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Animal Biotechnology Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/labt20
Construction of a CRISPR-Cas9 System for Pig Genome Targeting a
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Yu-Hsiu Su , Tai-Yun Lin , Chung-Lin Huang , Ching-Fu Tu & Chin-Kai Chuang a
Division of Animal Technology, Laboratories of Animal Technology, Agricultural Technology Research Institute, Xiangshan District, Hsinchu City, Taiwan Published online: 09 Jul 2015.
Click for updates To cite this article: Yu-Hsiu Su, Tai-Yun Lin, Chung-Lin Huang, Ching-Fu Tu & Chin-Kai Chuang (2015) Construction of a CRISPRCas9 System for Pig Genome Targeting, Animal Biotechnology, 26:4, 279-288 To link to this article: http://dx.doi.org/10.1080/10495398.2015.1027774
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Animal Biotechnology, 26:279–288, 2015 Copyright # Taylor & Francis Group, LLC ISSN: 1049-5398 print=1532-2378 online DOI: 10.1080/10495398.2015.1027774
Construction of a CRISPR-Cas9 System for Pig Genome Targeting Yu-Hsiu Su, Tai-Yun Lin, Chung-Lin Huang, Ching-Fu Tu, and Chin-Kai Chuang? Division of Animal Technology, Laboratories of Animal Technology, Agricultural Technology Research Institute, Xiangshan District, Hsinchu City, Taiwan
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A Cas9/sgRNA RNA-guided endonuclease expression system including a codon-optimized Streptococcus pyogenes A20 Cas9 recombinant protein expression vector and a spacer-guide chimeric RNA expression vector using the porcine U6 promoter was constructed for application in pigs. Only the Flag2-NLS1-Cas9-NLS2 recombinant protein in complex with sgRNA was translocated into the nucleus; the Flag2-NLS1-Cas9-NLS2 protein alone was excluded from the nucleus. Up to 13% of porcine PK1 cells targeted in vitro were observed, regardless of transfection efficiency. Keywords
Cas9; CRISPR; sgRNA
INTRODUCTION Clustered regularly interspaced short palindromic repeats (CRISPRs), which are tandem repeated arrays composed of genome-targeting sequences (spacers) interspaced with identical repeats, and CRISPR-associated (Cas) proteins build an RNA-mediated adaptive immune system against phages or plasmids that are broadly found in prokaryotes (1, 2). CRISPR repeat-spacer arrays are transcribed as a precursor CRISPR RNA (pre-crRNA), followed by enzymatic cleavage to yield a crRNA spacer-repeat unit that is associated with a transactivating crRNA (tracrRNA) via the repeat part and Cas proteins, to form a ribonucleoprotein complex. The target site (protospacer) adapted by the CRISPR on the genome of the invaded phages or plasmids can be recognized and cleaved by this ribonucleoprotein complex. The site-specific cleavage is determined by the base pairing complementarity between the crRNA and the protospacer DNA, as well as a short motif (protospaceradjacent motif, PAM) located on the DNA juxtaposed to the complementary region (3, 4). The CRISPR-Cas systems developed in bacteria and archaea have been classified into three types and further divided into several subtypes according to the specific combinations of Cas genes. In type I and III systems, multi-Cas proteins are involved in the crRNA-tracrRNA-Cas complex; however, only the Cas9 protein is necessary in the type II system (5, 6). Address correspondence to Chin-Kai Chuang, Division of Animal Technology, Laboratories of Animal Technology, Agricultural Technology Research Institute. No. 1, Lane 51, Dahu Rd., Xiangshan District, Hsinchu City 30093, Taiwan. E-mail: [email protected] Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/labt.
The crRNA=tracrRNA heteroduplex can be replaced by a single crRNA-shortened tracrRNA chimeric RNA with Cas9 of Streptococcus pyogenes SF370 to resume site-specific deoxyribonuclease activity (7). A modified single-stranded RNA that mimics the configuration of the crRNA repeat region and tracrRNA portion was reported and termed guide RNA (gRNA). The single-stranded spacer-gRNA chimeric RNA can efficiently replace the original crRNA= tracrRNA heteroduplex (8). Cas9 is a large multidomain protein; for instance, Sp SF370 Cas9 comprises 1368 amino acids. It can be roughly divided into N terminus RuvC, middle HNH, and C-terminus domain. The RuvC domain is involved in the single-stranded DNA nickase activity of Cas9 on the sense strand, and the HNH domain is on the complementary strand to crRNA (4, 9). The X-ray crystal structure of fulllength Sp Cas9 in complex with a 98 nt sg RNA and a 23 nt target DNA was recently resolved. Cas9 is composed of a recognition lobe and a nuclease lobe, which comprises the RuvC and HNH nuclease domains, as well as the PAM-interacting domain (10). As the CRISPR-Cas9 system is found in prokaryotes, its application as an RNA-guided engineered nuclease (RGEN) (11) in eukaryotic cells meets some problems. Codons should be optimized according to the target cells, and a nucleus-localization sequence (NLS) is necessary for the effective transfer of the Cas9 protein into the nucleus. A short RNA expression system that allows the production of a spacer-gRNA chimeric RNA is also critical (8, 12). In this study, we developed a CRISPR-Cas9 system that worked well in mammalian cells, and was especially adaptive in porcine cells. The Streptococcus pyogenes A20 Cas9 with humanized codons was flanked by the SV40 T-antigen NLS
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(PKKKRKVG, NLS1) and the Daxx NLS (KKSRKEKK, NLS2) (13) at the N and C termini, respectively. An overlapped Flag2 tag (EYKDDDGDYKDDDDK) was added at the end of the N terminus. The CMV enhancer-chicken b-actin promoter was used to derive the Flag2-NLS1-Cas9NLS2mRNA, and the porcine U6 promoter (14) was used to transcribe the spacer-gRNA chimeric RNA. MATERIALS AND METHODS
Construction of the pCX-Flag2-NLS1-Cas9-NLS2 Expression Vector The synthesized cDNA composed of humanized codons encoding the Cas9 protein of Streptococcus pyrogenes A20 and the NLS of Daxx (KKSRKEKK)(13) flanked with an MluI site and an Acc65I site at the 50 and the 30 ends, (Appendix 1) respectively, was inserted into the pCX-Flag2-NLS-MCS1(SNX=N) vector, between the MluI and Acc65I sites, to build the pCX-Flag2NLS1-Cas9-NLS2 construct (Fig. 1c).
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Construction of the pCX-Flag2-NLS-MCS1 Expression Vector The oligonucleotide pair encoding the Flag2 tag (EYKDDDGDYKDDDDK) and the SV40T-antigen
NLS (PKKKRKVG) was inserted at the EcoRI site of pCX-MCS1(SNX=N) (Fig. 1a) to construct the pCX-Flag2NLS-MCS1(SNX=N) expression vector (Fig. 1b).
FIG. 1. Linear maps of the pCX-MCS1(SNX=N), pCX-Flag2-NLS-MCS1(SNX=N), and pCX-Flag2-NLS1-Cas9-NLS2 vectors. (a) Linear map of the pCX-MCS1(SNX=N) vector, which was derived from the pCAGGS-NLS-Cre vector. A SalI=NotI=XbaI oligonucleotide was inserted in front of the CMV enhancer, and the HindIII site located at the 30 end of the polyA signal was replaced by a NotI site. A set of restriction enzyme cutting sites (MCS1) was created for cDNA insertion. (b) An oligonucleotide composed of a Kozak sequence, a hybrid Flag tag (Flag2), and the SV40T antigen NLS was inserted into the EcoRI site of pCX-MCS1(SNX=N), to obtain the pCX-Flag2-NLS-MCS1(SNX=N) plasmid. (c) A synthesized Cas9-NLS2 cDNA with a humanized codon was inserted between the MluI and Acc65I sites of pCX-Flag2-NLS-MCS1(SNX=N), to construct pCX-Flag2-NLS1-Cas9-NLS2.
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IF to Confirm Flag2-NLS1-Cas9-NLS2 Expression The pCX-Flag2-NLS1-Cas9-NLS2 was transfected into HeLa cells using Turbofect (#R0531, Fermentas=Thermo) according to the manufacturer’s instructions. Two days after transfection, the cells were fixed in 4% paraformaldehyde for 10 min at room temperature. The M2 monoclonal antibody (Cat. # F1804, Sigma) (1:50) against the Flag tag was used as the primary antibody, and DyLight488conjugated AffiniPure donkey anti-mouse IgG (715-485151, Jackson Lab.) (1:100) was used as the secondary antibody to perform IF.
Construction of the ppU6-BsaI2-gRNA Vector A DNA fragment composed of the pig U6 promoter (14), a pair of type II restriction enzyme BsaI sites, and the CRISPR spacer-guide-RNA flanked by a BamHI site at the 50 end, as well as by BglII and XbaI sites at the 30 end, was synthesized as shown in Appendix 2 and inserted into pUC57, in which a unique BsaI site had been removed by site-directed mutagenesis, to obtain the ppU6-BsaI2gRNA plasmid (Fig. 2a).
Genomic DNA Target Sites and Preparation of the ppU6-Target-gRNA Vector The target sites of this CRISPR-Cas9 system should follow the GN19NGG rule (Fig. 2b). It is suggested that there is no site with less than three mismatches that prevents off-targeting. When the target site is determined, a pair of oligonucleotides ought to be synthesized and annealed before ligation with BsaI-treated ppU6-BsaI2-gRNA vector, to prepare the ppU6-Target-gRNA vector (Fig. 2c).
Pig GGTA1 Gene Targeting The 371 amino acid coding region of the pig GGTA1 gene (NCBI access number, NC_010443) comprises six exons, the last of which covers most of the protein (amino acids 141 to 371), including the active domain with a-1,3-galactosyl transferase activity. The GGTA1 gene region, including parts of the last intron and last exon, used in this study is shown in Appendix 2. A primer pair, CAGCAACAGACGTCTCTCATC (GGTA1F) and CTCTCGTAGG TGAACTCGTCA (GGTA1 R), via a 511 bp DNA fragment, which can be specifically amplified by PCR, was selected for the subsequent analysis. Four target sites were picked up and the oligonucleotide pairs synthesized for these sites are shown in Tables 1 and 2, respectively. After an annealing reaction, the four oligonucleotide pairs were ligated with BsaI-treated ppU6-BsaI2-gRNA vector to prepare ppU6-GGTA1(38–60)-gRNA, ppU6GGTA1 (187–209)-gRNA, ppU6-GGTA1 (181–159) -gRNA, and ppU6-GGTA1(223–201)-gRNA.
FIG. 2.
The Spacer-gRNA expression vector. (a) Linear map of ppU6-(BsaI)2-gRNA, which was constructed using a pUC57 vehicle from which a BsaI site located in the Amp region had been removed. (b) The candidate target sites on genomic DNA were screened by the GN19NGG [GN19 spacer þNGG protospacer adjacent spacer (PAM)] rule. (c) The procedures used to construct ppU6-Spacer-gRNA are shown. The annealed primer pair was inserted between the two BsaI sites of the ppU6-(BsaI)2gRNA vector.
Transfection of the pCX-Flag2-NLS1-Cas9-NLS2 and ppU6-GGTA1-gRNA Vectors into PK1 Cells All four ppU6-GGTA1(38–60)-gRNA, ppU6-GGTA1 (187–209)-gRNA, ppU6-GGTA1(181–159)-gRNA, and ppU6GGTA1(223–201)-gRNA plasmids (2 mg of each), and 2 mg of pCX-Flag2-NLS1-Cas9-NLS2 were mixed with 1 106 pig PK1 cells in a 100 mL tip connected to a Micro-Porator (MP-100, Digital Bio.) and treated with a single pulse at
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TABLE 1 Target sites picked up for pig GGTA1 gene editing
Reverse 159–181 201–223
CCGAGAAGAGGTGGCAAGACATC CCATCGGGGAGCACATCCTGGCC
Forward (sense) 38–60 GCAAATACATACTTCATGGTTGG 187–209 GATGCGCATGAAGACCATCGGGG Forward (antisense) 181–159 GATGTCTTGCCACCTCTTCTCGG 223–201 GGCCAGGATGTGCTCCCCGATGG
TABLE 2 Oligonucleotide pairs synthesized for pig GGTA1 gene editing
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Oligonucleotide GGTA1 F 38–60 GGTA1 R 38–60 GGTA1 F 187–209 GGTA1 R 187–209 GGTA1 F 181–159 GGTA1 R 181–159 GGTA1 F 223–201 GGTA1 R 223–201
Sequence CGTC GCAAATACATACTTCATGGT GTTTTAGAGCTAGAAAT TGCTATTTCTAGCTCTAAAAC ACCATGAAGTATGTATTTGC CGTCGATGCGCATGAAGACCATCG GTTTTAGAGCTAGAAAT TGCTATTTCTAGCTCTAAAAC CGATGGTCTTCATGCGCATC CGTC GATGTCTTGCCACCTCTTCT GTTTTAGAGCTAGAAAT TGCTATTTCTAGCTCTAAAAC AGAAGAGGTGGCAAGACATC CGTC GGCCAGGATGTGCTCCCCGA GTTTTAGAGCTAGAAAT TGCTATTTCTAGCTCTAAAAC TCGGGGAGCACATCCTGGCC
1300 V for 30 ms. To obtain significant results, three rounds of transfection were performed every third day. To test the individual GGTA1-gRNA activity, 5 mg each of ppU6-GGTA1(38–60)-gRNA, ppU6-GGTA1(187–209)gRNA, ppU6-GGTA1(181–159)-gRNA, or ppU6-GGTA1 (223–201)-gRNA were mixed with 5 mg of pCX-Flag2-NLS1Cas9-NLS2 before transfection into PK1 cells via a single round of microporation, as previously described.
1 mL of PBS supplemented with 1 mg of isolectin-B4-FITC (Cat. # L2895, Sigma), and incubated on ice for 1 h. Cells were washed with PBS as previously described and suspended in PBS for FACS analysis. Normal PK1 cells served as controls. Data were processed using the WinMDI software.
Analysis of the Cas9/GGTA1-gRNA Activities by DNA Sequencing Three days after the last transfection, genomic DNA samples were isolated using a genomic DNA purification kit (#K0721, Fermentas=Thermo). A DNA fragment including the GGTA1 restriction sites was amplified by PCR using 0.1 mg of genomic DNA, 0.25 mM each of GGTA1 F and GGTA1 R primers, and 5 U of Taq DNA polymerase in a 50 mL reaction with a program of 95 C for 3 min, 35 cycles of 94 C for 30 s=60 C for 40 s=72 C for 30 s, and 72 C for 3 min, followed by soaking at 4 C. The DNA PCR products were purified using a PCR clean-up kit (Cat. # DF300, Geneaid) and subcloned into the pGEM-T Easy vector (Cat. # A1360, Promega). The DNA sequences of the plasmids that were isolated from individual colonies were determined using the T7 primer on an ABI 3730 sequencer. Flow Cytometry Analysis Transfected PK1 cells were trypsinized and fixed in 70% alcohol overnight. The fixed cells were washed with PBS by centrifugation and suspension twice, and then suspended in
FIG. 3.
Localization of the Flag2-NLS1-Cas9-NLS2 recombinant protein, which was overexpressed in HeLa cells. Either Flag2-NLS1-Cas9NLS2 (a) or the Flag2-NLS1-Cas9-NLS2=GGTA1(38–60)-gRNA complex (b) was overexpressed in HeLa cells. The Cas9 protein component was detected via the Flag tag using the M2 monoclonal antibody.
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RESULTS Construction of the pCX-Flag2-NLS1-Cas9-NLS2/ PpU6-(BsaI)2-gRNA System The expression vector pCX-MCS1(SNX=N) (Fig. 1a) used in this study was modified from the backbone of the pCAGGS-NLS-Cre vector (which was a kind gift from Andras Nagy, Mount Sinai Hospital, Toronto). A SalI= NotI=XbaI oligonucleotide was inserted in front of the CMV enhancer, and the HindIII site at the 30 end of the polyA signal was replaced by a NotI site. A set of restriction enzyme cutting sites (MCS1) was used to replace the NLSCre coding region. An oligonucleotide composed of a Kozak sequence, hybrid Flag tag (Flag2), and the SV40 T antigen NLS was inserted into the EcoRI site of pCXMCS1(SNX=N), to obtain the pCX-Flag2-NLS-MCS1(SNX=N) plasmid (Fig. 1b). A synthesized Cas9-NLS2 cDNA with humanized codons was then inserted between MluI and Acc65I site of pCX-Flag2-NLS-MCS1(SNX=N) to build pCXFlag2-NLS1-Cas9-NLS2 (Fig. 1c). The DNA sequence of the entire coding region is shown in Appendix 3. The DNA composed of the pig U6 promoter, BsaI=NotI=MluI=XhoI=BsaI
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restriction enzyme cutting sites, and gRNA was synthesized (see Appendix 2) and inserted between the BamHI and XbaI sites of a modified pUC57 vehicle in which the BsaI site, which was within the Amp region, had been removed to prepare ppU6-(BsaI)2-gRNA vector (Fig. 2a). The candidate target sites, which comprise a GN19 protospacer and an NGG protospacer-adjacent motif (PAM) on genomic DNA were screened according to the GN19NGG rule. The primer pair encoding the spacer and the 50 end of gRNA were designed as shown in Fig. 2b, and annealed before ligation with BsaI-treated ppU6-Spacer-gRNA vector, to build the ppU6-Spacer-gRNA (Fig. 2c). Expression of the Flag2-NLS1-Cas9-NLS2 Recombinant Protein in HeLa Cells After pCX-Flag2-NLS1-Cas9-NLS2 DNA transfection, the expression and localization of the Flag2-NLS1-Cas9NLS2 recombinant protein was performed by immunofluorescence assay using the Flag tag-specific M2 mAb as the primary antibody and DyLight488-conjugated AffiniPure donkey anti-mouse IgG as the secondary antibody. As
FIG. 4. Targeting the pig GGTA1 gene in PK1 cells. (a) Four protospacers within exon 6 of the pig GGTA1 gene were selected. (b) After three successive transfections, this locus was amplified by PCR using the GGTA1 F and GGTA1 R primer pair. Two bands of DNA were observed (left panel) and subcloned into the pGEM-T Easy vector. Twenty clones were picked up, 19 of which contained inserts of the expected size (right panel). (c) The DNA sequences of the 19 inserts were aligned with the standard one. Predicted insertion and deletion sites are indicated by the ^ and – symbols, respectively. The protospacer þ PAM sequences are indicated by blue arrows, and the expected cutting sites are pointed out by red arrow heads.
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FIG. 4. Continued. shown in Fig. 3a, the overexpressed flag-tagged recombinant protein was located around the nuclei, rather than within them. We speculated whether the Cas9 protein assembles with the crRNA=tracrRNA complex or spacer-gRNA to adopt a conformation that is able to be transported into the nucleus. A GGTA1(38–60) spacer-gRNA was used to test this assumption. After cotransfection of pCX-Flag2-NLS1Cas9-NLS2 and ppU6-GGTA1(38–60)-gRNA, the amount of the Cas9 component, as monitored using the M2 monoclonal antibody, was significantly increased in nuclei, even though signals were still detected mainly in the cytoplasm (Fig. 3b). Targeting the Pig GGTA1 Gene with the Flag2-NLS1Cas9-NLS2/GGTA1-gRNA Complex Four ppU6-GGTA1-gRNA plasmids, ppU6-GGTA1(38–60)gRNA, ppU6-GGTA1(187–209)-gRNA, ppU6-GGTA1(181–159)gRNA, and ppU6-GGTA1(223–201)-gRNA, were prepared to target the protospacers on pig GGTA1 exon 6 (Fig. 4a). These four plasmids were cotransfected with pCX-Flag2NLS1-Cas9-NLS2 into pig PK1 cells, as described in the
Materials and Methods section. Three days after the last transfection, genomic DNA was isolated and GGTA1 PCR was performed. Two bands of DNA were observed (Fig. 4b, left panel) and were subcloned into the pGEM-T Easy vector. Twenty clones were picked up, 19 of which contained inserts of the expected size (Fig. 4b, right panel). The DNA sequences of the 19 inserts were aligned with the standard one. Three of the 19 clones were the same as the standard. The remaining 16 clones contained insertions or deletions. Site 54 (GGTA1(38–60)) was involved in 13 mutant clones, site 164 (GGTA1(181–159)) was involved in four mutant clones, site 203 (GGTA1(187–209)) was involved in four mutant clones, and site 206 (GGTA1(223–201) was involved in 10 mutant clones (Fig. 4c). Expression Levels of the a-1,3-Gal Epitope Were Reduced upon GGTA-1 Gene Knockout by the Flag2NLS1-Cas9-NLS2/GGTA1-gRNA Complex The expression levels of the a-1,3-Gal epitope on pig PK1 cells were detected by FITC-conjugated isolectin-B4. Compared with the control, in which 91.65% of cells exhibited
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high FITC fluorescence signals, the ratio of cells with a high level of expression of the a-1,3-Gal epitope was reduced to 75.29%, 89.19%, 81.71%, and 80.40% after cotransfection with pCX-Flag2-NLS1-Cas9-NLS2=ppU6-GGTA1(38–60)gRNA, pCX-Flag2-NLS1-Cas9-NLS2=ppU6-GGTA1 pCX-Flag2-NLS1-Cas9-NLS2=ppU6(187–209)-gRNA, GGTA1(181–159)-gRNA, and pCX-Flag2-NLS1-Cas9-NLS2= ppU6-GGTA1(223–201)-gRNA plasmid pair, respectively.
FIG. 5. Flow cytometry analysis of the expression level of the a-1,3-Gal epitope. The expression levels of the a-1,3-Gal epitope on pig PK1 cells were detected by FITC-conjugated isolectine-B4. Untreated PK1 cells were used as a control. The levels of expression of the a-1,3-Gal epitope in PK1 cells cotransfected with pCX-Flag2-NLS1-Cas9-NLS2=ppU6-GGTA1(38–60)gRNA, pCX-Flag 2-NLS1 -Cas9-NLS 2=ppU6-GGTA1(187–209)-gRNA, pCX-Flag2-NLS1-Cas9-NLS2=ppU6-GGTA1(181–159)-gRNA, and pCXFlag2-NLS1-Cas9-NLS2=ppU6-GGTA1(233-201)-gRNA plasmid pairs, respectively, were analyzed using a flow cytometer. The ratios of cells with high and low levels of fluorescence signals were determined.
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Moreover, a group of cells that expressed a lower level of the a-1,3-Gal epitope corresponding to the quantities of reduction of the high-expression population was identified (Fig. 5).
DISCUSSION The gene encoding 1368 amino acids of Cas9 used in this study was extracted from the genome of Streptococcus pyrogenes strain A20, which was isolated in south Taiwan. The amino acid sequence of Sp A20 Cas9 is nearly the same as that of Sp SF370 Cas9, with only one difference: the 24th amino acid is Asp instead of Glu. Therefore, it is reasonable to think that the strategy of spacer-gRNA (8, 15) can be applied to Sp A20 Cas9. To build a spacer-gRNA expression vector for mammalian cells, the porcine U6 promoter, which has been proven to work more effectively than the human promoter in porcine cells (14), was used to transcribe the chimeric RNA. Three prototypes of the ppU6-BsaI2-gRNA vector were tested, each of them adapted with a 24 nt oligonucleotide pair (20 nt for the spacer þ 4 nt of gRNA), a 32 nt oligonucleotide pair (20 nt for the spacer þ 12 nt of gRNA), or a 41 nt oligonucleotide pair (20 nt for the spacer þ 21 nt of gRNA) insert (Fig. 2c). It was found that only the 41 nt oligonucleotide pair could be effectively ligated to the BsaI-treated vector using the 1 h quick ligation protocol (data not shown). Therefore, the third prototype was chosen for further experiments. When HeLa cells were transfected with pCX-Flag2NLS1-Cas9-NLS2 alone, the Flag2-NLS1-Cas9-NLS2 recombinant protein was detected unsymmetrically outside the boundary of nuclei (Fig. 3a). pCX-HA-NLS1-Cas9NLS2 and pCX-Myc-NLS1-Cas9-NLS2 were also generated, and the HA-NLS1-Cas9-NLS2 and Myc-NLS1-Cas9NLS2 recombinant proteins were located at the same place as Flag2-NLS1-Cas9-NLS2 (data not shown). However, when cells were cotransfected with pCX-Flag2-NLS1Cas9-NLS2 and ppU6-spacer-gRNA, a significant amount of the Flag2-NLS1-Cas9-NLS2 recombinant protein was detected by M2 monoclonal antibody staining in the nuclei, even though most of the signals were mainly detected in the cytoplasm (Fig. 3b). These results indicate that only the Flag2-NLS1-Cas9-NLS2=sgRNA complex could be translocated into the nucleus. Two recent papers (10, 15) reported the crystal structure of Cas9 and the Cas9=sgRNA=target DNA triple complex, respectively. The architectures of Cas9 enzymes define nucleic acid binding clefts, and single-particle electron microscopy reconstructions showed that the two structural lobes that harbor these clefts undergo guide RNA-induced reorientation to form a central channel, to which DNA substrates bind. The observation that extensive structural rearrangements occurred before target DNA duplex binding implicates that guide RNA loading is a key step in Cas9 activation (7).
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To analyze the RNA-guided endonuclease activity of our Cas9=sgRNA system, the pig GGTA1 gene was used as a target, because the enzymatic activity of its product, a-1, 3-galactotransferase, can simply monitor the amount of the a-1,3-Gal epitope on the cell surface by isolectin-B4. Four target sites on pig GGTA1 exon 6 (Fig. 4a) were selected, and two strategies were used. First, all of the four GGTA1 sg-RNA expression vectors were cotransfected with pCX-Flag2-NLS1-Cas9-NLS2 for three rounds, to increase the populations of transfected pig PK1 cells. Three days after the last transfection, genomic DNA was isolated and GGTA1 PCR was performed. The PCR-amplified DNA fragments were subcloned into the pGEM-T Easy vector. The DNA sequences of the 19 clones were aligned with the standard one. Sixteen of the 19 clones contained insertions or deletions. Site 54 (GGTA1(38–60)) was involved in 13 mutant clones, site 164 (GGTA1(181–159)) was involved in four mutant clones, site 203 (GGTA1(187–209)) was involved in four mutant clones, and site 206 (GGTA1(223–201) was involved in 10 mutant clones (Fig. 4c). The relative efficiencies of the four sites, sites 54, 164, 203, and 206, were roughly estimated as 13:4:4:10. In the second strategy, individual GGTA1 sg-RNA expression vectors were cotransfected with pCXFlag2-NLS1-Cas9-NLS2 into pig PK1 cells. The expression levels of the a-1,3-Gal epitope at the cell surface were monitored using a flow cytometer and FITC-conjugated isolectinB4. A group of cells expressed a lower level of the a-1,3-Gal epitope, corresponding to the quantities of reduction of the high expression population (Fig. 5). The reduction percentages of the four cases were 13.4% (24.12%-7.72%), 2.86% (10.58%-7.72%), 10.26% (17.98%-7.72%), and 11.39% (19.11%-7.72%) for site 54 (GGTA1(38–60)), site 164 (GGTA1(181–159)), site 203 (GGTA1(187–209)), and site 206 (GGTA1(223–201), respectively. Even though the efficiency of transfection was not considered, the targeting efficiencies observed in both strategies were quite high. FUNDING This work was supported by grants NSC 99-2313-B-059004-MY3 and MOST 102-2320-B-059-001-MY3 from the Ministry of Science and Technology of Taiwan to C. K. Chuang. REFERENCES 1. Horvath P, Barrangou R. CRISPR=Cas, the immune system of bacteria and archaea. Science 2010; 327:167–170.
2. Terns MP, Terns RM. CRISPR-based adaptive immune system. Curr Opin Microbiol 2011; 14:321–327. 3. Mojica FJ, Dı´ez-Villasen˜or C, Garcı´a-Martı´nez J, Almendros C. Short motif sequences determine the targets of the prokaryotic CRISPR defence system. Microbiol 2009; 155: 733–740. 4. Gasiunas G, Barrangou R, Horvath P, Siksnys V. Cas9crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria. Proc Natl Acad Sci USA 2012; 109:E2579–E2586. 5. Bhava D, Davison M, Barrangou R. CRISPR-Cas systems in bacteria and archaea: versatile small RNAs for adaptive defense and regulation. Annu Rev Genet 2011; 45:273–297. 6. Chylinski K, Le Rhun A, Charpentier E. The tracrRNA and Cas9 families of type II CRISPR-Cas immunity systems. RNA Biol 2013; 10:726–737. 7. Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 2012; 337:816–821. 8. Mali P, Yang L, Esvelt KM, Aach J, Guell M, DiCarlo JE, Norville JE, Church GM. RNA-guided human genome engineering via Cas9. Science 2013; 339:823–826. 9. Ran FA, Hsu PD, Lin CY, Gootenberg JS, Konermann S, Trevino AE, Scott DA, Inoue A, Matoba S, Zhang Y, Zhang F. Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity. Cell 2013; 154: 1380–1389. 10. Nishimasu H, Ran FA, Hsu PD, Konermann S, Shehata SI, Dohmae N, Ishitani R, Zhang F, Nureki O. Crystal structure of Cas9 in complex with guide RNA and target DNA. Cell 2014; 156:935–949. 11. Bae S, Park J, Kim JS. Cas-OFFinder: A fast and versatile algorithm that searches for potential off-target sites of Cas9 RNA-guided endonucleases. Bioinformatics 2014; 30:1473– 1475. 12. Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu X, Jiang W, Marraffini LA, Zhang F. Multiplex genome engineering using CRISPR=Cas systems. Science 2013; 339:819–923. 13. Yeung PL, Chen LY, Tsai SC, Zhang A, Chen JD. Daxx contains two nuclear localization signals and interacts with importin alpha3. J Cell Biochem 2008; 103:456–70. 14. Chuang CK, Lee KH, Fan CT, Su YS. Porcine type III RNA polymerase III promoters for short hairpin RNA expression. Anim Biotechnol 2009; 20:34–39. 15. Jinek M, Jiang F, Taylor DW, Sternberg SH, Kaya E, Ma E, Anders C, Hauer M, Zhou K, Lin S, Kaplan M, Iavarone AT, Charpentier E, Nogales E, Doudna JA. Structures of Cas9 endonucleases reveal RNA-mediated conformational activation. Science 2014; 343:1247997.
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APPENDIX 1. DNA SEQUENCE OF FLAG2-NLS1-CAS9-NLS2
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Flag2-NLS1-Cas9-NLS2 ATGGACTACA AAGAAACGCA CTGGACATTG AGCAAGAAGT GGAGCCCTGC CGGAGAAGGT GAAATGGCCA GAAGATAAGA CACGAAAAGT GCAGACCTGC CTGATCGAGG GTCCAGACAT AAAGCCATCC CTGCCAGGGG ACACCCAACT GACACTTATG CTGTTCCTGG AACACCGAGA CATCAGGATC GAAATCTTCT CAGGAGGAGT CTGCTGGTGA AGCATCCCTC TTCTACCCAT CCCTACTATG TCTGAGGAAA CAGTCTTTCA CCCAAGCACA TACGTCACTG GTGGACCTGC TTCAAGAAAA GCCAGCCTGG AACGAGGAAA AGGGAAATGA AAACAGCTGA GGGATTCGCG GCCAACCGGA CAGAAGGCAC GGCTCTCCTG AAAGTCATGG ACCACACAGA AAGGAACTGG GAGAAGCTGT GATATTAACC GATGACAGCA AATGTCCCCT GCCAAACTGA AGCGAACTGG AAGCACGTCG CTGATCAGAG
AAGATGACGA AAGTGGGTGA GAACAAACTC TCAAGGTGCT TGTTCGACTC ATACTCGCCG AGGTGGACGA AACACGAGCG ACCCTACTAT GCCTGATCTA GGGATCTGAA ACAATCAGCT TGAGTGCCCG AAAAGAAAAA TCAAGAGCAA ACGATGACCT CCGCTAAGAA TTACAAAAGC TGACCCTGCT TTGATCAGTC TCTACAAGTT AACTGAATAG ACCAGATTCA TTCTGAAGGA TGGGACCTCT CAATCACTCC TTGAGCGGAT GCCTGCTGTA AGGGGATGAG TGTTTAAAAC TTGAATGTTT GGACCTACCA ATGAGGATAT TCGAGGAACG AGCGACGGAG ACAAACAGAG ACTTCATGCA AGGTGTCCGG CCATCAAGAA GAAGACATAA AAGGCCAGAA GCTCTCAGAT ATCTGTACTA GACTGAGTGA TTGACAATAA CAGAGGAAGT TCACCCAGCG ACAAAGCAGG CCCAGATTCT AAGTGAAGGT
TGGTGATTAT ATTCCTCGAG TGTGGGCTGG GGGCAACACC CGGCGAGACA AAAGAATAGG TAGTTTCTTT GCATCCCATC CTATCATCTG TCTGGCCCTG CCCAGACAAT GTTTGAGGAA GCTGTCTAAG CGGCCTGTTT TTTTGATCTG GGATAACCTG TCTGTCTGAC CCCCCTGTCA GAAGGCTCTG TAAGAACGGA TATCAAACCC GGAAGACCTG TCTGGGAGAG TAACAGGGAG GGCCCGGGGC CTGGAACTTC GACAAACTTC CGAGTATTTC AAAGCCTGCC CAATAGGAAG CGATTCTGTG CGATCTGCTG CCTGGAAGAC CCTGAAGACT ATACACCGGA TGGAAAGACT GCTGATTCAC GCAGGGAGAC AGGGATTCTG GCCAGAAAAC GAACTCCAGG CCTGAAAGAG TCTGCAGAAT TTATGACGTG GGTGCTGACC GGTCAAGAAA AAAGTTTGAT CTTCATTAAG GGATTCCAGA CATTACTCTG
AAAGACGATG ACGCGTATGG GCTGTGATTA GATAGACACA GCTGAAGCAA ATCTGCTACC CATCGCCTGG TTTGGCAACA AGGAAGAAAC GCTCACATGA AGCGATGTGG AACCCCATTA AGTCGGAGAC GGGAATCTGA GCCGAGGACG CTGGCTCAGA GCCATCCTGC GCTAGCATGA GTGAGGCAGC TACGCCGGCT ATTCTGGAGA CTGAGGAAGC CTGCACGCAA AAGATCGAAA AACAGCCGGT GAGGAAGTGG GACAAGAACC ACCGTCTATA TTCCTGAGCG GTGACAGTCA GAGATCAGTG AAGATCATTA ATTGTGCTGA TATGCCCATC TGGGGCCGAC ATCCTGGACT GATGACTCCC TCTCTGCACG CAGACCGTGA ATCGTGATTG GAGCGCATGA CACCCCGTGG GGGCGCGATA GATCATATCG CGCTCAGACA ATGAAGAACT AACCTGACAA CGACAGCTGG ATGAACACAA AAGTCAAAAC
ACGATAAGAA ATAAAAAGTA CCGACGATTA GCATCAAGAA CTCGGCTGAA TGCAGGAGAT AGGAATCATT TTGTGGACGA TGGTGGACAG TTAAGTTCCG ACAAGCTGTT ATGCATCTGG TGGAGAACCT TTGCACTGTC CTAAACTGCA TCGGGGATCA TGAGTGATAT TCAAGAGATA AGCTGCCTGA ATATTGACGG AGATGGACGG AGCGCACATT TCCTGAGGCG AAATTCTGAC TTGCATGGAT TCGATAAGGG TGCCAAACGA ACGAACTGAC GAGAACAGAA AGCAGCTGAA GCGTCGAAGA AGGATAAAGA CCCTGACACT TGTTCGATGA TGTCTCGGAA TTCTGAAATC TGACCTTCAA AGCATATCGC AGGTGGTCGA AGATGGCCAG AACGCATCGA AAAACACTCA TGTACGTGGA TCCCACAGTC AAAACCGAGG ACTGGCGGCA AAGCTGAGCG TGGAGACCCG AGTACGATGA TGGTGAGCGA
TTTGCCGAAG TAGTATTGGG CAAGGTGCCT AAATCTGATT AAGAACAGCT TTTCAGCAAC CCTGGTCGAG GGTCGCTTAT CACCGATAAA GGGCCATTTT CATCCAGCTG CGTGGACGCA GATCGCTCAG ACTGGGACTG GCTGAGCAAG GTACGCAGAC TCTGAGAGTG TGACGAGCAC GAAGTACAAG CGGGGCTAGT CACCGAGGAA TGATAACGGA CCAGGAAGAC CTTCCGCATT GACACGCAAA CGCTTCCGCA AAAAGTGCTG AAAGGTGAAA GAAAGCTATC AGAGGACTAT CCGGTTCAAC CTTCCTGGAC GTTTGAGGAT CAAAGTGATG GCTGATCAAT AGATGGCTTC AGAGGATATC AAACCTGGCC CGAGCTGGTG GGAAAATCAG GGAAGGAATT GCTGCAGAAT CCAGGAGCTG ATTCCTGAAA AAAGAGTGAT GCTGCTGAAT GGGAGGCCTG GCAGATCACA GAATGACAAA CTTTCGGAAA
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288 GATTTCCAGT CTGAACGCAG GTGTACGGGG ATTGGCAAAG GAGATCACCC ACCGGAGAAA ATGCCCCAAG TCAATTCTGC AAGAAGTATG GTGGAGAAGG ATGGAGAGGA GAAGTGAAGA GGGAGAAAGA CCCAGTAAGT CCTGAGGATA ATCATTGAAC AAGGTCCTGT ATCATTCATC ACTACCATCG CACCAGAGCA GGGGGAAGCG
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TTTATAAGGT TGGTCGGCAC ACTATAAGGT CCACCGCTAA TGGCAAATGG TCGTGTGGGA TGAATATTGT CTAAACGCAA GCGGGTTCGA GAAAAAGCAA GCTCCTTCGA AAGACCTGAT GGATGCTGGC ACGTGAACTT ACGAACAGAA AGATTAGCGA CTGCATACAA TGTTCACTCT ATCGCAAACG TTACTGGCCT GCGGGAAGAA
CAGAGAGATC CGCCCTGATT GTACGATGTC GTATTTCTTT GGAAATCCGA CAAAGGAAGA CAAGAAAACA CAGTGATAAG CTCCCCAACA GAAACTGAAA AAAGAATCCT CATCAAGCTG ATCTGCCGGG CCTGTATCTG ACAGCTGTTT GTTCTCCAAG CAAACACCGG GACCAACCTG ATACACATCA GTACGAGACC GAGCAGGAAG
AACAACTACC AAGAAATACC AGAAAAATGA TACAGCAACA AAGCGGCCAC GATTTTGCTA GAGGTGCAGA CTGATCGCCC GTGGCTTACT TCCGTCAAGG ATCGATTTTC CCAAAGTACT GAGCTGCAGA GCTAGCCACT GTGGAGCAGC AGAGTGATCC GACAAGCCAA GGAGCCCCCG ACTAAGGAGG CGGATCGACC GAGAAAAAAC
ACCATGCTCA CTAAACTGGA TCGCCAAGTC TCATGAATTT TGATTGAGAC CCGTGAGGAA CTGGGGGATT GAAAGAAAGA CTGTCCTGGT AACTGCTGGG TGGAGGCCAA CCCTGTTTGA AAGGAAATGA ACGAGAAGCT ACAAGCATTA TGGCTGACGC TCAGAGAGCA CAGCCTTCAA TGCTGGATGC TGAGTCAGCT TCGAGTGAGG
TGACGCATAC GTCCGAGTTC AGAGCAGGAA CTTTAAGACT TAACGGGGAG GGTCCTGTCT CAGTAAGGAA CTGGGACCCC GGTCGCAAAG CATCACTATT AGGCTATAAG GCTGGAAAAC ACTGGCCCTG GAAAGGCTCC TCTGGACGAG AAATCTGGAT GGCCGAAAAT GTATTTTGAC TACCCTGATC GGGGGGGGAC TACC
GCAGACCCGC AGTAAGGCGC ATATAATATG TATGATGACA AAAATTACTC CCCTCTGAGA AGTTAAAATA TT
AATAGTGAGA GCGGAGGTCG TAGGAAGGTT GTATGTGTGA TTCTATAGTC AGACCCAGCC AGGCTAGTCC
GAAGCGCTAG GGCGGGCAGA AAAGAGACAA CATAGAAGGT ACCATAATCA GTCGGAGACC GTTATCAACT
APPENDIX 2. DNA SEQUENCE OF PU6-BSAI2-GRNA pU6-BsaI2-gRNA GGATCC AGGAGGACTC GGGAACGAGC GGGCCAATTT TTCTACTTAA CATAAATCCT AAAAAGTTTT GCGGCCGCAC TGAAAAAGTG AGATCT
CAGGGACAAC ACCACGTGAC CCCATGGTCC CTCTCCCAAA CGAATACTTT TATGTTCTTG GCGTCTCGAG GCACCGAGTC TCTAGA
CGCTGGGCTC CGAGCGTGAC CTTCATTTGC ATCACAAAGA TAAATTGAAA GCTTTATATA GTCTCTAGCA GGTGCTTTTT
Animal Biotechnology Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/labt20
Construction of a CRISPR-Cas9 System for Pig Genome Targeting a
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Yu-Hsiu Su , Tai-Yun Lin , Chung-Lin Huang , Ching-Fu Tu & Chin-Kai Chuang a
Division of Animal Technology, Laboratories of Animal Technology, Agricultural Technology Research Institute, Xiangshan District, Hsinchu City, Taiwan Published online: 09 Jul 2015.
Click for updates To cite this article: Yu-Hsiu Su, Tai-Yun Lin, Chung-Lin Huang, Ching-Fu Tu & Chin-Kai Chuang (2015) Construction of a CRISPRCas9 System for Pig Genome Targeting, Animal Biotechnology, 26:4, 279-288 To link to this article: http://dx.doi.org/10.1080/10495398.2015.1027774
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Animal Biotechnology, 26:279–288, 2015 Copyright # Taylor & Francis Group, LLC ISSN: 1049-5398 print=1532-2378 online DOI: 10.1080/10495398.2015.1027774
Construction of a CRISPR-Cas9 System for Pig Genome Targeting Yu-Hsiu Su, Tai-Yun Lin, Chung-Lin Huang, Ching-Fu Tu, and Chin-Kai Chuang? Division of Animal Technology, Laboratories of Animal Technology, Agricultural Technology Research Institute, Xiangshan District, Hsinchu City, Taiwan
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A Cas9/sgRNA RNA-guided endonuclease expression system including a codon-optimized Streptococcus pyogenes A20 Cas9 recombinant protein expression vector and a spacer-guide chimeric RNA expression vector using the porcine U6 promoter was constructed for application in pigs. Only the Flag2-NLS1-Cas9-NLS2 recombinant protein in complex with sgRNA was translocated into the nucleus; the Flag2-NLS1-Cas9-NLS2 protein alone was excluded from the nucleus. Up to 13% of porcine PK1 cells targeted in vitro were observed, regardless of transfection efficiency. Keywords
Cas9; CRISPR; sgRNA
INTRODUCTION Clustered regularly interspaced short palindromic repeats (CRISPRs), which are tandem repeated arrays composed of genome-targeting sequences (spacers) interspaced with identical repeats, and CRISPR-associated (Cas) proteins build an RNA-mediated adaptive immune system against phages or plasmids that are broadly found in prokaryotes (1, 2). CRISPR repeat-spacer arrays are transcribed as a precursor CRISPR RNA (pre-crRNA), followed by enzymatic cleavage to yield a crRNA spacer-repeat unit that is associated with a transactivating crRNA (tracrRNA) via the repeat part and Cas proteins, to form a ribonucleoprotein complex. The target site (protospacer) adapted by the CRISPR on the genome of the invaded phages or plasmids can be recognized and cleaved by this ribonucleoprotein complex. The site-specific cleavage is determined by the base pairing complementarity between the crRNA and the protospacer DNA, as well as a short motif (protospaceradjacent motif, PAM) located on the DNA juxtaposed to the complementary region (3, 4). The CRISPR-Cas systems developed in bacteria and archaea have been classified into three types and further divided into several subtypes according to the specific combinations of Cas genes. In type I and III systems, multi-Cas proteins are involved in the crRNA-tracrRNA-Cas complex; however, only the Cas9 protein is necessary in the type II system (5, 6). Address correspondence to Chin-Kai Chuang, Division of Animal Technology, Laboratories of Animal Technology, Agricultural Technology Research Institute. No. 1, Lane 51, Dahu Rd., Xiangshan District, Hsinchu City 30093, Taiwan. E-mail: [email protected] Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/labt.
The crRNA=tracrRNA heteroduplex can be replaced by a single crRNA-shortened tracrRNA chimeric RNA with Cas9 of Streptococcus pyogenes SF370 to resume site-specific deoxyribonuclease activity (7). A modified single-stranded RNA that mimics the configuration of the crRNA repeat region and tracrRNA portion was reported and termed guide RNA (gRNA). The single-stranded spacer-gRNA chimeric RNA can efficiently replace the original crRNA= tracrRNA heteroduplex (8). Cas9 is a large multidomain protein; for instance, Sp SF370 Cas9 comprises 1368 amino acids. It can be roughly divided into N terminus RuvC, middle HNH, and C-terminus domain. The RuvC domain is involved in the single-stranded DNA nickase activity of Cas9 on the sense strand, and the HNH domain is on the complementary strand to crRNA (4, 9). The X-ray crystal structure of fulllength Sp Cas9 in complex with a 98 nt sg RNA and a 23 nt target DNA was recently resolved. Cas9 is composed of a recognition lobe and a nuclease lobe, which comprises the RuvC and HNH nuclease domains, as well as the PAM-interacting domain (10). As the CRISPR-Cas9 system is found in prokaryotes, its application as an RNA-guided engineered nuclease (RGEN) (11) in eukaryotic cells meets some problems. Codons should be optimized according to the target cells, and a nucleus-localization sequence (NLS) is necessary for the effective transfer of the Cas9 protein into the nucleus. A short RNA expression system that allows the production of a spacer-gRNA chimeric RNA is also critical (8, 12). In this study, we developed a CRISPR-Cas9 system that worked well in mammalian cells, and was especially adaptive in porcine cells. The Streptococcus pyogenes A20 Cas9 with humanized codons was flanked by the SV40 T-antigen NLS
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(PKKKRKVG, NLS1) and the Daxx NLS (KKSRKEKK, NLS2) (13) at the N and C termini, respectively. An overlapped Flag2 tag (EYKDDDGDYKDDDDK) was added at the end of the N terminus. The CMV enhancer-chicken b-actin promoter was used to derive the Flag2-NLS1-Cas9NLS2mRNA, and the porcine U6 promoter (14) was used to transcribe the spacer-gRNA chimeric RNA. MATERIALS AND METHODS
Construction of the pCX-Flag2-NLS1-Cas9-NLS2 Expression Vector The synthesized cDNA composed of humanized codons encoding the Cas9 protein of Streptococcus pyrogenes A20 and the NLS of Daxx (KKSRKEKK)(13) flanked with an MluI site and an Acc65I site at the 50 and the 30 ends, (Appendix 1) respectively, was inserted into the pCX-Flag2-NLS-MCS1(SNX=N) vector, between the MluI and Acc65I sites, to build the pCX-Flag2NLS1-Cas9-NLS2 construct (Fig. 1c).
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Construction of the pCX-Flag2-NLS-MCS1 Expression Vector The oligonucleotide pair encoding the Flag2 tag (EYKDDDGDYKDDDDK) and the SV40T-antigen
NLS (PKKKRKVG) was inserted at the EcoRI site of pCX-MCS1(SNX=N) (Fig. 1a) to construct the pCX-Flag2NLS-MCS1(SNX=N) expression vector (Fig. 1b).
FIG. 1. Linear maps of the pCX-MCS1(SNX=N), pCX-Flag2-NLS-MCS1(SNX=N), and pCX-Flag2-NLS1-Cas9-NLS2 vectors. (a) Linear map of the pCX-MCS1(SNX=N) vector, which was derived from the pCAGGS-NLS-Cre vector. A SalI=NotI=XbaI oligonucleotide was inserted in front of the CMV enhancer, and the HindIII site located at the 30 end of the polyA signal was replaced by a NotI site. A set of restriction enzyme cutting sites (MCS1) was created for cDNA insertion. (b) An oligonucleotide composed of a Kozak sequence, a hybrid Flag tag (Flag2), and the SV40T antigen NLS was inserted into the EcoRI site of pCX-MCS1(SNX=N), to obtain the pCX-Flag2-NLS-MCS1(SNX=N) plasmid. (c) A synthesized Cas9-NLS2 cDNA with a humanized codon was inserted between the MluI and Acc65I sites of pCX-Flag2-NLS-MCS1(SNX=N), to construct pCX-Flag2-NLS1-Cas9-NLS2.
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IF to Confirm Flag2-NLS1-Cas9-NLS2 Expression The pCX-Flag2-NLS1-Cas9-NLS2 was transfected into HeLa cells using Turbofect (#R0531, Fermentas=Thermo) according to the manufacturer’s instructions. Two days after transfection, the cells were fixed in 4% paraformaldehyde for 10 min at room temperature. The M2 monoclonal antibody (Cat. # F1804, Sigma) (1:50) against the Flag tag was used as the primary antibody, and DyLight488conjugated AffiniPure donkey anti-mouse IgG (715-485151, Jackson Lab.) (1:100) was used as the secondary antibody to perform IF.
Construction of the ppU6-BsaI2-gRNA Vector A DNA fragment composed of the pig U6 promoter (14), a pair of type II restriction enzyme BsaI sites, and the CRISPR spacer-guide-RNA flanked by a BamHI site at the 50 end, as well as by BglII and XbaI sites at the 30 end, was synthesized as shown in Appendix 2 and inserted into pUC57, in which a unique BsaI site had been removed by site-directed mutagenesis, to obtain the ppU6-BsaI2gRNA plasmid (Fig. 2a).
Genomic DNA Target Sites and Preparation of the ppU6-Target-gRNA Vector The target sites of this CRISPR-Cas9 system should follow the GN19NGG rule (Fig. 2b). It is suggested that there is no site with less than three mismatches that prevents off-targeting. When the target site is determined, a pair of oligonucleotides ought to be synthesized and annealed before ligation with BsaI-treated ppU6-BsaI2-gRNA vector, to prepare the ppU6-Target-gRNA vector (Fig. 2c).
Pig GGTA1 Gene Targeting The 371 amino acid coding region of the pig GGTA1 gene (NCBI access number, NC_010443) comprises six exons, the last of which covers most of the protein (amino acids 141 to 371), including the active domain with a-1,3-galactosyl transferase activity. The GGTA1 gene region, including parts of the last intron and last exon, used in this study is shown in Appendix 2. A primer pair, CAGCAACAGACGTCTCTCATC (GGTA1F) and CTCTCGTAGG TGAACTCGTCA (GGTA1 R), via a 511 bp DNA fragment, which can be specifically amplified by PCR, was selected for the subsequent analysis. Four target sites were picked up and the oligonucleotide pairs synthesized for these sites are shown in Tables 1 and 2, respectively. After an annealing reaction, the four oligonucleotide pairs were ligated with BsaI-treated ppU6-BsaI2-gRNA vector to prepare ppU6-GGTA1(38–60)-gRNA, ppU6GGTA1 (187–209)-gRNA, ppU6-GGTA1 (181–159) -gRNA, and ppU6-GGTA1(223–201)-gRNA.
FIG. 2.
The Spacer-gRNA expression vector. (a) Linear map of ppU6-(BsaI)2-gRNA, which was constructed using a pUC57 vehicle from which a BsaI site located in the Amp region had been removed. (b) The candidate target sites on genomic DNA were screened by the GN19NGG [GN19 spacer þNGG protospacer adjacent spacer (PAM)] rule. (c) The procedures used to construct ppU6-Spacer-gRNA are shown. The annealed primer pair was inserted between the two BsaI sites of the ppU6-(BsaI)2gRNA vector.
Transfection of the pCX-Flag2-NLS1-Cas9-NLS2 and ppU6-GGTA1-gRNA Vectors into PK1 Cells All four ppU6-GGTA1(38–60)-gRNA, ppU6-GGTA1 (187–209)-gRNA, ppU6-GGTA1(181–159)-gRNA, and ppU6GGTA1(223–201)-gRNA plasmids (2 mg of each), and 2 mg of pCX-Flag2-NLS1-Cas9-NLS2 were mixed with 1 106 pig PK1 cells in a 100 mL tip connected to a Micro-Porator (MP-100, Digital Bio.) and treated with a single pulse at
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TABLE 1 Target sites picked up for pig GGTA1 gene editing
Reverse 159–181 201–223
CCGAGAAGAGGTGGCAAGACATC CCATCGGGGAGCACATCCTGGCC
Forward (sense) 38–60 GCAAATACATACTTCATGGTTGG 187–209 GATGCGCATGAAGACCATCGGGG Forward (antisense) 181–159 GATGTCTTGCCACCTCTTCTCGG 223–201 GGCCAGGATGTGCTCCCCGATGG
TABLE 2 Oligonucleotide pairs synthesized for pig GGTA1 gene editing
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Oligonucleotide GGTA1 F 38–60 GGTA1 R 38–60 GGTA1 F 187–209 GGTA1 R 187–209 GGTA1 F 181–159 GGTA1 R 181–159 GGTA1 F 223–201 GGTA1 R 223–201
Sequence CGTC GCAAATACATACTTCATGGT GTTTTAGAGCTAGAAAT TGCTATTTCTAGCTCTAAAAC ACCATGAAGTATGTATTTGC CGTCGATGCGCATGAAGACCATCG GTTTTAGAGCTAGAAAT TGCTATTTCTAGCTCTAAAAC CGATGGTCTTCATGCGCATC CGTC GATGTCTTGCCACCTCTTCT GTTTTAGAGCTAGAAAT TGCTATTTCTAGCTCTAAAAC AGAAGAGGTGGCAAGACATC CGTC GGCCAGGATGTGCTCCCCGA GTTTTAGAGCTAGAAAT TGCTATTTCTAGCTCTAAAAC TCGGGGAGCACATCCTGGCC
1300 V for 30 ms. To obtain significant results, three rounds of transfection were performed every third day. To test the individual GGTA1-gRNA activity, 5 mg each of ppU6-GGTA1(38–60)-gRNA, ppU6-GGTA1(187–209)gRNA, ppU6-GGTA1(181–159)-gRNA, or ppU6-GGTA1 (223–201)-gRNA were mixed with 5 mg of pCX-Flag2-NLS1Cas9-NLS2 before transfection into PK1 cells via a single round of microporation, as previously described.
1 mL of PBS supplemented with 1 mg of isolectin-B4-FITC (Cat. # L2895, Sigma), and incubated on ice for 1 h. Cells were washed with PBS as previously described and suspended in PBS for FACS analysis. Normal PK1 cells served as controls. Data were processed using the WinMDI software.
Analysis of the Cas9/GGTA1-gRNA Activities by DNA Sequencing Three days after the last transfection, genomic DNA samples were isolated using a genomic DNA purification kit (#K0721, Fermentas=Thermo). A DNA fragment including the GGTA1 restriction sites was amplified by PCR using 0.1 mg of genomic DNA, 0.25 mM each of GGTA1 F and GGTA1 R primers, and 5 U of Taq DNA polymerase in a 50 mL reaction with a program of 95 C for 3 min, 35 cycles of 94 C for 30 s=60 C for 40 s=72 C for 30 s, and 72 C for 3 min, followed by soaking at 4 C. The DNA PCR products were purified using a PCR clean-up kit (Cat. # DF300, Geneaid) and subcloned into the pGEM-T Easy vector (Cat. # A1360, Promega). The DNA sequences of the plasmids that were isolated from individual colonies were determined using the T7 primer on an ABI 3730 sequencer. Flow Cytometry Analysis Transfected PK1 cells were trypsinized and fixed in 70% alcohol overnight. The fixed cells were washed with PBS by centrifugation and suspension twice, and then suspended in
FIG. 3.
Localization of the Flag2-NLS1-Cas9-NLS2 recombinant protein, which was overexpressed in HeLa cells. Either Flag2-NLS1-Cas9NLS2 (a) or the Flag2-NLS1-Cas9-NLS2=GGTA1(38–60)-gRNA complex (b) was overexpressed in HeLa cells. The Cas9 protein component was detected via the Flag tag using the M2 monoclonal antibody.
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RESULTS Construction of the pCX-Flag2-NLS1-Cas9-NLS2/ PpU6-(BsaI)2-gRNA System The expression vector pCX-MCS1(SNX=N) (Fig. 1a) used in this study was modified from the backbone of the pCAGGS-NLS-Cre vector (which was a kind gift from Andras Nagy, Mount Sinai Hospital, Toronto). A SalI= NotI=XbaI oligonucleotide was inserted in front of the CMV enhancer, and the HindIII site at the 30 end of the polyA signal was replaced by a NotI site. A set of restriction enzyme cutting sites (MCS1) was used to replace the NLSCre coding region. An oligonucleotide composed of a Kozak sequence, hybrid Flag tag (Flag2), and the SV40 T antigen NLS was inserted into the EcoRI site of pCXMCS1(SNX=N), to obtain the pCX-Flag2-NLS-MCS1(SNX=N) plasmid (Fig. 1b). A synthesized Cas9-NLS2 cDNA with humanized codons was then inserted between MluI and Acc65I site of pCX-Flag2-NLS-MCS1(SNX=N) to build pCXFlag2-NLS1-Cas9-NLS2 (Fig. 1c). The DNA sequence of the entire coding region is shown in Appendix 3. The DNA composed of the pig U6 promoter, BsaI=NotI=MluI=XhoI=BsaI
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restriction enzyme cutting sites, and gRNA was synthesized (see Appendix 2) and inserted between the BamHI and XbaI sites of a modified pUC57 vehicle in which the BsaI site, which was within the Amp region, had been removed to prepare ppU6-(BsaI)2-gRNA vector (Fig. 2a). The candidate target sites, which comprise a GN19 protospacer and an NGG protospacer-adjacent motif (PAM) on genomic DNA were screened according to the GN19NGG rule. The primer pair encoding the spacer and the 50 end of gRNA were designed as shown in Fig. 2b, and annealed before ligation with BsaI-treated ppU6-Spacer-gRNA vector, to build the ppU6-Spacer-gRNA (Fig. 2c). Expression of the Flag2-NLS1-Cas9-NLS2 Recombinant Protein in HeLa Cells After pCX-Flag2-NLS1-Cas9-NLS2 DNA transfection, the expression and localization of the Flag2-NLS1-Cas9NLS2 recombinant protein was performed by immunofluorescence assay using the Flag tag-specific M2 mAb as the primary antibody and DyLight488-conjugated AffiniPure donkey anti-mouse IgG as the secondary antibody. As
FIG. 4. Targeting the pig GGTA1 gene in PK1 cells. (a) Four protospacers within exon 6 of the pig GGTA1 gene were selected. (b) After three successive transfections, this locus was amplified by PCR using the GGTA1 F and GGTA1 R primer pair. Two bands of DNA were observed (left panel) and subcloned into the pGEM-T Easy vector. Twenty clones were picked up, 19 of which contained inserts of the expected size (right panel). (c) The DNA sequences of the 19 inserts were aligned with the standard one. Predicted insertion and deletion sites are indicated by the ^ and – symbols, respectively. The protospacer þ PAM sequences are indicated by blue arrows, and the expected cutting sites are pointed out by red arrow heads.
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FIG. 4. Continued. shown in Fig. 3a, the overexpressed flag-tagged recombinant protein was located around the nuclei, rather than within them. We speculated whether the Cas9 protein assembles with the crRNA=tracrRNA complex or spacer-gRNA to adopt a conformation that is able to be transported into the nucleus. A GGTA1(38–60) spacer-gRNA was used to test this assumption. After cotransfection of pCX-Flag2-NLS1Cas9-NLS2 and ppU6-GGTA1(38–60)-gRNA, the amount of the Cas9 component, as monitored using the M2 monoclonal antibody, was significantly increased in nuclei, even though signals were still detected mainly in the cytoplasm (Fig. 3b). Targeting the Pig GGTA1 Gene with the Flag2-NLS1Cas9-NLS2/GGTA1-gRNA Complex Four ppU6-GGTA1-gRNA plasmids, ppU6-GGTA1(38–60)gRNA, ppU6-GGTA1(187–209)-gRNA, ppU6-GGTA1(181–159)gRNA, and ppU6-GGTA1(223–201)-gRNA, were prepared to target the protospacers on pig GGTA1 exon 6 (Fig. 4a). These four plasmids were cotransfected with pCX-Flag2NLS1-Cas9-NLS2 into pig PK1 cells, as described in the
Materials and Methods section. Three days after the last transfection, genomic DNA was isolated and GGTA1 PCR was performed. Two bands of DNA were observed (Fig. 4b, left panel) and were subcloned into the pGEM-T Easy vector. Twenty clones were picked up, 19 of which contained inserts of the expected size (Fig. 4b, right panel). The DNA sequences of the 19 inserts were aligned with the standard one. Three of the 19 clones were the same as the standard. The remaining 16 clones contained insertions or deletions. Site 54 (GGTA1(38–60)) was involved in 13 mutant clones, site 164 (GGTA1(181–159)) was involved in four mutant clones, site 203 (GGTA1(187–209)) was involved in four mutant clones, and site 206 (GGTA1(223–201) was involved in 10 mutant clones (Fig. 4c). Expression Levels of the a-1,3-Gal Epitope Were Reduced upon GGTA-1 Gene Knockout by the Flag2NLS1-Cas9-NLS2/GGTA1-gRNA Complex The expression levels of the a-1,3-Gal epitope on pig PK1 cells were detected by FITC-conjugated isolectin-B4. Compared with the control, in which 91.65% of cells exhibited
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high FITC fluorescence signals, the ratio of cells with a high level of expression of the a-1,3-Gal epitope was reduced to 75.29%, 89.19%, 81.71%, and 80.40% after cotransfection with pCX-Flag2-NLS1-Cas9-NLS2=ppU6-GGTA1(38–60)gRNA, pCX-Flag2-NLS1-Cas9-NLS2=ppU6-GGTA1 pCX-Flag2-NLS1-Cas9-NLS2=ppU6(187–209)-gRNA, GGTA1(181–159)-gRNA, and pCX-Flag2-NLS1-Cas9-NLS2= ppU6-GGTA1(223–201)-gRNA plasmid pair, respectively.
FIG. 5. Flow cytometry analysis of the expression level of the a-1,3-Gal epitope. The expression levels of the a-1,3-Gal epitope on pig PK1 cells were detected by FITC-conjugated isolectine-B4. Untreated PK1 cells were used as a control. The levels of expression of the a-1,3-Gal epitope in PK1 cells cotransfected with pCX-Flag2-NLS1-Cas9-NLS2=ppU6-GGTA1(38–60)gRNA, pCX-Flag 2-NLS1 -Cas9-NLS 2=ppU6-GGTA1(187–209)-gRNA, pCX-Flag2-NLS1-Cas9-NLS2=ppU6-GGTA1(181–159)-gRNA, and pCXFlag2-NLS1-Cas9-NLS2=ppU6-GGTA1(233-201)-gRNA plasmid pairs, respectively, were analyzed using a flow cytometer. The ratios of cells with high and low levels of fluorescence signals were determined.
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Moreover, a group of cells that expressed a lower level of the a-1,3-Gal epitope corresponding to the quantities of reduction of the high-expression population was identified (Fig. 5).
DISCUSSION The gene encoding 1368 amino acids of Cas9 used in this study was extracted from the genome of Streptococcus pyrogenes strain A20, which was isolated in south Taiwan. The amino acid sequence of Sp A20 Cas9 is nearly the same as that of Sp SF370 Cas9, with only one difference: the 24th amino acid is Asp instead of Glu. Therefore, it is reasonable to think that the strategy of spacer-gRNA (8, 15) can be applied to Sp A20 Cas9. To build a spacer-gRNA expression vector for mammalian cells, the porcine U6 promoter, which has been proven to work more effectively than the human promoter in porcine cells (14), was used to transcribe the chimeric RNA. Three prototypes of the ppU6-BsaI2-gRNA vector were tested, each of them adapted with a 24 nt oligonucleotide pair (20 nt for the spacer þ 4 nt of gRNA), a 32 nt oligonucleotide pair (20 nt for the spacer þ 12 nt of gRNA), or a 41 nt oligonucleotide pair (20 nt for the spacer þ 21 nt of gRNA) insert (Fig. 2c). It was found that only the 41 nt oligonucleotide pair could be effectively ligated to the BsaI-treated vector using the 1 h quick ligation protocol (data not shown). Therefore, the third prototype was chosen for further experiments. When HeLa cells were transfected with pCX-Flag2NLS1-Cas9-NLS2 alone, the Flag2-NLS1-Cas9-NLS2 recombinant protein was detected unsymmetrically outside the boundary of nuclei (Fig. 3a). pCX-HA-NLS1-Cas9NLS2 and pCX-Myc-NLS1-Cas9-NLS2 were also generated, and the HA-NLS1-Cas9-NLS2 and Myc-NLS1-Cas9NLS2 recombinant proteins were located at the same place as Flag2-NLS1-Cas9-NLS2 (data not shown). However, when cells were cotransfected with pCX-Flag2-NLS1Cas9-NLS2 and ppU6-spacer-gRNA, a significant amount of the Flag2-NLS1-Cas9-NLS2 recombinant protein was detected by M2 monoclonal antibody staining in the nuclei, even though most of the signals were mainly detected in the cytoplasm (Fig. 3b). These results indicate that only the Flag2-NLS1-Cas9-NLS2=sgRNA complex could be translocated into the nucleus. Two recent papers (10, 15) reported the crystal structure of Cas9 and the Cas9=sgRNA=target DNA triple complex, respectively. The architectures of Cas9 enzymes define nucleic acid binding clefts, and single-particle electron microscopy reconstructions showed that the two structural lobes that harbor these clefts undergo guide RNA-induced reorientation to form a central channel, to which DNA substrates bind. The observation that extensive structural rearrangements occurred before target DNA duplex binding implicates that guide RNA loading is a key step in Cas9 activation (7).
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To analyze the RNA-guided endonuclease activity of our Cas9=sgRNA system, the pig GGTA1 gene was used as a target, because the enzymatic activity of its product, a-1, 3-galactotransferase, can simply monitor the amount of the a-1,3-Gal epitope on the cell surface by isolectin-B4. Four target sites on pig GGTA1 exon 6 (Fig. 4a) were selected, and two strategies were used. First, all of the four GGTA1 sg-RNA expression vectors were cotransfected with pCX-Flag2-NLS1-Cas9-NLS2 for three rounds, to increase the populations of transfected pig PK1 cells. Three days after the last transfection, genomic DNA was isolated and GGTA1 PCR was performed. The PCR-amplified DNA fragments were subcloned into the pGEM-T Easy vector. The DNA sequences of the 19 clones were aligned with the standard one. Sixteen of the 19 clones contained insertions or deletions. Site 54 (GGTA1(38–60)) was involved in 13 mutant clones, site 164 (GGTA1(181–159)) was involved in four mutant clones, site 203 (GGTA1(187–209)) was involved in four mutant clones, and site 206 (GGTA1(223–201) was involved in 10 mutant clones (Fig. 4c). The relative efficiencies of the four sites, sites 54, 164, 203, and 206, were roughly estimated as 13:4:4:10. In the second strategy, individual GGTA1 sg-RNA expression vectors were cotransfected with pCXFlag2-NLS1-Cas9-NLS2 into pig PK1 cells. The expression levels of the a-1,3-Gal epitope at the cell surface were monitored using a flow cytometer and FITC-conjugated isolectinB4. A group of cells expressed a lower level of the a-1,3-Gal epitope, corresponding to the quantities of reduction of the high expression population (Fig. 5). The reduction percentages of the four cases were 13.4% (24.12%-7.72%), 2.86% (10.58%-7.72%), 10.26% (17.98%-7.72%), and 11.39% (19.11%-7.72%) for site 54 (GGTA1(38–60)), site 164 (GGTA1(181–159)), site 203 (GGTA1(187–209)), and site 206 (GGTA1(223–201), respectively. Even though the efficiency of transfection was not considered, the targeting efficiencies observed in both strategies were quite high. FUNDING This work was supported by grants NSC 99-2313-B-059004-MY3 and MOST 102-2320-B-059-001-MY3 from the Ministry of Science and Technology of Taiwan to C. K. Chuang. REFERENCES 1. Horvath P, Barrangou R. CRISPR=Cas, the immune system of bacteria and archaea. Science 2010; 327:167–170.
2. Terns MP, Terns RM. CRISPR-based adaptive immune system. Curr Opin Microbiol 2011; 14:321–327. 3. Mojica FJ, Dı´ez-Villasen˜or C, Garcı´a-Martı´nez J, Almendros C. Short motif sequences determine the targets of the prokaryotic CRISPR defence system. Microbiol 2009; 155: 733–740. 4. Gasiunas G, Barrangou R, Horvath P, Siksnys V. Cas9crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria. Proc Natl Acad Sci USA 2012; 109:E2579–E2586. 5. Bhava D, Davison M, Barrangou R. CRISPR-Cas systems in bacteria and archaea: versatile small RNAs for adaptive defense and regulation. Annu Rev Genet 2011; 45:273–297. 6. Chylinski K, Le Rhun A, Charpentier E. The tracrRNA and Cas9 families of type II CRISPR-Cas immunity systems. RNA Biol 2013; 10:726–737. 7. Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 2012; 337:816–821. 8. Mali P, Yang L, Esvelt KM, Aach J, Guell M, DiCarlo JE, Norville JE, Church GM. RNA-guided human genome engineering via Cas9. Science 2013; 339:823–826. 9. Ran FA, Hsu PD, Lin CY, Gootenberg JS, Konermann S, Trevino AE, Scott DA, Inoue A, Matoba S, Zhang Y, Zhang F. Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity. Cell 2013; 154: 1380–1389. 10. Nishimasu H, Ran FA, Hsu PD, Konermann S, Shehata SI, Dohmae N, Ishitani R, Zhang F, Nureki O. Crystal structure of Cas9 in complex with guide RNA and target DNA. Cell 2014; 156:935–949. 11. Bae S, Park J, Kim JS. Cas-OFFinder: A fast and versatile algorithm that searches for potential off-target sites of Cas9 RNA-guided endonucleases. Bioinformatics 2014; 30:1473– 1475. 12. Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu X, Jiang W, Marraffini LA, Zhang F. Multiplex genome engineering using CRISPR=Cas systems. Science 2013; 339:819–923. 13. Yeung PL, Chen LY, Tsai SC, Zhang A, Chen JD. Daxx contains two nuclear localization signals and interacts with importin alpha3. J Cell Biochem 2008; 103:456–70. 14. Chuang CK, Lee KH, Fan CT, Su YS. Porcine type III RNA polymerase III promoters for short hairpin RNA expression. Anim Biotechnol 2009; 20:34–39. 15. Jinek M, Jiang F, Taylor DW, Sternberg SH, Kaya E, Ma E, Anders C, Hauer M, Zhou K, Lin S, Kaplan M, Iavarone AT, Charpentier E, Nogales E, Doudna JA. Structures of Cas9 endonucleases reveal RNA-mediated conformational activation. Science 2014; 343:1247997.
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APPENDIX 1. DNA SEQUENCE OF FLAG2-NLS1-CAS9-NLS2
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Flag2-NLS1-Cas9-NLS2 ATGGACTACA AAGAAACGCA CTGGACATTG AGCAAGAAGT GGAGCCCTGC CGGAGAAGGT GAAATGGCCA GAAGATAAGA CACGAAAAGT GCAGACCTGC CTGATCGAGG GTCCAGACAT AAAGCCATCC CTGCCAGGGG ACACCCAACT GACACTTATG CTGTTCCTGG AACACCGAGA CATCAGGATC GAAATCTTCT CAGGAGGAGT CTGCTGGTGA AGCATCCCTC TTCTACCCAT CCCTACTATG TCTGAGGAAA CAGTCTTTCA CCCAAGCACA TACGTCACTG GTGGACCTGC TTCAAGAAAA GCCAGCCTGG AACGAGGAAA AGGGAAATGA AAACAGCTGA GGGATTCGCG GCCAACCGGA CAGAAGGCAC GGCTCTCCTG AAAGTCATGG ACCACACAGA AAGGAACTGG GAGAAGCTGT GATATTAACC GATGACAGCA AATGTCCCCT GCCAAACTGA AGCGAACTGG AAGCACGTCG CTGATCAGAG
AAGATGACGA AAGTGGGTGA GAACAAACTC TCAAGGTGCT TGTTCGACTC ATACTCGCCG AGGTGGACGA AACACGAGCG ACCCTACTAT GCCTGATCTA GGGATCTGAA ACAATCAGCT TGAGTGCCCG AAAAGAAAAA TCAAGAGCAA ACGATGACCT CCGCTAAGAA TTACAAAAGC TGACCCTGCT TTGATCAGTC TCTACAAGTT AACTGAATAG ACCAGATTCA TTCTGAAGGA TGGGACCTCT CAATCACTCC TTGAGCGGAT GCCTGCTGTA AGGGGATGAG TGTTTAAAAC TTGAATGTTT GGACCTACCA ATGAGGATAT TCGAGGAACG AGCGACGGAG ACAAACAGAG ACTTCATGCA AGGTGTCCGG CCATCAAGAA GAAGACATAA AAGGCCAGAA GCTCTCAGAT ATCTGTACTA GACTGAGTGA TTGACAATAA CAGAGGAAGT TCACCCAGCG ACAAAGCAGG CCCAGATTCT AAGTGAAGGT
TGGTGATTAT ATTCCTCGAG TGTGGGCTGG GGGCAACACC CGGCGAGACA AAAGAATAGG TAGTTTCTTT GCATCCCATC CTATCATCTG TCTGGCCCTG CCCAGACAAT GTTTGAGGAA GCTGTCTAAG CGGCCTGTTT TTTTGATCTG GGATAACCTG TCTGTCTGAC CCCCCTGTCA GAAGGCTCTG TAAGAACGGA TATCAAACCC GGAAGACCTG TCTGGGAGAG TAACAGGGAG GGCCCGGGGC CTGGAACTTC GACAAACTTC CGAGTATTTC AAAGCCTGCC CAATAGGAAG CGATTCTGTG CGATCTGCTG CCTGGAAGAC CCTGAAGACT ATACACCGGA TGGAAAGACT GCTGATTCAC GCAGGGAGAC AGGGATTCTG GCCAGAAAAC GAACTCCAGG CCTGAAAGAG TCTGCAGAAT TTATGACGTG GGTGCTGACC GGTCAAGAAA AAAGTTTGAT CTTCATTAAG GGATTCCAGA CATTACTCTG
AAAGACGATG ACGCGTATGG GCTGTGATTA GATAGACACA GCTGAAGCAA ATCTGCTACC CATCGCCTGG TTTGGCAACA AGGAAGAAAC GCTCACATGA AGCGATGTGG AACCCCATTA AGTCGGAGAC GGGAATCTGA GCCGAGGACG CTGGCTCAGA GCCATCCTGC GCTAGCATGA GTGAGGCAGC TACGCCGGCT ATTCTGGAGA CTGAGGAAGC CTGCACGCAA AAGATCGAAA AACAGCCGGT GAGGAAGTGG GACAAGAACC ACCGTCTATA TTCCTGAGCG GTGACAGTCA GAGATCAGTG AAGATCATTA ATTGTGCTGA TATGCCCATC TGGGGCCGAC ATCCTGGACT GATGACTCCC TCTCTGCACG CAGACCGTGA ATCGTGATTG GAGCGCATGA CACCCCGTGG GGGCGCGATA GATCATATCG CGCTCAGACA ATGAAGAACT AACCTGACAA CGACAGCTGG ATGAACACAA AAGTCAAAAC
ACGATAAGAA ATAAAAAGTA CCGACGATTA GCATCAAGAA CTCGGCTGAA TGCAGGAGAT AGGAATCATT TTGTGGACGA TGGTGGACAG TTAAGTTCCG ACAAGCTGTT ATGCATCTGG TGGAGAACCT TTGCACTGTC CTAAACTGCA TCGGGGATCA TGAGTGATAT TCAAGAGATA AGCTGCCTGA ATATTGACGG AGATGGACGG AGCGCACATT TCCTGAGGCG AAATTCTGAC TTGCATGGAT TCGATAAGGG TGCCAAACGA ACGAACTGAC GAGAACAGAA AGCAGCTGAA GCGTCGAAGA AGGATAAAGA CCCTGACACT TGTTCGATGA TGTCTCGGAA TTCTGAAATC TGACCTTCAA AGCATATCGC AGGTGGTCGA AGATGGCCAG AACGCATCGA AAAACACTCA TGTACGTGGA TCCCACAGTC AAAACCGAGG ACTGGCGGCA AAGCTGAGCG TGGAGACCCG AGTACGATGA TGGTGAGCGA
TTTGCCGAAG TAGTATTGGG CAAGGTGCCT AAATCTGATT AAGAACAGCT TTTCAGCAAC CCTGGTCGAG GGTCGCTTAT CACCGATAAA GGGCCATTTT CATCCAGCTG CGTGGACGCA GATCGCTCAG ACTGGGACTG GCTGAGCAAG GTACGCAGAC TCTGAGAGTG TGACGAGCAC GAAGTACAAG CGGGGCTAGT CACCGAGGAA TGATAACGGA CCAGGAAGAC CTTCCGCATT GACACGCAAA CGCTTCCGCA AAAAGTGCTG AAAGGTGAAA GAAAGCTATC AGAGGACTAT CCGGTTCAAC CTTCCTGGAC GTTTGAGGAT CAAAGTGATG GCTGATCAAT AGATGGCTTC AGAGGATATC AAACCTGGCC CGAGCTGGTG GGAAAATCAG GGAAGGAATT GCTGCAGAAT CCAGGAGCTG ATTCCTGAAA AAAGAGTGAT GCTGCTGAAT GGGAGGCCTG GCAGATCACA GAATGACAAA CTTTCGGAAA
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288 GATTTCCAGT CTGAACGCAG GTGTACGGGG ATTGGCAAAG GAGATCACCC ACCGGAGAAA ATGCCCCAAG TCAATTCTGC AAGAAGTATG GTGGAGAAGG ATGGAGAGGA GAAGTGAAGA GGGAGAAAGA CCCAGTAAGT CCTGAGGATA ATCATTGAAC AAGGTCCTGT ATCATTCATC ACTACCATCG CACCAGAGCA GGGGGAAGCG
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TTTATAAGGT TGGTCGGCAC ACTATAAGGT CCACCGCTAA TGGCAAATGG TCGTGTGGGA TGAATATTGT CTAAACGCAA GCGGGTTCGA GAAAAAGCAA GCTCCTTCGA AAGACCTGAT GGATGCTGGC ACGTGAACTT ACGAACAGAA AGATTAGCGA CTGCATACAA TGTTCACTCT ATCGCAAACG TTACTGGCCT GCGGGAAGAA
CAGAGAGATC CGCCCTGATT GTACGATGTC GTATTTCTTT GGAAATCCGA CAAAGGAAGA CAAGAAAACA CAGTGATAAG CTCCCCAACA GAAACTGAAA AAAGAATCCT CATCAAGCTG ATCTGCCGGG CCTGTATCTG ACAGCTGTTT GTTCTCCAAG CAAACACCGG GACCAACCTG ATACACATCA GTACGAGACC GAGCAGGAAG
AACAACTACC AAGAAATACC AGAAAAATGA TACAGCAACA AAGCGGCCAC GATTTTGCTA GAGGTGCAGA CTGATCGCCC GTGGCTTACT TCCGTCAAGG ATCGATTTTC CCAAAGTACT GAGCTGCAGA GCTAGCCACT GTGGAGCAGC AGAGTGATCC GACAAGCCAA GGAGCCCCCG ACTAAGGAGG CGGATCGACC GAGAAAAAAC
ACCATGCTCA CTAAACTGGA TCGCCAAGTC TCATGAATTT TGATTGAGAC CCGTGAGGAA CTGGGGGATT GAAAGAAAGA CTGTCCTGGT AACTGCTGGG TGGAGGCCAA CCCTGTTTGA AAGGAAATGA ACGAGAAGCT ACAAGCATTA TGGCTGACGC TCAGAGAGCA CAGCCTTCAA TGCTGGATGC TGAGTCAGCT TCGAGTGAGG
TGACGCATAC GTCCGAGTTC AGAGCAGGAA CTTTAAGACT TAACGGGGAG GGTCCTGTCT CAGTAAGGAA CTGGGACCCC GGTCGCAAAG CATCACTATT AGGCTATAAG GCTGGAAAAC ACTGGCCCTG GAAAGGCTCC TCTGGACGAG AAATCTGGAT GGCCGAAAAT GTATTTTGAC TACCCTGATC GGGGGGGGAC TACC
GCAGACCCGC AGTAAGGCGC ATATAATATG TATGATGACA AAAATTACTC CCCTCTGAGA AGTTAAAATA TT
AATAGTGAGA GCGGAGGTCG TAGGAAGGTT GTATGTGTGA TTCTATAGTC AGACCCAGCC AGGCTAGTCC
GAAGCGCTAG GGCGGGCAGA AAAGAGACAA CATAGAAGGT ACCATAATCA GTCGGAGACC GTTATCAACT
APPENDIX 2. DNA SEQUENCE OF PU6-BSAI2-GRNA pU6-BsaI2-gRNA GGATCC AGGAGGACTC GGGAACGAGC GGGCCAATTT TTCTACTTAA CATAAATCCT AAAAAGTTTT GCGGCCGCAC TGAAAAAGTG AGATCT
CAGGGACAAC ACCACGTGAC CCCATGGTCC CTCTCCCAAA CGAATACTTT TATGTTCTTG GCGTCTCGAG GCACCGAGTC TCTAGA
CGCTGGGCTC CGAGCGTGAC CTTCATTTGC ATCACAAAGA TAAATTGAAA GCTTTATATA GTCTCTAGCA GGTGCTTTTT
