pKLV2-U6gRNA5(BbsI)-PGKpuro2ABFP-W

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	For goldengate reaction, there is a low and a high concentration mixture available for T4 ligase. For single inserts, the low concentration is just fine, but the follow-up article on golden gate cloning (PMID: 19436741) found that the high-concentration T4 was better for multiple insert cloning.

Protocol tips
For goldengate reaction, there is a low and a high concentration mixture available for T4 ligase. For single inserts, the low concentration is just fine, but the follow-up article on golden gate cloning (PMID: 19436741) found that the high-concentration T4 was better for multiple insert cloning.

Publication protocol

Plasmids, Cell Lines, Mouse Lines, and Reagents
Guide RNA expression vectors with the improved scaffold, pKLV2-U6gRNA5(BbsI)-PKGpuro2ABFP-W and pKLV2.2-h7SKgRNA5(SapI)-U6gRNA5(BbsI)-PGKpruo2ABFP-W, for a single and dual gRNA expression, respectively, were generated in this study and have been deposited with Addgene. The optimized human and murine CRISPR libraries were also available through Addgene. Guide RNA sequences used in a gene-by-gene approach are listed in Table S6. All AML cell lines (MOLM-13, MV4-11, HL-60, OCI-AML2, and OCI-AML3), colon cancer cell line HT-29, and fibrosarcoma cell line HT-1080 were obtained from the Sanger Institute Cancer Cell Line Panel and were mycoplasma free. Cas9-expressing cell lines were generated by lentiviral transduction using pKLV2-EF1aBsd2ACas9-W, and Cas9 activity in individual subclones was tested using a lentiviral reporter pKLV2-U6gRNA(gGFP)-PGKBFP2AGFP-W. A Cas9-expressing mouse line was generated by inserting the human EF1a promoter-driven Cas9 expression cassette into the Rosa26 locus in mouse ESC line JM8 (Pettitt et al., 2009) and is kept in the C57BL/6N background. See also Supplemental Information. All animal studies were carried out in accordance with the Animals (Scientific Procedures) Act 1986 (UK) and approved by the Ethics Committee at the Sanger Institute.

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Reviews

Discussion

Papers

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Manufacturer protocol

Download the product protocol from Addgene for pKLV2-U6gRNA5(BbsI)-PGKpuro2ABFP-W below.

Download manufacturer protocol

Videos

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