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Lenticrispr v2 blast vector

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The LentiCRISPR v2-Blast vector is a plasmid designed for CRISPR-Cas9 genome editing. It contains the Cas9 gene and a blasticidin resistance gene, allowing for selection of transduced cells. The vector can be used to express single guide RNAs (sgRNAs) for targeted gene knockout or modification experiments.

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Lab products found in correlation

Pmirglo luciferase expression vector, by Promega (2 mentions) Plenti ddk puro egfp vector, by Addgene (2 mentions) Lenticrispr v2 vector, by Addgene (2 mentions) Psbbi bla, by Addgene (1 mentions) Plenti6.2 v5 dest, by Thermo Fisher Scientific (1 mentions) Plko 1 blast vector, by Addgene (1 mentions)

Close spelling variants of this product

LentiCRISPR v2-Blast LentiCRISPR v2 vector LentiCRISPR v2 LentiCRISPR vector LentiCRISPR

5 protocols using lenticrispr v2 blast vector

1

Molecular Cloning for Genetic Manipulation

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shRNAs were cloned into the pHIV7-GFP lentiviral vector. sgRNAs were cloned into lentiCRISPR v2 vector (Addgene plasmid # 52961) or lentiCRISPR v2-Blast vector (Addgene plasmid # 83480). The sequences for shRNAs and sgRNAs were listed in Supplemental table 11. The WT or mutant PUS7 (D256A)47 was cloned into the CSC lentiviral vector. The reporter plasmids were prepared by inserting 6 x CGG or 6 x CGA sequences before the firefly luciferase coding region in the pmirGlo luciferase expression vector (Promega) to obtain the 6 x Arg (CGG) or the 6 x Arg (CGA) reporter plasmid. The TYK2 fragment plasmids were cloned by replacing the eGFP sequences in the pLENTI-DDK-puro-eGFP vector (Addgene plasmid #123299) with the WT or mutant TYK2 fragment (aa 100- aa 264).
Cui Q., Yin K., Zhang X., Ye P., Chen X., Chao J., Meng H., Wei J., Daniel R., Li L., Qin Y., Sun G., Zhang M., Klein J., Huynhle M., Wang C., Zhang L., Badie B., Kalkum M., He C., Yi C, & Shi Y. (2021). Targeting PUS7 suppresses tRNA pseudouridylation and glioblastoma tumorigenesis. Nature cancer, 2(9), 932-949.
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2

CRISPR Lentiviral Cloning Workflow

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sgRNAs were cloned into the lentiCRISPR v2-Blast vector (Addgene #83480). Three sgRNAs per gene were designed. For BRCA2 exon 11 was targeted and for ARID1A exon 1 was targeted within a 250 bp region (key resources table). sgRNAs were cloned into the vector using BsmBI digest. Lentiviral particles were produced by transient transfection of HEK293T cells with sgRNA vectors along with the packing constructs PAX2 and VSV-G. Virus-containing supernatants were harvested on Day 4-5 after transfection and passed through a 0.45um filter. Viral supernatant was ultra-centrifuged at 10’500 rpm for 2 h and afterward stored at −80°C.
Hirt C.K., Booij T.H., Grob L., Simmler P., Toussaint N.C., Keller D., Taube D., Ludwig V., Goryachkin A., Pauli C., Lenggenhager D., Stekhoven D.J., Stirnimann C.U., Endhardt K., Ringnalda F., Villiger L., Siebenhüner A., Karkampouna S., De Menna M., Beshay J., Klett H., Kruithof-de Julio M., Schüler J, & Schwank G. (2022). Drug screening and genome editing in human pancreatic cancer organoids identifies drug-gene interactions and candidates for off-label therapy. Cell Genomics, 2(2), 100095.
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3

Molecular Cloning for Genetic Manipulation

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shRNAs were cloned into the pHIV7-GFP lentiviral vector. sgRNAs were cloned into lentiCRISPR v2 vector (Addgene plasmid # 52961) or lentiCRISPR v2-Blast vector (Addgene plasmid # 83480). The sequences for shRNAs and sgRNAs were listed in Supplemental table 11. The WT or mutant PUS7 (D256A)47 was cloned into the CSC lentiviral vector. The reporter plasmids were prepared by inserting 6 x CGG or 6 x CGA sequences before the firefly luciferase coding region in the pmirGlo luciferase expression vector (Promega) to obtain the 6 x Arg (CGG) or the 6 x Arg (CGA) reporter plasmid. The TYK2 fragment plasmids were cloned by replacing the eGFP sequences in the pLENTI-DDK-puro-eGFP vector (Addgene plasmid #123299) with the WT or mutant TYK2 fragment (aa 100- aa 264).
Cui Q., Yin K., Zhang X., Ye P., Chen X., Chao J., Meng H., Wei J., Daniel R., Li L., Qin Y., Sun G., Zhang M., Klein J., Huynhle M., Wang C., Zhang L., Badie B., Kalkum M., He C., Yi C, & Shi Y. (2021). Targeting PUS7 suppresses tRNA pseudouridylation and glioblastoma tumorigenesis. Nature cancer, 2(9), 932-949.
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4

Lentiviral CRISPR Screening Protocol

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sgRNAs were cloned into the lentiCRISPR v2-Blast vector (Addgene #83480). Three sgRNAs per gene were designed. For BRCA2 exon 11 was targeted and for ARID1A exon 1 was targeted within a 250 bp region (Star Methods Table). sgRNAs were cloned into the vector using BsmBI digest. Lentiviral particles were produced by transient transfection of HEK293T cells with sgRNA vectors along with the packing constructs PAX2 and VSV-G. Virus-containing supernatants were harvested on Day 4-5 after transfection and passed through a 0.45um filter. Viral supernatant was ultra-centrifuged at 10’500 rpm for 2h and afterwards stored at -80°C.
Hirt C.K., Booij T.H., Grob L., Simmler P., Toussaint N.C., Keller D., Taube D., Ludwig V., Goryachkin A., Pauli C., Lenggenhager D., Stekhoven D.J., Stirnimann C.U., Endhardt K., Ringnalda F., Villiger L., Siebenhüner A., Karkampouna S., De Menna M., Beshay J., Klett H., Kruithof-de Julio M., Schüler J, & Schwank G. (2022). Drug screening and genome editing in human pancreatic cancer organoids identifies drug-gene interactions and candidates for off-label therapy. Cell Genomics, 2(2), 100095.
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5

Optimizing CRISPR/Cas9 Plasmid Systems

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We utilized the pX330-U6-Chimeric_BB-CBh-hSpCas9 plasmid, obtained from Feng Zhang (Addgene #42230), as the basis for constructing CRISPR/Cas vectors. To generate the mAID donor plasmids, we modified constructs of the Kanemaki lab (Addgene #72827 and #121180). In order to incorporate mRuby2, we replaced mCherry2 in the donor plasmid (Addgene #121180).
For the rescue experiments, wild-type (WT) UHRF1 and each of the point mutants (M8R/F46V, Y188A, DAEA, G448D, and H741A) were cloned into pLenti6.2/V5-DEST (invitrogen). Likewise, WT DNMT1 and each of the point mutants (H170V, D381A/E382A/S392A, W464A/W465A, C1226W) were cloned into pSBbi-Bla (Addgene: #60526). To target DNMT3A and DNMT3B, we cloned the oligonucleotide sequences for gRNA into the lentiCRISPR v2-Blast vector (Addgene #83480). Additionally, we cloned the shRNA targeting TET2 into the pLKO.1-blast vector (Addgene #26655). Plasmids were generated using PCR, restriction enzymes, or Gibson Assembly Cloning techniques. All plasmids underwent sequencing prior to their utilization. The oligonucleotide sequences inserted into the lentiCRISPR v2-Blast vector and pLKO.1-blast vector are available in Supplementary Table 1.
Yamaguchi K., Chen X., Rodgers B., Miura F., Bashtrykov P., Bonhomme F., Salinas-Luypaert C., Haxholli D., Gutekunst N., Aygenli B.Ö., Ferry L., Kirsh O., Laisné M., Scelfo A., Ugur E., Arimondo P.B., Leonhardt H., Kanemaki M.T., Bartke T., Fachinetti D., Jeltsch A., Ito T, & Defossez P.A. (2024). Non-canonical functions of UHRF1 maintain DNA methylation homeostasis in cancer cells. Nature Communications, 15, 2960.
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