hCas9

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

hCas9 (a gift from George Church (Addgene plasmid # 41815))15 was PCR-amplified and placed into the second exon of CD4 in a construct (CD4-DEPE, gift from Marc Schmidt-Supprian) consisting of the CD4 promoter, the distal and the proximal enhancer, exon 1 and parts of exon 2 but lacking the intronic silencer, thus supporting expression in all αβ T cells. Subsequently, the plasmid was digested with NotI. The Crispr design tool at crispr.mit.edu was used to identify the protospacer specific for CD2 Exon 2 (5′-GACTAGGCTGGAGAAGGACC-3′), which was cloned via BbsI into a modified px330 vector (px330ccdBChR), thus replacing a ccdB/chloramphenicol cassette. The transgene was cut out by BciVI and XbaI. Both constructs were injected into FVB/N oocytes. The founders were screened by PCR using the following primers: CD4 Cas9 typ fwd: 5′- tgc tca caa ccc ttt agt tt-3′, CD4 Cas9 typ rev: 5′-ctt ttt atc ctc ctc cac c-3′ (product length: 835bp); U6 fwd: 5′-gag ggc cta ttt ccc atg att cc-3′, T7 gRNA rev: 5′-gca cgc gct aaa aac gga-3′ (product length: 407bp). Animals were kept in barrier-SPF level animal facilities at Technische Universität München according to German Animal Protection Law. Experiments were conducted under the license number 55.2-1-54-2532-2-12 and approved by the Regierung von Oberbayern.

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Papers

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Manufacturer protocol

Download the product protocol from Addgene for hCas9 below.

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