure. We selected two target sites (N1 and N2) located near the SapI and AatII restriction endonuclease sites in the pUC18 plasmid and, using the plasmid bearing the
CRISPR: Evolution, Mechanisms and Infection
Figure 1 In vitro assembly and functional analysis of St-Cas9t complex (A) Schematic representation of the CRISPR3–Cas system of S. thermophilus DGCC7710. Four cas genes (cas9, cas1, cas2 and csn2) are located upstream of the CRISPR repeat–spacer array, consisting of 13 repeat (R) sequences and 12 unique spacers (S1–S12). The tracrRNA is located upstream of the cas9 gene and encoded on the opposite DNA strand (shown by an arrow) with respect to the other elements of this system. (B) In vitro assembly of the Cas9t complex. The Cas9 protein is first pre-incubated with 150 nt pre-crRNA and 105 nt tracrRNA transcripts, followed by subsequent treatment with RNase III to generate a catalytically competent Cas9t complex. (C) The cassette for a new spacer insertion into the CRISPR region and crRNA synthesis. A synthetic oligoduplex encoding a desired spacer sequence and containing SapI- and Eco31I-compatible ends is inserted between two repeats. The PCR fragment of the CRISPR region is used as a template for crRNA synthesis by in vitro transcription. T7 RNA pol, T7 RNA polymerase. (D) DNA oligoduplex cleavage by Cas9t complex. The oligoduplex strand which is complementary to crRNA is marked as ( + ) strand, whereas the other strand is marked as ( − ) strand. M1 and M2 are synthetic oligonucleotide markers corresponding to the 37 nt of the ( − ) strand and 18 nt of the ( + ) strand used to map the cleavage position. C, non-specific oligoduplex lacking a protospacer SP1. Reaction time varied between 0 and 30 min. Sizes are indicated in nt. (E) Plasmid DNA cleavage. Cas9t complex was incubated for 5 min with pSP1 or pUC18 plasmids and reaction products analysed in the agarose gel. pSP1 plasmid contained a protospacer SP1 sequence flanked by the 5 -NGGNG-3 PAM sequence. Protospacer SP1 sequence was not present in pUC18. FLL, full-length linear; OC, open circle; SC, supercoiled.
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Figure 2 St-Cas9t complex as a tool for DNA manipulation (A) Schematic representation of the target sites in the pUC18 plasmid. The distance between SapI and AatII restriction enzymes sites is 775 bp, whereas the distance between two protospacer sites Cas9t-N1 and Cas9t-N2 is 612 bp. (B) pUC18 plasmid cleavage by reprogrammed Cas9t complexes. Cleavage products resulting from the double digestion with SapI and AatII restriction enzymes are also shown. Sizes are indicated in kbp. (C) Run-off sequencing data of pUC18 pre-cleaved with Cas9t-N1 (upper panel) and Cas9t-N2 (lower panel) complexes. Cleavage of plasmid DNA at both sites occurred 3 nt upstream of the PAM. (D) Schematic representation of the cloning experiment. Plasmid vector pre-cleaved with Cas9t Cas9t-N1 and Cas9t-N2 complexes was ligated with a PCR fragment encoding a promoter and a tetracycline resistance gene. Clones were selected on media containing tetracycline (Tc) and ampicillin (Ap).
interchangeable spacer cassette (described above), generated crRNAs matching protospacers N1 and N2. The theoretical length of the fragment located between Cas9t-N1/Cas9tN2 sites is 612 bp (Figure 2A). Double digestion of pUC18 plasmid with Cas9t-N1 and Cas9t-N2 (Figure 2B) yielded two DNA fragments (∼2000 and ∼600 bp respectively), indicating that the plasmid was cut at sites targeted by crRNAs incorporated in Cas9t-N1/Cas9t-N2 complexes. Sequencing data confirmed that plasmid DNA cleavage occurred at both sites 3 nt upstream of the PAM sequence generating a blunt-ended plasmid vector (Figure 2C). To C The
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demonstrate that the plasmid vector pre-cleaved with Cas9t is suitable for further manipulation, we ligated a PCR fragment amplified from the region containing a promoter and a tetracycline-resistance gene in the pACYC184 plasmid (Figure 2D), and used a resulting ligation mixture to transform E. coli cells. The resulting clones were selected on medium containing tetracycline and ampicillin. Sequencing of DNA isolated from the selected clones confirmed that the PCR fragment was inserted at the desired position and no mutations or indels were introduced upon Cas9t cleavage. Taken together, these experiments demonstrate that
CRISPR: Evolution, Mechanisms and Infection
Figure 3 DNA cleavage in vitro by the Cas9t complex (A) λ DNA cleavage. Phage λ DNA was incubated with the Cas9t complex for various times and reaction products were analysed by agarose gel electrophoresis. The Cas9t is programmed for a target located 8 kb away from the cos site. Sizes of markers (M) are indicated in kbp. (B) E. coli genomic DNA cleavage. DNA extracted from the E. coli BL21 strain was fragmented by PstI enzyme. The Cas9t is programmed for a target located between two PstI sites. If genomic DNA is cleaved by the Cas9t, a 466 bp fragment should be detected by probe hybridization; in the absence of cleavage, the hybridization probe should detect the 1499 bp fragment. C, E. coli genomic DNA fragmented with PstI. Genomic DNA was incubated for 30 min with Cas9t complex before fragmentation with PstI. Sizes of markers (M) are indicated in kbp. (C) Human genomic DNA cleavage by Cas9. Following Cas9t–HS1 and Cas9t–HS2 cleavage, the relative amounts of uncleaved DNA targets (HS1 or HS2) were estimated by qPCR. The ratio of cleaved to uncleaved DNA target quantified by qPCR is at least 25:1.
(i) Cas9t can be used as a programmable DNA endonuclease suitable for cloning experiments, and (ii) multiple cleavage can be performed simultaneously by different Cas9t complexes.
In vitro cleavage of genomic DNA by the Cas9t complex The predicted length of the DNA target recognized by the St-Cas9 is determined by the 20 nt sequence complementary to the crRNA plus 3 nt of the PAM sequence. Plasmid interference experiments in vivo indicate that 6 nt at the PAM distal end of a protospacer are not important for interference, suggesting that only 14 nt matching the crRNA and the PAM sequence are absolutely required for DNA interference by the St-Cas9t [19]. In theory, the 17 nt recognition sequence allows to design St-Cas9t complexes that target unique sequences in genomic DNA. To find out whether Cas9t is able to locate and cleave target sites in vitro in the context of genomic DNA, we programmed the Cas9t complex for unique sites in bacteriophage λ (49 kbp), E. coli (4.6 Mbp) and human (3.2 Gbp) DNA. The analysis of phage λ genomic DNA cleavage products by agarose gel electrophoresis confirmed that the 49 kbp DNA is cleaved only once, yielding 42 kbp and 7 kbp products as predicted (Figure 3A). To identify cleavage products of the E. coli genomic DNA, we used Southern blot analysis (see the Supplementary Online Data). More specifically, we first treated genomic DNA with an excess of Cas9t, followed by PstI restriction enzyme digestion, and then analysed reaction products by Southern blotting using a probe designed against the DNA fragment located between the Cas9t cleavage site and a downstream PstI target
(Figure 3B). The distance between two PstI recognition sites is ∼1500 bp, whereas the distance between a protospacer and the downstream PstI site is ∼500 bp. After Cas9t cleavage, we detected by probe hybridization only a ∼500 bp DNA fragment, indicating that E. coli genomic DNA was cleaved by Cas9t at the desired position. In the next set of experiments, we analysed human DNA cleavage by the Cas9t–HS1 and Cas9t–HS2 complexes targeted to RASGEF1C (HS1) and ARL15 (HS2) loci respectively. The cleavage reaction was monitored by qPCR (quantitative real-time PCR) (Figure 3C, and see the Supplementary Online Data). After treatment with Cas9t– HS1 and Cas9t–HS2, the amount of intact DNA targets in both loci decreased more than 25-fold, indicating that human genomic DNA was cleaved at the desired loci. Taken together, these data demonstrate that St-Cas9t effectively finds and cleaves its targets in the context of genomic DNA of different complexity, including viral, bacterial and mammalian DNA.
Acknowledgements We thank Dr Arturas Petronis from the Centre for Addiction and Mental Health, Toronto, Canada, for providing human DNA samples, advice on target site selection and discussion.
Funding This work on CRISPR systems in V.S.’s laboratory is funded by the European Social Fund under Global Grant Measure.
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CRISPR: Evolution, Mechanisms and Infection
SUPPLEMENTARY ONLINE DATA
Programmable DNA cleavage in vitro by Cas9 Tautvydas Karvelis*1 , Giedrius Gasiunas*1 and Virginijus Siksnys*2 *Institute of Biotechnology, Vilnius University, Graiciuno 8, LT-02241, Vilnius, Lithuania
DNA manipulations Construction of Cas9 expression plasmid and plasmid pSP1 containing a protospacer was described previously [1,2]. To obtain a cassette for a spacer insertion, the DNA fragment was amplified from pCas9 plasmid [2] using GG116 and GG-567 primers (Table S1) designed to introduce a SapI site after the first repeat. The PCR fragment was inserted into the pCas9 plasmid vector pre-cleaved with Eco31I and BamHI. The resulting pCas9-1R plasmid contained the SapI site after the first repeat, and a partially truncated CRISPR region. Next, the cas9 gene was deleted by cutting the pCas9-1R plasmid with Eco105I and StuI to generate the pcas-1R plasmid. The resulting pcas-1R plasmid was then cleaved with SapI and BamHI and ligated with oligoduplex assembled from GG-580 and GG-581 oligonucleotides (Table S1) (designed to introduce the second repeat and Eco31I site), resulting in the pcas-2R plasmid. The new spacer cassettes were assembled from synthetic oligoduplexes (Table S2) containing the sticky ends for SapI and Eco31I restriction endonucleases. Oligoduplexes were cloned into pcas-2R plasmid through SapI and Eco31I sites to yield plasmids containing CRISPR regions with engineered spacers (see Figure 1C in the main text). Sequencing of CRISPR regions of obtained plasmids revealed that required spacers have been inserted between two repeats.
Expression and purification of Cas9 protein Purification of His6 -tagged variant of St-Cas9 protein was performed as described in [1]. Briefly, Cas9 was expressed in E. coli DH10B strain. Harvested cells were disrupted by sonication, and supernatant was loaded on to a Ni2 + -charged chelating column, followed by a heparin column. Fractions containing the purified protein were pooled, transferred to storage buffer and stored at − 20 ◦ C.
RNA production RNA fragments required for Cas9t complex assembly were either produced by in vitro transcription using TranscriptAid T7 High Yield Transcription Kit (Thermo Fisher Scientific) or 1 2
These authors contributed equally to this work. To whom correspondence should be addressed (email [email protected]).
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purchased as synthetic oligoribonucleotides (Metabion). For in vitro transcription, a fragment containing a T7 promoter at the proximal end of the RNA coding sequence was PCRgenerated using primers and templates provided in Table S3. Transcribed RNA fragments were purified using RNeasy® MinElute® Cleanup Kit (Qiagen). Sequences of synthetic oligoribonucleotides are provided in Table S4.
Assembly of the Cas9t complex For Cas9t complex assembly using 42 nt synthetic crRNA and 78 nt tracrRNA, His6 -tagged Cas9 protein was mixed with a pre-annealed crRNA and tracrRNA duplex at 1:1 molar ratio and incubated in a buffer containing 10 mM Tris/HCl (pH 7.5 at 37 ◦ C), 100 mM NaCl and 1 mM DTT at 37 ◦ C for 1 h. For in vitro assembly of a catalytically competent Cas9t complex (0.1–2 μM) the His6 -tagged Cas9 (0.1–2 μM) protein was mixed with 150 nt pre-crRNA (0.1–2 μM) and 105 nt tracrRNA (0.2–4 μM) transcripts at 1:1:2 molar ratio and pre-incubated in a buffer containing 10 mM Tris/HCl (pH 7.5 at 37 ◦ C) and 100 mM NaCl at 37 ◦ C for 30 min followed by addition of RNAse III (0.1–2 μM; Ambion) and subsequent incubation for additional 30 min at 37 ◦ C in 10 mM Tris/HCl (pH 7.5 at 37 ◦ C), 100 mM NaCl, 10 mM MgCl2 and 1 mM DTT.
Reactions with oligonucleotide substrates The 5 -ends of oligonucleotides (Table S5) were radiolabelled using T4 PNK (polynucleotide kinase) (Thermo Fisher Scientific) and [γ -33 P]ATP (Hartmann Analytic). Duplexes were made by annealing two oligonucleotides with complementary sequences at 95 ◦ C following slow cooling to room temperature. A radioactive label was introduced at the 5 end of individual DNA strands before the annealing with unlabelled strands. Reactions were typically carried out by mixing labelled oligoduplex with Cas9t complex (1:1 v/v ratio) and incubating at 37 ◦ C. The final reaction mixture contained 1 nM labelled duplex, 50 nM Cas9t complex, 10 mM Tris/HCl (pH 7.5 at 37 ◦ C), 100 mM NaCl, 1 mM DTT and 10 mM MgCl2 in a 100 μl reaction volume. Aliquots of 6 μl were removed from the reaction mixture at timed intervals, quenched with 10 μl of loading dye [95 % (v/v) formamide, 0.01 % Bromophenol Blue and C The
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Table S1 Plasmid construction for new spacer insertion Restriction endonuclease sites are in bold and underlined.
Table S2 Oligonucleotides for new spacer insertion Spacer sequences are in upper case. Name
Sequence
Description
GG-564 GG-565
5΄-aacTGTCATGATAATAATGGTTTCTTAGACGTC-3΄ 3΄-ACAGTACTATTATTACCAAAGAATCTGCAGcaaa-5΄
Construction of pΔcas-2R plasmid with N1 spacer
GG-566 GG-567
5΄-aacACGAGCCGGAAGCATAAAGTGTAAAGCCTG-3΄ 3΄-TGCTCGGCCTTCGTATTTCACATTTCGGACcaaa-5΄
Construction of pΔcas-2R plasmid with N2 spacer
TK-43 TK-44
5΄-aacTCAAGGGAGAATAGAGGCTCTCGTTGCATT-3΄ 3΄-AGTTCCCTCTTATCTCCGAGAGCAACGTAAcaaa-5΄
Construction of pΔcas-2R plasmid with spacer targeting E. coli genomic DNA
TK-47 TK-48
5΄-aacCGGGAGGGAAGCTGCATGATGCGATGTTAT-3΄ 3΄-GCCCTCCCTTCGACGTACTACGCTACAATAcaaa-5΄
Construction of pΔcas-2R plasmid with spacer targeting λ genomic DNA
Table S3 Oligonucleotides for generation of in vitro transcription (IVT) templates T7 promoters are in bold and underlined. Name
Sequence
Description
TK-4
5 -aaaaacaccgaatcggtgccac-3
Generation of PCR template from pCRISPR3
TK-5
5 -taatacgactcactatagggtaataataattgtggtttgaaaccattc-3
TK-15
5 -taatacgactcactatagggtagaaaagatatcctacgagg-3
TK-2
5 -caacaaccaagctaatacagcag-3
TK-27
5 -cacgatgcgtccggcgtagaggatcc-3
plasmid for IVT of 105 nt tracrRNA Generation of PCR template from pCRISPR3 plasmid for IVT of pre-crRNA Generation of PCR template with TK-15 from pcas-2R plasmid with inserted spacer for IVT of pre-crRNA
TK-4
5 -aaaaacaccgaatcggtgccac-3
TK-52
5 -taatacgactcactatagggcgaaacaacacagcgagttaaaataagg-3
Generation of PCR template from pCRISPR3 plasmid for IVT of 78 nt tracrRNA
Table S4 crRNAs for human genomic DNA targeting and oligonucleotides used for qPCR Name
Sequence
Description
pr-hs1f pr-hs1r pr-hs2f
5 -tgctgctcgatgcacaggt-3 5 -catcttcaccttcctgctgag-3 5 -ccaaattataagacagatgcctag-3
Primers for qPCR to amplify RASGEF1C (HS1) locus
pr-hs2r GG-625 GG-627
5 -gccaacttctgtgaaactacact-3 5 -gcucccggggcucgaugaagguuuuagagcuguguuguuucg-3 5 -ugaaucgugaaaucugcucaguuuuagagcuguguuguuucg-3
25 mM EDTA (pH 9.0)] and subjected to denaturing gel electrophoresis (20 % polyacrylamide containing 8.5 M urea). Gels were dried and visualized by phosphorimaging (FLA5100; Fujifilm).
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Primers for qPCR to amplify ARL15 (HS2) locus 42 nt crRNA targeting RASGEF1C (HS1) 42 nt crRNA targeting ARL15 (HS2)
Reactions with plasmid substrates Reactions were initiated by mixing a supercoiled plasmid DNA with Cas9t complex (1:1 v/v ratio) and incubation at 37 ◦ C. The final reaction mixture contained 3 nM (500 ng)
CRISPR: Evolution, Mechanisms and Infection
Table S5 Oligonucleotide substrates The spacer is in upper case and the PAM is in bold and underlined. Name
Sequence
Description
GG-316 GG-317
5΄-gctcgaattgAAATTCTAAACGCTAAAGAGGAAGAGGACAtGGtGaattcgtaat-3΄ 3΄-cgagcttaacTTTAAGATTTGCGATTTCTCCTTCTCCTGTaCCaCttaagcatta-5΄
SP1 duplex
GG-494 GG-495
5΄-gctcgaattgTACTGCTGTATTAGCTTGGTTGTTGGTTTGtGGtGaattcgtaat-3΄ 3΄-cgagcttaacATGACGACATAATCGAACCAACAACCAAACaCCaCttaagcatta-5΄
Control duplex
GG-371
5΄-attacgaattcaccatgt-3΄
18 nt marker for (+) strand cleavage
GG-516
5΄-gctcgaattgaaattctaaacgctaaagaggaagagg-3΄
37 nt marker for (-) strand cleavage
plasmid, 50 nM Cas9t complex, 10 mM Tris/HCl (pH 7.5 at 37 ◦ C), 100 mM NaCl, 1 mM DTT and 10 mM MgCl2 in a 100 μl reaction volume. Aliquots were removed at timed intervals and quenched with phenol/chloroform. The aqueous phase was mixed with 3× loading dye solution (0.01 % Bromophenol Blue and 75 mM EDTA in 50 % v/v glycerol) and reaction products were analysed by agarose gel electrophoresis and ethidium bromide staining.
λ DNA cleavage
The reactions were initiated by mixing λ DNA (Thermo Fisher Scientific) with Cas9t complex (1:1 v/v ratio) and incubation at 37 ◦ C. The final reaction mixture contained 2 μg of λ DNA, 50 nM Cas9t complex, 10 mM Tris/HCl (pH 7.5 at 37 ◦ C), 100 mM NaCl, 1 mM DTT and 10 mM MgCl2 in a 100 μl reaction volume. Aliquots were removed at timed intervals and quenched with phenol/chloroform. The aqueous phase was mixed with 3× loading dye solution and reaction products were analysed by agarose gel electrophoresis and ethidium bromide staining.
E. coli genomic DNA cleavage and Southern hybridization Genomic DNA from E. coli (BL21) strain was isolated using the Genomic DNA purification kit (Thermo Fisher Scientific). For the cleavage assay, genomic DNA was combined with Cas9t complex (1:1 v/v ratio) and incubated for 3 h at 37 ◦ C. The final reaction mixture contained 30 μg of genomic DNA, 1 μM Cas9, 10 mM Tris/HCl (pH 7.5 at 37 ◦ C), 100 mM NaCl, 1 mM DTT and 10 mM MgCl2 in a 300 μl reaction volume. Following incubation, PstI (Thermo Fisher Scientific) was added and the reaction mixture was incubated at 37 ◦ C for an additional 16 h. The reaction was terminated by heating the mixture at 55 ◦ C for 30 min with proteinase K (0.5 mg/ml; Thermo Fisher Scientific) and SDS (0.5 % w/v) following 30 min of incubation at room temperature with RNase A (0.25 mg/ml; Thermo Fisher Scientific). After phenol/chloroform treatment, DNA was precipitated by propan-2-ol and dissolved in TE buffer (10 mM Tris/HCl, pH 8.0, and 1 mM EDTA). Then, 10 μg of DNA was mixed with 3× loading dye solution
and subjected to agarose gel electrophoresis (1 % agarose gel). Southern blot analysis was performed as described in [3] with the following modifications. Fractionated DNA was transferred from the agarose gel on to SensiBlot Plus Nylon membrane (Thermo Fisher Scientific) via semi-dry transfer. DNA was denatured and fixed on the membrane by placing it on a paper towel saturated with 0.4 M NaOH for 10 min following rinsing with 2× SSC (1× SSC is 0.15 M NaCl/0.015 M sodium citrate) and air drying. The membrane was prehybridized with 6× SSC buffer containing 0.5 % SDS and 100 μg/ml denatured salmon sperm DNA (Amresco) for 1 h at 65 ◦ C. The hybridization probe was generated by PCR using genomic E. coli BL21(DE3) DNA as template yielding a 397 bp product (Table S6). 5 -ends were radiolabelled by incubating with [γ -32 P]ATP and T4 PNK. The labelled probe was purified using GeneJET PCR purification kit (Thermo Fisher Scientific), denatured by heating to 95 ◦ C for 5 min, rapidly cooled on ice and added directly to the prehybridization solution. The membrane was probed for 16 h at 65 ◦ C and washed twice with 2× SSC/0.5 % SDS and twice with 2× SSC/0.1 % SDS at room temperature, air-dried and visualized by phosphorimaging (FLA-5100; Fujifilm).
Human genomic DNA cleavage Human genomic DNA extracted from brain tissue was provided by Dr Arturas Petronis. Cas9t–HS1 and Cas9t–HS2 complexes were assembled using 78 nt tracrRNA obtained by in vitro transcription and synthetic 42 nt crRNAs (Tables S3 and S4) targeting RASGEF1C or ARL15 loci respectively. Cas9t complexes were mixed with human genomic DNA and incubated for 30 min at 37 ◦ C. The final reaction mixture contained 1 μg of genomic DNA, 100 nM Cas9t complex, 10 mM Tris/HCl (pH 7.5 at 37 ◦ C), 100 mM NaCl, 1 mM DTT and 10 mM MgCl2 in a 100 μl reaction volume. Cleavage products were analysed by qPCR using 30 ng of DNA as a template for qPCR (25 μl) with Maxima SYBR Green Master Mix 2× (Thermo Fisher Scientific). Relative quantification of DNA was performed using the CT method [4]. C The
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Table S6 Generation of the hybridization probe for Southern blotting Name
Sequence
Description
TK-36
5 -cagcaattataagagatgtatcagaagaagatgc-3
Generation of 397 bp hybridization probe from E. coli BL21(DE3) genomic DNA
TK-34
5 -gcacctttattcccaactgttctttgatttaga-3
Table S7 Cloning the tetracycline-resistance gene into pUC18 Name
Sequence
Description
GG-574 GG-575
5 -gtaattctcatgtttgacagcttatcatc-3 5 -attcaggtcgaggtggcccg-3
Amplification of 1282 bp fragment coding tetracycline-resistance gene from pACYC184 plasmid
Cloning of tetracycline-resistance gene in pUC18 plasmid with reprogrammed Cas9t complexes pUC18 (2 μg) was incubated with two different Cas9t complexes (250 nM each) containing different crRNAs for 1 h at 37 ◦ C in 100 μl of reaction buffer containing 10 mM Tris/HCl (pH 7.5 at 37 ◦ C), 100 mM NaCl, 1 mM DTT and 10 mM MgCl2 . The linear vector fragment was purified from agarose gel using GeneJET gel extraction kit (Thermo Fisher Scientific) and divided into two equal parts. One part of pre-cleaved vector was dephosphorylated with FastAP alkaline phosphatase (Thermo Fisher Scientific), whereas the other part remained untreated. A 1282 bp insert containing promoter and tetracycline-resistance gene was obtained by PCR (primers are listed in Table S7) using the pACYC184 plasmid as a template. After purification with GeneJET PCR purification kit, the PCR fragment was divided into two parts. One part was untreated, whereas the other was phosphorylated with T4 PNK. Untreated vector was ligated with untreated PCR fragment, whereas dephosphorylated
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vector was ligated with phosphorylated fragment using T4 DNA ligase (Thermo Fisher Scientific). After transformation, the recombinant E. coli DH5α clones were selected on a medium supplemented with 100 μg/ml ampicillin and 25 μg/ml tetracycline. Plasmid DNA was purified from resulting transformants and subjected to sequencing.
References 1 Karvelis, T., Gasiunas, G., Miksys, A., Barrangou, R., Horvath, P. and Siksnys, V. (2013) crRNA and tracrRNA guide Cas9-mediated DNA interference in Streptococcus thermophilus. RNA Biol. 10, 841–851 2 Sapranauskas, R., Gasiunas, G., Fremaux, C., Barrangou, R., Horvath, P. and Siksnys, V. (2011) The Streptococcus thermophilus CRISPR/Cas system provides immunity in Escherichia coli. Nucleic Acids Res. 39, 9275–9282 3 Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning: a Laboratory Manual, 2nd edn., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 4 Livak, K.J. and Schmittgen, T.D. (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2 − C T method. Methods 25, 402–408 Received 24 July 2013 doi:10.1042/BST20130164
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Programmable DNA cleavage in vitro by Cas9 Tautvydas Karvelis*1 , Giedrius Gasiunas*1 and Virginijus Siksnys*2 *Institute of Biotechnology, Vilnius University, Graiciuno 8, LT-02241, Vilnius, Lithuania
Biochemical Society Transactions
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Abstract The ternary Cas9–crRNA–tracrRNA complex (Cas9t) of the Type II CRISPR (clustered regularly interspaced short palindromic repeats)–Cas (CRISPR-associated) system functions as an Mg2 + -dependent RNA-directed DNA endonuclease that locates its DNA target guided by the crRNA (CRISPR RNA) in the tracrRNA–crRNA structure and introduces a double-strand break at a specific site in DNA. The simple modular organization of Cas9t, where specificity for the DNA target is encoded by a small crRNA and the cleavage reaction is executed by the Cas9 endonuclease, provides a versatile platform for the engineering of universal RNA-directed DNA endonucleases. By altering the crRNA sequence within the Cas9t complex, programmable endonucleases can be designed for both in vitro and in vivo applications. Cas9t has been recently employed as a geneediting tool in various eukaryotic cell types. Using Streptococcus thermophilus Cas9t as a model system, we demonstrate the feasibility of Cas9t as a programmable molecular tool for in vitro DNA manipulations.
Introduction CRISPR (clustered regularly interspaced short palindromic repeats) is a microbial adaptive immune system which provides acquired resistance against viruses and plasmids [1]. The CRISPR array consists of short conserved repeat sequences interspaced by unique spacer sequences, which often originate from phage or plasmid DNA. The cas (CRISPR-associated) genes are located adjacent to the CRISPR array. CRISPR–Cas systems hijack fragments of the invading nucleic acid to incorporate them as spacers into a host genome and later use them as templates to generate small RNA molecules [crRNA (CRISPR RNA)] which together with Cas proteins are assembled into an effector complex which silences foreign nucleic acids in the subsequent rounds of infection [2–7]. CRISPR–Cas systems have been categorized into three main types, on the basis of core element content and sequences [8]. The composition and stoichiometry of effector complexes differ strikingly between different CRISPR subtypes [9,10]. In the Type I and Type III systems, effector complexes are arranged as large multisubunit ribonucleoprotein complexes [11–16]. In Type II CRISPR–Cas systems, the effector complex required for DNA interference comprises a sole Cas9 protein bound to a dual RNA molecule [17–20]. Cas9 is a large multidomain protein which contains two nuclease domains, an RuvC-like nuclease domain near the N-terminus and a HNH (His-AsnHis)-like nuclease domain in the middle of the protein [17– 19,21]. Cas9 forms a ternary complex (Cas9t) with two RNA
Key words: cloning, clustered regularly interspaced short palindromic repeats (CRISPR), CRISPR RNA (crRNA), RNA-guided endonuclease, site-specific cleavage, targeted genome modification. Abbreviations used: CRISPR, clustered regularly interspaced short palindromic repeats; crRNA, CRISPR RNA; PAM, protospacer-adjacent motif; qPCR, quantitative real-time PCR; St-Cas9t, Cas9– crRNA–tracrRNA ternary complex of the Streptococcus thermophilus; tracrRNA, transactivating crRNA. 1 2
These authors contributed equally to this work. To whom correspondence should be addressed (email [email protected]).
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molecules: crRNA and tracrRNA (transactivating crRNA) [18–20]. The 42 nt mature crRNA consists of the 22-nt 3 -handle originating from the repeat sequence and 20-nt unique spacer fragment, which guides Cas9t to the matching DNA target [22]. tracrRNA is partially complementary to crRNA and is essential for the crRNA maturation [20,22] and DNA cleavage by Cas9t [18–20]. Cas9t binding to the target site (a protospacer) complementary to the crRNA occurs only if a short nucleotide sequence, called a PAM (protospacer-adjacent motif), is located in the vicinity of a protospacer [18,19]. If both the protospacer and PAM are present, Cas9t binds to the target site generating the Rloop, where one DNA strand is displaced while another is engaged into a heteroduplex with crRNA. DNA cleavage is mediated by a pair of Cas9 active sites that act on opposite DNA strands: RuvC cuts the displaced DNA strand, whereas HNH cuts DNA strand paired with RNA [18,19]. Doublestrand breaks introduced by the Cas9 protein of Streptococcus thermophilus occurs within a protospacer, 3 nt away from the PAM sequence, to produce blunt-end DNA products [18,19,23,24]. Thus the Cas9t complex functions as an Mg2 + dependent RNA-directed DNA endonuclease that requires five obligatory components for a site-specific DNA cleavage: the Cas9 protein, crRNA, tracrRNA, a protospacer sequence complementary to the crRNA and a PAM. The simple modular organization of the Cas9t complex, where specificity for DNA targets is encoded by a small crRNA and the cleavage machinery consists of a single multidomain Cas protein, provides a versatile platform for the engineering of universal RNA-guided DNA endonucleases. Indeed, by altering the RNA sequence within the Cas9t complex, programmable endonucleases can be designed for both in vitro and in vivo applications. Cas9t has been recently employed as a gene-editing tool in various eukaryotic cells, including human, mouse, zebrafish, yeast and Drosophila [25–36]. In the present article, we show that C The
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Cas9t provides a versatile programmable molecular tool for in vitro applications.
In vitro assembly of a programmable Cas9t complex We have shown previously that the Cas9–crRNA–tracrRNA ternary complex of the S. thermophilus DGCC7710 CRISPR3–Cas system (St-Cas9t) (Figure 1A) isolated from the recombinant host cleaves both a synthetic oligodeoxynucleotide duplex and a plasmid DNA bearing a nucleotide sequence complementary to the crRNA, in a PAM-dependent manner [18,20]. However, the low yield of the St-Cas9t complex isolated from the recombinant Escherichia coli host and traces of nucleases contaminating the St-Cas9t preparation hindered further applications. We therefore have developed a method to obtain in vitro an active St-Cas9t from individual components [20] (see the Supplementary Online Data at http://www. biochemsoctrans.org/bst/041/bst0411401add.htm). Two different experimental procedures have been used for the StCas9t complex assembly: (i) mixing of the Cas9 apoprotein with ‘mature’ forms of tracrRNA (78 nt) and crRNA (42 nt), and (ii) mixing of the Cas9 apoprotein with primary transcripts of tracrRNA (105 nt) and pre-crRNA (150 nt) followed by a subsequent incubation with RNase III [20]. RNase III together with tracrRNA are required for the long pre-crRNA processing in the Type II systems, including Streptococcus pyogenes and S. thermophilus [20,22]; however, RNase III turned out to be dispensable if pre-crRNA is transcribed from a minimal R-SP-R (where R is a repeat unit and SP is a spacer) CRISPR region [20]. In the latter case, the unprocessed crRNA carrying flanking repeat sequences together with tracrRNA and Cas9 protein assemble into a functionally active Cas9t complex. Subsequent RNase III treatment results in a ∼2-fold increase in Cas9t DNA cleavage activity. Therefore, in our study of Cas9t complex assembly, we first incubated the 105 nt tracrRNA transcript (101nt-long putative tracrRNA transcript + 4 nt for in vitro transcription using T7 RNA polymerase) with 150-nt-long pre-crRNA (20 nt leader, R-SP-R unit and downstream flanking sequence) in the presence of St-Cas9 protein to promote tracrRNA–pre-crRNA duplex formation and then added RNase III for the crRNA end-processing to generate a functional complex (Figure 1B). The finding that an active Cas9t can be assembled using crRNA and tracrRNA obtained by in vitro transcription prompted us to engineer a recombinant plasmid, pcas-2R, by inserting a cassette encoding a spacer sequence flanked by two repeat units between SapI and Eco31I restriction sites (Figure 1C). This plasmid allows an easy reprogramming of the Cas9t complex by ligating a synthetic oligoduplex carrying a new spacer sequence into the SapI/Eco31I precleaved plasmid followed by in vitro transcription of the PCR product carrying the R-SP-R unit to generate a desired precrRNA. C The
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DNA cleavage by the in vitro assembled St-Cas9t complex First, the DNA cleavage activity of the in vitro assembled St-Cas9t complex was assayed on oligodeoxynucleotide and plasmid substrates containing protospacer and PAM sequences. Cas9t loaded by the dual tracrRNA–crRNA matching the protospacer SP1 in the 55 bp oligoduplex cleaved both strands at a fixed position, 3 nt upstream of the PAM (5 -NGGNG-3 ) sequence, leaving blunt ends (Figure 1D), identically with Cas9t complex purified from E. coli [18]. As expected, an oligoduplex lacking a protospacer SP1 was resistant to the Cas9t cleavage. Furthermore, upon Cas9t treatment the pSP1 plasmid containing a protospacer SP1 and PAM sequence was converted into a linear form (Figure 1E), indicating that both DNA strands were cleaved specifically within the protospacer. Re-ligation of the linear pSP1 form by DNA ligase followed by a transformation of E. coli cells yielded multiple ampicillin-resistant clones. Sequencing of DNA purified from selected clones revealed no changes in the protospacer SP1 sequence. In a control experiment, E. coli transformation by the ligation mixture in the absence of T4 DNA ligase produced no transformants, indicating that a supercoiled plasmid does not co-purify with a linear reaction product. Taken together, these results demonstrate that (i) no mutations or indels are introduced upon Cas9t cleavage of plasmid DNA, and (ii) DNA ends generated by Cas9t cleavage are substrates for T4 DNA ligase, and therefore must contain a phosphate at the 5 terminus and a free hydroxy group at the 3 terminus [37]. In addition, using the procedure described above for the Cas9 CRISPR3–Cas system, we were able to assemble in vitro an active ternary Cas9 complex of the S. thermophilus CRISPR1–Cas [1,23,38] system (results not shown). Unlike the Cas9 from CRISPR3–Cas which depends on the 5 -NGGNG-3 PAM sequence, Cas9t of CRISPR1–Cas requires a 5 -AGAAW-3 PAM located in the vicinity of the target complimentary to the crRNA [38,39]. The availability of functional Cas9t variants with different PAM requirements expands the range of targets accessible for the Cas9t cleavage.
St-Cas9t complex as a tool for DNA manipulation Restriction endonucleases recognize short nucleotide sequences usually 4–8 bp in length and, in the presence of Mg2 + , ions cut both DNA strands within or close to the target site. Owing to their unique specificity, restriction enzymes became indispensable tools for DNA cloning [40]. However, the strict specificity of restriction enzymes limits flexibility when inserting gene fragments into a plasmid vector. To demonstrate that Cas9t could be used as a universal RNA-guided DNA endonuclease programmed by the crRNA to target any DNA site, we adapted it for a cloning procedure. We selected two target sites (N1 and N2) located near the SapI and AatII restriction endonuclease sites in the pUC18 plasmid and, using the plasmid bearing the
CRISPR: Evolution, Mechanisms and Infection
Figure 1 In vitro assembly and functional analysis of St-Cas9t complex (A) Schematic representation of the CRISPR3–Cas system of S. thermophilus DGCC7710. Four cas genes (cas9, cas1, cas2 and csn2) are located upstream of the CRISPR repeat–spacer array, consisting of 13 repeat (R) sequences and 12 unique spacers (S1–S12). The tracrRNA is located upstream of the cas9 gene and encoded on the opposite DNA strand (shown by an arrow) with respect to the other elements of this system. (B) In vitro assembly of the Cas9t complex. The Cas9 protein is first pre-incubated with 150 nt pre-crRNA and 105 nt tracrRNA transcripts, followed by subsequent treatment with RNase III to generate a catalytically competent Cas9t complex. (C) The cassette for a new spacer insertion into the CRISPR region and crRNA synthesis. A synthetic oligoduplex encoding a desired spacer sequence and containing SapI- and Eco31I-compatible ends is inserted between two repeats. The PCR fragment of the CRISPR region is used as a template for crRNA synthesis by in vitro transcription. T7 RNA pol, T7 RNA polymerase. (D) DNA oligoduplex cleavage by Cas9t complex. The oligoduplex strand which is complementary to crRNA is marked as ( + ) strand, whereas the other strand is marked as ( − ) strand. M1 and M2 are synthetic oligonucleotide markers corresponding to the 37 nt of the ( − ) strand and 18 nt of the ( + ) strand used to map the cleavage position. C, non-specific oligoduplex lacking a protospacer SP1. Reaction time varied between 0 and 30 min. Sizes are indicated in nt. (E) Plasmid DNA cleavage. Cas9t complex was incubated for 5 min with pSP1 or pUC18 plasmids and reaction products analysed in the agarose gel. pSP1 plasmid contained a protospacer SP1 sequence flanked by the 5 -NGGNG-3 PAM sequence. Protospacer SP1 sequence was not present in pUC18. FLL, full-length linear; OC, open circle; SC, supercoiled.
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Figure 2 St-Cas9t complex as a tool for DNA manipulation (A) Schematic representation of the target sites in the pUC18 plasmid. The distance between SapI and AatII restriction enzymes sites is 775 bp, whereas the distance between two protospacer sites Cas9t-N1 and Cas9t-N2 is 612 bp. (B) pUC18 plasmid cleavage by reprogrammed Cas9t complexes. Cleavage products resulting from the double digestion with SapI and AatII restriction enzymes are also shown. Sizes are indicated in kbp. (C) Run-off sequencing data of pUC18 pre-cleaved with Cas9t-N1 (upper panel) and Cas9t-N2 (lower panel) complexes. Cleavage of plasmid DNA at both sites occurred 3 nt upstream of the PAM. (D) Schematic representation of the cloning experiment. Plasmid vector pre-cleaved with Cas9t Cas9t-N1 and Cas9t-N2 complexes was ligated with a PCR fragment encoding a promoter and a tetracycline resistance gene. Clones were selected on media containing tetracycline (Tc) and ampicillin (Ap).
interchangeable spacer cassette (described above), generated crRNAs matching protospacers N1 and N2. The theoretical length of the fragment located between Cas9t-N1/Cas9tN2 sites is 612 bp (Figure 2A). Double digestion of pUC18 plasmid with Cas9t-N1 and Cas9t-N2 (Figure 2B) yielded two DNA fragments (∼2000 and ∼600 bp respectively), indicating that the plasmid was cut at sites targeted by crRNAs incorporated in Cas9t-N1/Cas9t-N2 complexes. Sequencing data confirmed that plasmid DNA cleavage occurred at both sites 3 nt upstream of the PAM sequence generating a blunt-ended plasmid vector (Figure 2C). To C The
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demonstrate that the plasmid vector pre-cleaved with Cas9t is suitable for further manipulation, we ligated a PCR fragment amplified from the region containing a promoter and a tetracycline-resistance gene in the pACYC184 plasmid (Figure 2D), and used a resulting ligation mixture to transform E. coli cells. The resulting clones were selected on medium containing tetracycline and ampicillin. Sequencing of DNA isolated from the selected clones confirmed that the PCR fragment was inserted at the desired position and no mutations or indels were introduced upon Cas9t cleavage. Taken together, these experiments demonstrate that
CRISPR: Evolution, Mechanisms and Infection
Figure 3 DNA cleavage in vitro by the Cas9t complex (A) λ DNA cleavage. Phage λ DNA was incubated with the Cas9t complex for various times and reaction products were analysed by agarose gel electrophoresis. The Cas9t is programmed for a target located 8 kb away from the cos site. Sizes of markers (M) are indicated in kbp. (B) E. coli genomic DNA cleavage. DNA extracted from the E. coli BL21 strain was fragmented by PstI enzyme. The Cas9t is programmed for a target located between two PstI sites. If genomic DNA is cleaved by the Cas9t, a 466 bp fragment should be detected by probe hybridization; in the absence of cleavage, the hybridization probe should detect the 1499 bp fragment. C, E. coli genomic DNA fragmented with PstI. Genomic DNA was incubated for 30 min with Cas9t complex before fragmentation with PstI. Sizes of markers (M) are indicated in kbp. (C) Human genomic DNA cleavage by Cas9. Following Cas9t–HS1 and Cas9t–HS2 cleavage, the relative amounts of uncleaved DNA targets (HS1 or HS2) were estimated by qPCR. The ratio of cleaved to uncleaved DNA target quantified by qPCR is at least 25:1.
(i) Cas9t can be used as a programmable DNA endonuclease suitable for cloning experiments, and (ii) multiple cleavage can be performed simultaneously by different Cas9t complexes.
In vitro cleavage of genomic DNA by the Cas9t complex The predicted length of the DNA target recognized by the St-Cas9 is determined by the 20 nt sequence complementary to the crRNA plus 3 nt of the PAM sequence. Plasmid interference experiments in vivo indicate that 6 nt at the PAM distal end of a protospacer are not important for interference, suggesting that only 14 nt matching the crRNA and the PAM sequence are absolutely required for DNA interference by the St-Cas9t [19]. In theory, the 17 nt recognition sequence allows to design St-Cas9t complexes that target unique sequences in genomic DNA. To find out whether Cas9t is able to locate and cleave target sites in vitro in the context of genomic DNA, we programmed the Cas9t complex for unique sites in bacteriophage λ (49 kbp), E. coli (4.6 Mbp) and human (3.2 Gbp) DNA. The analysis of phage λ genomic DNA cleavage products by agarose gel electrophoresis confirmed that the 49 kbp DNA is cleaved only once, yielding 42 kbp and 7 kbp products as predicted (Figure 3A). To identify cleavage products of the E. coli genomic DNA, we used Southern blot analysis (see the Supplementary Online Data). More specifically, we first treated genomic DNA with an excess of Cas9t, followed by PstI restriction enzyme digestion, and then analysed reaction products by Southern blotting using a probe designed against the DNA fragment located between the Cas9t cleavage site and a downstream PstI target
(Figure 3B). The distance between two PstI recognition sites is ∼1500 bp, whereas the distance between a protospacer and the downstream PstI site is ∼500 bp. After Cas9t cleavage, we detected by probe hybridization only a ∼500 bp DNA fragment, indicating that E. coli genomic DNA was cleaved by Cas9t at the desired position. In the next set of experiments, we analysed human DNA cleavage by the Cas9t–HS1 and Cas9t–HS2 complexes targeted to RASGEF1C (HS1) and ARL15 (HS2) loci respectively. The cleavage reaction was monitored by qPCR (quantitative real-time PCR) (Figure 3C, and see the Supplementary Online Data). After treatment with Cas9t– HS1 and Cas9t–HS2, the amount of intact DNA targets in both loci decreased more than 25-fold, indicating that human genomic DNA was cleaved at the desired loci. Taken together, these data demonstrate that St-Cas9t effectively finds and cleaves its targets in the context of genomic DNA of different complexity, including viral, bacterial and mammalian DNA.
Acknowledgements We thank Dr Arturas Petronis from the Centre for Addiction and Mental Health, Toronto, Canada, for providing human DNA samples, advice on target site selection and discussion.
Funding This work on CRISPR systems in V.S.’s laboratory is funded by the European Social Fund under Global Grant Measure.
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References 1 Barrangou, R., Fremaux, C., Deveau, H., Richards, M., Boyaval, P., Moineau, S., Romero, D.A. and Horvath, P. (2007) CRISPR provides acquired resistance against viruses in prokaryotes. Science 315, 1709–1712 2 Wiedenheft, B., Sternberg, S.H. and Doudna, J.A. (2012) RNA-guided genetic silencing systems in bacteria and archaea. Nature 482, 331–338 3 Westra, E.R., Swarts, D.C., Staals, R.H.J., Jore, M.M., Brouns, S.J.J. and van der Oost, J. (2012) The CRISPRs, they are a-changin’: how prokaryotes generate adaptive immunity. Annu. Rev. Genet. 46, 311–339 4 Sorek, R., Lawrence, C.M. and Wiedenheft, B. (2013) CRISPR-mediated adaptive immune systems in bacteria and archaea. Annu. Rev. Biochem. 1–30 5 Barrangou, R. (2013) CRISPR–Cas systems and RNA-guided interference. Wiley Interdiscip. Rev.: RNA 4, 267–278 6 Reeks, J., Naismith, J.H. and White, M.F. (2013) CRISPR interference: a structural perspective. Biochem. J. 453, 155–166 7 Gasiunas, G., Sinkunas, T. and Siksnys, V. (2013) Molecular mechanisms of CRISPR-mediated microbial immunity. Cell. Mol. Life Sci., doi:10.1007/s00018-013-1438-6 8 Makarova, K.S., Haft, D.H., Barrangou, R., Brouns, S.J. J., Charpentier, E., Horvath, P., Moineau, S., Mojica, F.J.M., Wolf, Y.I., Yakunin, A.F. et al. (2011) Evolution and classification of the CRISPR–Cas systems. Nat. Rev. Microbiol. 9, 467–477 9 Makarova, K.S., Wolf, Y.I. and Koonin, E.V. (2013) Comparative genomics of defense systems in archaea and bacteria. Nucleic Acids Res. 41, 4360–4377 10 Koonin, E. V. and Makarova, K.S. (2013) CRISPR–Cas: evolution of an RNA-based adaptive immunity system in prokaryotes. RNA Biol. 10, 679–686 11 Brouns, S.J.J., Jore, M.M., Lundgren, M., Westra, E.R., Slijkhuis, R.J.H., Snijders, A.P.L., Dickman, M.J., Makarova, K.S., Koonin, E.V. and van der Oost, J. (2008) Small CRISPR RNAs guide antiviral defense in prokaryotes. Science 321, 960–964 12 Wiedenheft, B., Lander, G.C., Zhou, K., Jore, M.M., Brouns, S.J. J., van der Oost, J., Doudna, J.A. and Nogales, E. (2011) Structures of the RNA-guided surveillance complex from a bacterial immune system. Nature 477, 486–489 13 Sinkunas, T., Gasiunas, G., Waghmare, S.P., Dickman, M.J., Barrangou, R., Horvath, P. and Siksnys, V. (2013) In vitro reconstitution of Cascade-mediated CRISPR immunity in Streptococcus thermophilus. EMBO J. 32, 385–394 14 Wiedenheft, B., van Duijn, E., Bultema, J., Waghmare, S., Zhou, K., Barendregt, A., Westphal, W., Heck, A., Boekema, E., Dickman, M. et al. (2011) RNA-guided complex from a bacterial immune system enhances target recognition through seed sequence interactions. Proc. Natl. Acad. Sci. U.S.A. 108, 10092–10097 15 Hale, C.R., Zhao, P., Olson, S., Duff, M.O., Graveley, B.R., Wells, L., Terns, R.M. and Terns, M.P. (2009) RNA-guided RNA cleavage by a CRISPR RNA–Cas protein complex. Cell 139, 945–956 16 Zhang, J., Rouillon, C., Kerou, M., Reeks, J., Brugger, K., Graham, S., Reimann, J., Cannone, G., Liu, H., Albers, S.-V. et al. (2012) Structure and mechanism of the CMR complex for CRISPR-mediated antiviral immunity. Mol. Cell 45, 303–313 17 Sapranauskas, R., Gasiunas, G., Fremaux, C., Barrangou, R., Horvath, P. and Siksnys, V. (2011) The Streptococcus thermophilus CRISPR/Cas system provides immunity in Escherichia coli. Nucleic Acids Res. 39, 9275–9282 18 Gasiunas, G., Barrangou, R., Horvath, P. and Siksnys, V. (2012) Cas9–crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria. Proc. Natl. Acad. Sci. U.S.A. 109, E2579–E2586 19 Jinek, M., Chylinski, K., Fonfara, I., Hauer, M., Doudna, J.A. and Charpentier, E. (2012) A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816– 821 20 Karvelis, T., Gasiunas, G., Miksys, A., Barrangou, R., Horvath, P. and Siksnys, V. (2013) crRNA and tracrRNA guide Cas9-mediated DNA interference in Streptococcus thermophilus. RNA Biol. 10, 841–851
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CRISPR: Evolution, Mechanisms and Infection
SUPPLEMENTARY ONLINE DATA
Programmable DNA cleavage in vitro by Cas9 Tautvydas Karvelis*1 , Giedrius Gasiunas*1 and Virginijus Siksnys*2 *Institute of Biotechnology, Vilnius University, Graiciuno 8, LT-02241, Vilnius, Lithuania
DNA manipulations Construction of Cas9 expression plasmid and plasmid pSP1 containing a protospacer was described previously [1,2]. To obtain a cassette for a spacer insertion, the DNA fragment was amplified from pCas9 plasmid [2] using GG116 and GG-567 primers (Table S1) designed to introduce a SapI site after the first repeat. The PCR fragment was inserted into the pCas9 plasmid vector pre-cleaved with Eco31I and BamHI. The resulting pCas9-1R plasmid contained the SapI site after the first repeat, and a partially truncated CRISPR region. Next, the cas9 gene was deleted by cutting the pCas9-1R plasmid with Eco105I and StuI to generate the pcas-1R plasmid. The resulting pcas-1R plasmid was then cleaved with SapI and BamHI and ligated with oligoduplex assembled from GG-580 and GG-581 oligonucleotides (Table S1) (designed to introduce the second repeat and Eco31I site), resulting in the pcas-2R plasmid. The new spacer cassettes were assembled from synthetic oligoduplexes (Table S2) containing the sticky ends for SapI and Eco31I restriction endonucleases. Oligoduplexes were cloned into pcas-2R plasmid through SapI and Eco31I sites to yield plasmids containing CRISPR regions with engineered spacers (see Figure 1C in the main text). Sequencing of CRISPR regions of obtained plasmids revealed that required spacers have been inserted between two repeats.
Expression and purification of Cas9 protein Purification of His6 -tagged variant of St-Cas9 protein was performed as described in [1]. Briefly, Cas9 was expressed in E. coli DH10B strain. Harvested cells were disrupted by sonication, and supernatant was loaded on to a Ni2 + -charged chelating column, followed by a heparin column. Fractions containing the purified protein were pooled, transferred to storage buffer and stored at − 20 ◦ C.
RNA production RNA fragments required for Cas9t complex assembly were either produced by in vitro transcription using TranscriptAid T7 High Yield Transcription Kit (Thermo Fisher Scientific) or 1 2
These authors contributed equally to this work. To whom correspondence should be addressed (email [email protected]).
Biochem. Soc. Trans. (2013) 41, 1401–1406; doi:10.1042/BST20130164
purchased as synthetic oligoribonucleotides (Metabion). For in vitro transcription, a fragment containing a T7 promoter at the proximal end of the RNA coding sequence was PCRgenerated using primers and templates provided in Table S3. Transcribed RNA fragments were purified using RNeasy® MinElute® Cleanup Kit (Qiagen). Sequences of synthetic oligoribonucleotides are provided in Table S4.
Assembly of the Cas9t complex For Cas9t complex assembly using 42 nt synthetic crRNA and 78 nt tracrRNA, His6 -tagged Cas9 protein was mixed with a pre-annealed crRNA and tracrRNA duplex at 1:1 molar ratio and incubated in a buffer containing 10 mM Tris/HCl (pH 7.5 at 37 ◦ C), 100 mM NaCl and 1 mM DTT at 37 ◦ C for 1 h. For in vitro assembly of a catalytically competent Cas9t complex (0.1–2 μM) the His6 -tagged Cas9 (0.1–2 μM) protein was mixed with 150 nt pre-crRNA (0.1–2 μM) and 105 nt tracrRNA (0.2–4 μM) transcripts at 1:1:2 molar ratio and pre-incubated in a buffer containing 10 mM Tris/HCl (pH 7.5 at 37 ◦ C) and 100 mM NaCl at 37 ◦ C for 30 min followed by addition of RNAse III (0.1–2 μM; Ambion) and subsequent incubation for additional 30 min at 37 ◦ C in 10 mM Tris/HCl (pH 7.5 at 37 ◦ C), 100 mM NaCl, 10 mM MgCl2 and 1 mM DTT.
Reactions with oligonucleotide substrates The 5 -ends of oligonucleotides (Table S5) were radiolabelled using T4 PNK (polynucleotide kinase) (Thermo Fisher Scientific) and [γ -33 P]ATP (Hartmann Analytic). Duplexes were made by annealing two oligonucleotides with complementary sequences at 95 ◦ C following slow cooling to room temperature. A radioactive label was introduced at the 5 end of individual DNA strands before the annealing with unlabelled strands. Reactions were typically carried out by mixing labelled oligoduplex with Cas9t complex (1:1 v/v ratio) and incubating at 37 ◦ C. The final reaction mixture contained 1 nM labelled duplex, 50 nM Cas9t complex, 10 mM Tris/HCl (pH 7.5 at 37 ◦ C), 100 mM NaCl, 1 mM DTT and 10 mM MgCl2 in a 100 μl reaction volume. Aliquots of 6 μl were removed from the reaction mixture at timed intervals, quenched with 10 μl of loading dye [95 % (v/v) formamide, 0.01 % Bromophenol Blue and C The
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Table S1 Plasmid construction for new spacer insertion Restriction endonuclease sites are in bold and underlined.
Table S2 Oligonucleotides for new spacer insertion Spacer sequences are in upper case. Name
Sequence
Description
GG-564 GG-565
5΄-aacTGTCATGATAATAATGGTTTCTTAGACGTC-3΄ 3΄-ACAGTACTATTATTACCAAAGAATCTGCAGcaaa-5΄
Construction of pΔcas-2R plasmid with N1 spacer
GG-566 GG-567
5΄-aacACGAGCCGGAAGCATAAAGTGTAAAGCCTG-3΄ 3΄-TGCTCGGCCTTCGTATTTCACATTTCGGACcaaa-5΄
Construction of pΔcas-2R plasmid with N2 spacer
TK-43 TK-44
5΄-aacTCAAGGGAGAATAGAGGCTCTCGTTGCATT-3΄ 3΄-AGTTCCCTCTTATCTCCGAGAGCAACGTAAcaaa-5΄
Construction of pΔcas-2R plasmid with spacer targeting E. coli genomic DNA
TK-47 TK-48
5΄-aacCGGGAGGGAAGCTGCATGATGCGATGTTAT-3΄ 3΄-GCCCTCCCTTCGACGTACTACGCTACAATAcaaa-5΄
Construction of pΔcas-2R plasmid with spacer targeting λ genomic DNA
Table S3 Oligonucleotides for generation of in vitro transcription (IVT) templates T7 promoters are in bold and underlined. Name
Sequence
Description
TK-4
5 -aaaaacaccgaatcggtgccac-3
Generation of PCR template from pCRISPR3
TK-5
5 -taatacgactcactatagggtaataataattgtggtttgaaaccattc-3
TK-15
5 -taatacgactcactatagggtagaaaagatatcctacgagg-3
TK-2
5 -caacaaccaagctaatacagcag-3
TK-27
5 -cacgatgcgtccggcgtagaggatcc-3
plasmid for IVT of 105 nt tracrRNA Generation of PCR template from pCRISPR3 plasmid for IVT of pre-crRNA Generation of PCR template with TK-15 from pcas-2R plasmid with inserted spacer for IVT of pre-crRNA
TK-4
5 -aaaaacaccgaatcggtgccac-3
TK-52
5 -taatacgactcactatagggcgaaacaacacagcgagttaaaataagg-3
Generation of PCR template from pCRISPR3 plasmid for IVT of 78 nt tracrRNA
Table S4 crRNAs for human genomic DNA targeting and oligonucleotides used for qPCR Name
Sequence
Description
pr-hs1f pr-hs1r pr-hs2f
5 -tgctgctcgatgcacaggt-3 5 -catcttcaccttcctgctgag-3 5 -ccaaattataagacagatgcctag-3
Primers for qPCR to amplify RASGEF1C (HS1) locus
pr-hs2r GG-625 GG-627
5 -gccaacttctgtgaaactacact-3 5 -gcucccggggcucgaugaagguuuuagagcuguguuguuucg-3 5 -ugaaucgugaaaucugcucaguuuuagagcuguguuguuucg-3
25 mM EDTA (pH 9.0)] and subjected to denaturing gel electrophoresis (20 % polyacrylamide containing 8.5 M urea). Gels were dried and visualized by phosphorimaging (FLA5100; Fujifilm).
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Primers for qPCR to amplify ARL15 (HS2) locus 42 nt crRNA targeting RASGEF1C (HS1) 42 nt crRNA targeting ARL15 (HS2)
Reactions with plasmid substrates Reactions were initiated by mixing a supercoiled plasmid DNA with Cas9t complex (1:1 v/v ratio) and incubation at 37 ◦ C. The final reaction mixture contained 3 nM (500 ng)
CRISPR: Evolution, Mechanisms and Infection
Table S5 Oligonucleotide substrates The spacer is in upper case and the PAM is in bold and underlined. Name
Sequence
Description
GG-316 GG-317
5΄-gctcgaattgAAATTCTAAACGCTAAAGAGGAAGAGGACAtGGtGaattcgtaat-3΄ 3΄-cgagcttaacTTTAAGATTTGCGATTTCTCCTTCTCCTGTaCCaCttaagcatta-5΄
SP1 duplex
GG-494 GG-495
5΄-gctcgaattgTACTGCTGTATTAGCTTGGTTGTTGGTTTGtGGtGaattcgtaat-3΄ 3΄-cgagcttaacATGACGACATAATCGAACCAACAACCAAACaCCaCttaagcatta-5΄
Control duplex
GG-371
5΄-attacgaattcaccatgt-3΄
18 nt marker for (+) strand cleavage
GG-516
5΄-gctcgaattgaaattctaaacgctaaagaggaagagg-3΄
37 nt marker for (-) strand cleavage
plasmid, 50 nM Cas9t complex, 10 mM Tris/HCl (pH 7.5 at 37 ◦ C), 100 mM NaCl, 1 mM DTT and 10 mM MgCl2 in a 100 μl reaction volume. Aliquots were removed at timed intervals and quenched with phenol/chloroform. The aqueous phase was mixed with 3× loading dye solution (0.01 % Bromophenol Blue and 75 mM EDTA in 50 % v/v glycerol) and reaction products were analysed by agarose gel electrophoresis and ethidium bromide staining.
λ DNA cleavage
The reactions were initiated by mixing λ DNA (Thermo Fisher Scientific) with Cas9t complex (1:1 v/v ratio) and incubation at 37 ◦ C. The final reaction mixture contained 2 μg of λ DNA, 50 nM Cas9t complex, 10 mM Tris/HCl (pH 7.5 at 37 ◦ C), 100 mM NaCl, 1 mM DTT and 10 mM MgCl2 in a 100 μl reaction volume. Aliquots were removed at timed intervals and quenched with phenol/chloroform. The aqueous phase was mixed with 3× loading dye solution and reaction products were analysed by agarose gel electrophoresis and ethidium bromide staining.
E. coli genomic DNA cleavage and Southern hybridization Genomic DNA from E. coli (BL21) strain was isolated using the Genomic DNA purification kit (Thermo Fisher Scientific). For the cleavage assay, genomic DNA was combined with Cas9t complex (1:1 v/v ratio) and incubated for 3 h at 37 ◦ C. The final reaction mixture contained 30 μg of genomic DNA, 1 μM Cas9, 10 mM Tris/HCl (pH 7.5 at 37 ◦ C), 100 mM NaCl, 1 mM DTT and 10 mM MgCl2 in a 300 μl reaction volume. Following incubation, PstI (Thermo Fisher Scientific) was added and the reaction mixture was incubated at 37 ◦ C for an additional 16 h. The reaction was terminated by heating the mixture at 55 ◦ C for 30 min with proteinase K (0.5 mg/ml; Thermo Fisher Scientific) and SDS (0.5 % w/v) following 30 min of incubation at room temperature with RNase A (0.25 mg/ml; Thermo Fisher Scientific). After phenol/chloroform treatment, DNA was precipitated by propan-2-ol and dissolved in TE buffer (10 mM Tris/HCl, pH 8.0, and 1 mM EDTA). Then, 10 μg of DNA was mixed with 3× loading dye solution
and subjected to agarose gel electrophoresis (1 % agarose gel). Southern blot analysis was performed as described in [3] with the following modifications. Fractionated DNA was transferred from the agarose gel on to SensiBlot Plus Nylon membrane (Thermo Fisher Scientific) via semi-dry transfer. DNA was denatured and fixed on the membrane by placing it on a paper towel saturated with 0.4 M NaOH for 10 min following rinsing with 2× SSC (1× SSC is 0.15 M NaCl/0.015 M sodium citrate) and air drying. The membrane was prehybridized with 6× SSC buffer containing 0.5 % SDS and 100 μg/ml denatured salmon sperm DNA (Amresco) for 1 h at 65 ◦ C. The hybridization probe was generated by PCR using genomic E. coli BL21(DE3) DNA as template yielding a 397 bp product (Table S6). 5 -ends were radiolabelled by incubating with [γ -32 P]ATP and T4 PNK. The labelled probe was purified using GeneJET PCR purification kit (Thermo Fisher Scientific), denatured by heating to 95 ◦ C for 5 min, rapidly cooled on ice and added directly to the prehybridization solution. The membrane was probed for 16 h at 65 ◦ C and washed twice with 2× SSC/0.5 % SDS and twice with 2× SSC/0.1 % SDS at room temperature, air-dried and visualized by phosphorimaging (FLA-5100; Fujifilm).
Human genomic DNA cleavage Human genomic DNA extracted from brain tissue was provided by Dr Arturas Petronis. Cas9t–HS1 and Cas9t–HS2 complexes were assembled using 78 nt tracrRNA obtained by in vitro transcription and synthetic 42 nt crRNAs (Tables S3 and S4) targeting RASGEF1C or ARL15 loci respectively. Cas9t complexes were mixed with human genomic DNA and incubated for 30 min at 37 ◦ C. The final reaction mixture contained 1 μg of genomic DNA, 100 nM Cas9t complex, 10 mM Tris/HCl (pH 7.5 at 37 ◦ C), 100 mM NaCl, 1 mM DTT and 10 mM MgCl2 in a 100 μl reaction volume. Cleavage products were analysed by qPCR using 30 ng of DNA as a template for qPCR (25 μl) with Maxima SYBR Green Master Mix 2× (Thermo Fisher Scientific). Relative quantification of DNA was performed using the CT method [4]. C The
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Table S6 Generation of the hybridization probe for Southern blotting Name
Sequence
Description
TK-36
5 -cagcaattataagagatgtatcagaagaagatgc-3
Generation of 397 bp hybridization probe from E. coli BL21(DE3) genomic DNA
TK-34
5 -gcacctttattcccaactgttctttgatttaga-3
Table S7 Cloning the tetracycline-resistance gene into pUC18 Name
Sequence
Description
GG-574 GG-575
5 -gtaattctcatgtttgacagcttatcatc-3 5 -attcaggtcgaggtggcccg-3
Amplification of 1282 bp fragment coding tetracycline-resistance gene from pACYC184 plasmid
Cloning of tetracycline-resistance gene in pUC18 plasmid with reprogrammed Cas9t complexes pUC18 (2 μg) was incubated with two different Cas9t complexes (250 nM each) containing different crRNAs for 1 h at 37 ◦ C in 100 μl of reaction buffer containing 10 mM Tris/HCl (pH 7.5 at 37 ◦ C), 100 mM NaCl, 1 mM DTT and 10 mM MgCl2 . The linear vector fragment was purified from agarose gel using GeneJET gel extraction kit (Thermo Fisher Scientific) and divided into two equal parts. One part of pre-cleaved vector was dephosphorylated with FastAP alkaline phosphatase (Thermo Fisher Scientific), whereas the other part remained untreated. A 1282 bp insert containing promoter and tetracycline-resistance gene was obtained by PCR (primers are listed in Table S7) using the pACYC184 plasmid as a template. After purification with GeneJET PCR purification kit, the PCR fragment was divided into two parts. One part was untreated, whereas the other was phosphorylated with T4 PNK. Untreated vector was ligated with untreated PCR fragment, whereas dephosphorylated
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vector was ligated with phosphorylated fragment using T4 DNA ligase (Thermo Fisher Scientific). After transformation, the recombinant E. coli DH5α clones were selected on a medium supplemented with 100 μg/ml ampicillin and 25 μg/ml tetracycline. Plasmid DNA was purified from resulting transformants and subjected to sequencing.
References 1 Karvelis, T., Gasiunas, G., Miksys, A., Barrangou, R., Horvath, P. and Siksnys, V. (2013) crRNA and tracrRNA guide Cas9-mediated DNA interference in Streptococcus thermophilus. RNA Biol. 10, 841–851 2 Sapranauskas, R., Gasiunas, G., Fremaux, C., Barrangou, R., Horvath, P. and Siksnys, V. (2011) The Streptococcus thermophilus CRISPR/Cas system provides immunity in Escherichia coli. Nucleic Acids Res. 39, 9275–9282 3 Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning: a Laboratory Manual, 2nd edn., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 4 Livak, K.J. and Schmittgen, T.D. (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2 − C T method. Methods 25, 402–408 Received 24 July 2013 doi:10.1042/BST20130164
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