Oligonucleotides corresponding to five A3A-targeting gRNA sequences were cloned into the BsaI site of pGL3-U6-sgRNA-PGK-puromycin (Addgene, #51133) to create plasmids expressing sgRNAs targeting A3A. All the sgRNA target sequences and oligonucleotides used to create these plasmids are listed in the S5 Table . AU565 derived A3A knockout cell lines were generated by transiently co-transfecting AU565 cells with pST1374-nls-flag-cas9 (Addgene, #44758) and all five plasmids expressing A3A specific gRNAs. Cells expressing both Cas9 and gRNA were transiently selected by the addition of puromycin and blasticidin to the cell culture media for 3 days. Surviving cells were plated at approximately 100 cells / 10 cm dish and clonal cell lines were isolated via cloning cylinders. A3A knockouts were confirmed by PCR and sequencing using primers oTM-499 and oTM-501 (S2 Fig ).
Pgl3 u6 sgrna pgk puromycin
Manufactured by Addgene
Sourced in United States
The PGL3-U6-sgRNA-PGK-puromycin is a plasmid vector that can be used for CRISPR-Cas9 genome editing applications. It contains a U6 promoter for expressing single-guide RNA (sgRNA) and a PGK promoter for expressing a puromycin resistance gene. This vector allows for the selection of successfully transfected cells.
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Lab products found in correlation
Neon transfection kit, by Thermo Fisher Scientific (3 mentions)
Puc57 sgrna expression vector, by Addgene (3 mentions)
Flag cas9, by Addgene (2 mentions)
Quantasoft, by Bio-Rad (2 mentions)
Pegfp n2, by Takara Bio (2 mentions)
Qx200, by Bio-Rad (1 mentions)
Ye1 be3, by Addgene (1 mentions)
Yee be3, by Addgene (1 mentions)
Pgl3 u6 sgrna egfp, by Addgene (1 mentions)
Pet28b, by GenScript (1 mentions)
Px330, by Addgene (1 mentions)
Pcmv be3, by Addgene (1 mentions)
Megashortscript kit, by Thermo Fisher Scientific (1 mentions)
Pst1374 nls flag linker cas9, by Addgene (1 mentions)
T7 ultra kit, by Thermo Fisher Scientific (1 mentions)
Lenticrispr v2, by Addgene (1 mentions)
Puromycin, by New England Biolabs (1 mentions)
Pcmv ha3a be3 y130f, by Addgene (1 mentions)
Endofree plasmid maxi kit, by Qiagen (1 mentions)
13 protocols using pgl3 u6 sgrna pgk puromycin
1
Generation of A3A Knockout Cell Lines
2
Quantifying Gene Conversion Efficiency
3
CRISPR-Cas9 Mediated Gene Conversion
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Cell lines used in the assay are indicated in the figure. 2 × 106 cells were transfected with 5 μg pGL3-U6-sgRNA-PGK-puromycin (A gift of Xingxu Huang, Addgene # 51133), 5 μg Flag-Cas9 (A gift of Xingxu Huang, Addgene # 44758), with or without 10 μg HR long donor10 (link) and 1 μg pEGFP-N2 (Takara) by Neon transfection kit (Invitrogen) using a 1350 V, 30 ms pulse in a 100 μL chamber. Forty-eight hours post transfection, a portion of the cells were analyzed by flow cytometry to quantify the transfection efficiency, and the remaining cells were harvested for genomic DNA extraction (Qiagen). Digital PCR (QX-200, Bio-Rad) was performed to quantify the frequency of gene conversion events using the primers and Taqman probes listed in Supplementary Data 7 . The repair signal was normalized to 5000 copies of genomic DNA, measured using a Chromosome 6 control dPCR assay, using primers/probes sequences listed in Supplementary Data 7 . Analysis of dPCR data was performed using QuantaSoft (Bio-Rad).
4
Construction of hA3A-BE3-NG Vector
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The hA3A-BE3-NG vector was constructed in this study through in-fusion cloning to transfer the DNA fragment containing VRVRFRR variants of SpCas9-NG from Target-AID-NG (119861#; Addgene, Watertown, MA, United States) to hA3A-BE3 (113410#; Addgene, Watertown, MA, United States). For construction of sgRNAs, oligos were synthesized, annealed, and cloned into the BsaI site of the sgRNA-expressing vector, pGL3-U6-sgRNA-PGK-puromycin (51133#; Addgene, Watertown, MA, United States). The fragment pCAG-tdTomato was cloned into the BspQI linearized pGL3-U6-sgRNA-PGK-puromycin to construct the sgRNA-tdTomato-expressing vector, pGL3-U6-sgRNA-tdTomato. The sgRNAs used in this study are summarized in Supplementary Table S1 . The primers used in the construction of hA3A-BE3-NG are listed in Supplementary Table S2 .
5
CRISPR/Cas9-based gene correction protocol
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The oligos for mutation and correctional sgRNA were synthesized, cloned into the pGL3-U6-sgRNA-PGK-puromycin (51133; Addgene) and pUC57-sgRNA expression vector (51132; Addgene), and in vitro transcribed as the reported protocol:17 (link) mutation sgRNA, 5′-CGCCAATGGTGTTAACACA TAG G -3′; correctional sgRNA, 5′-CCG CCAATGGTGTTAACACg TAG-3′. Besides, the correctional sgRNA was also cloned into the pGL3-U6-sgRNA-PGK-GFP plasmid for the comparison of BE3, YE1-BE3, and YEE-BE3. The plasmids of Cas9 (44758; Addgene), BE3 (73021; Addgene), YE1-BE3 (85174; Addgene), and YEE-BE3 (85177; Addgene) were used, and these plasmids were transcribed in vitro as the reported protocol.17 (link) All of the transcribed RNA were stored at −80°C. The ssODN was synthesized at Sangon Biotech (http://www.sangon.com/ ) as the following sequence: 5′-GACGTATGGTGTTGGGTAAATCCGGGAGGACATTTGCATGTGAAGCCGCCAATGGTGTTAACACgTAGGAACTGGCAGTTGTGTTGCTTGGTTGCACACTCATCAAGATC-3′.
6
Plasmid-based CRISPR-Cas9 System
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For construction of sgRNAs, oligos were synthesized, annealed, and cloned into BsaI site of the sgRNA expression vector. Plasmids used include pGL3-U6-sgRNA-PGK-puromycin (Addgene, 51133), pUC57-sgRNA expression vector (Addgene, 51132), pCMV-SaBE3 (Addgene, 85169), pGFP-N1 (Addgene, 54712), pUC57-Sa sgRNA expression vector, pGL3-U6-sgRNA-EGFP, pGL3-U6-sgRNA-BFP, and pCMV-ABE.
7
Recombinant SPR Protein Production
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The pLVX-Puro-SPR expression vector was obtained from Public Protein/Plasmid Library. To produce recombinant SPR or SPRD257G with histidine tags, the SPR gene or SPRD257G gene was cloned into a pET-28b (GenScript, Piscataway, Nanjing, China) expression plasmid. For the construction of sgRNAs, oligos were synthesized, annealed, and cloned into the BsaI site of pGL3-U6-sgRNA-PGK-puromycin (Addgene, 51133) vector. pCMV-ABE and pST1374-N-NLS-flag-linker-Cas9 plasmid were kindly provided by Pro. Xingxv Huang (ShanghaiTech University, Shanghai, China).
8
Generating PrP Knockout Cell Lines
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To generate PrP knock-out M2 and BxPC-3 cell lines, we used two different CRISPR/Cas systems. The primers for PrP knock-out in M2 and BxPC-3 cells were listed in Table 1 . To knock-out PRNP in M2 cells, the primers (0.1 μm ) were annealed and ligated to PX330 (Addgene, number 42230). To generate PRNP null BxPC-3 cells, the primers (0.1 μm ) were annealed and ligated to pGL3-U6-sgRNA-PGK-puromycin (Addgene, number 51133). The plasmids were sequenced and transfected into M2 or BxPC-3 cancer cells, respectively. Single clones of M2 or BxPC-3 cells were selected and subjected to DNA sequencing and Western blotting. The sense primer for screening PrP knock-out clones was listed in Table 1 .
To knockdown PrP in M2 cells, three pairs of small interfering RNA oligos (listed inTable 4 ) were generated by the GenePharma (Shanghai, China). 20 nm siRNA was transfected into a well of a 6-well plate using 4 μl of PepMute (catalog number SL100571) siRNA transfection reagent. 24 h later, cells were treated by 20 ng/ml of TNFα for 0, 10, 15, 30, and 40 min. Then the cell lysate were collected and subjected to Western blot analysis.
To knockdown PrP in M2 cells, three pairs of small interfering RNA oligos (listed in
9
CRISPR-Cas9 Plasmid Construction for Targeted Editing
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Plasmids encoding BE3 (pCMV-BE3) were obtained from Addgene (73021). The small-guide RNA (sgRNA) (the guide RNA of CRISPR-Cas9 protein) for rs75356281 base editing was designed with an online sgRNA design tool, CHOPCHOP (87 ), and synthesized by Tsingke Biological Technology. Then the DNA oligos were annealed and cloned into the BsaI site of the sgRNA-expressing vector, pGL3-U6-sgRNA-PGK-puromycin (51133, Addgene). The sequence of the sgRNA used in this study is 5′-TATGGTCATAGGCCCACAGGTGG -3′ (the underlined sites are the short protospacer adjacent motif flanking the target DNA site).
10
Generation of Hipk4 Knockout Mice
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Approval was granted by Animal Ethical and Welfare Committee of Nanjing Medical University before animal studies. To generate Hipk4 KO mice, paired single guide RNAs (sgRNAs, Fig. S2 B) were designed to target exon 3 of Hipk4. The oligonucleotides used to generate the sgRNA expression plasmid were annealed and cloned into the pGL3-U6-sgRNA-PGK-puromycin (Addgene plasmid # 51133). Transcription and microinjection of CRISPR/Cas9 were performed in vitro as described previously (51 (link)). Briefly, the Cas9 plasmid (pST1374-NLS-flag-linker-Cas9, Addgene plasmid # 44758) was linearized using AgeI and transcribed using a T7 Ultra Kit (Ambion), pGL3-T7-sgRNA-PGK-puromycin expression vectors were linearized by BsaI and transcribed using the MEGAshortscript Kit in vitro (Ambion). A mixture of Cas9 mRNA and two sgRNAs were injected into the cytoplasm and male pronucleus of the zygote via electroporation. Embryos were implanted into pseudo-pregnant C57BL/6J females according to standard procedures. Founder mice were backcrossed to C57BL/6J. All animal experiments in this study were approved by the Institutional Animal Care and Use Committee of Nanjing Medical University, Nanjing, China. All mice were housed in a specific pathogen-free animal facility under standard conditions.
The cell line HEK-293T (Homo sapiens) cells were used by experiments for this paper.
The cell line HEK-293T (Homo sapiens) cells were used by experiments for this paper.
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