■ 基本信息

■ 質粒屬性

■ 質粒簡介

慢病毒基因敲除空載體

(Empty Backbone) Introduce sgRNA into a lentiviral vector (LentiCRISPR V2) which contains eSpCas9 and puromycin cassette

Greetings!

This plasmid is derived from the Feng Zhang lab’s pLentiCRISPR V2 plasmid (the one that has a highly active puromycin variant, Addgene #52961), and the eSpCas9(1.1) plasmid (Addgene #71814).

Cloning method:

A Fragment of eSpCas9 was amplified from #71814 using primers:

#2eSpCas9-F-EcoRVTTCTGGAAGATATCGTGCTGACCCTGACAC

#2eSpCas9-R-BamHIaaCGGtGGATCCCTTTTTCTTTTTTGCCTGGCCGGC

The reverse primer introduces a BamHI site which is present in #52961 but not #71814.  Primers amplify the 3’ half of eSpCas9 which contains all 3 point mutations described in Slaymaker et al (Science 351:84).

Plasmid #52961 and the PCR product were digested with BamHI and EcoRV. #52961 was also exposed to phosphatase. Fragments were ligated, screened for insert, and sequenced.

How to use:

You can clone sgRNAs into the vector just like you would using pLentiCRISPR V2 using the enzyme BsmBI as previously described by the Zhang lab. This will liberate a 2.2kb fragment on agarose gel. The larger fragment can be gel-purified to use as backbone, or the circular plasmid can be used in Golden Gate reactions.

Notes:

eSpCas9 is reportedly very sensitive to mismatches, including mismatches created by adding a 5’G to increase expression from a U6 promoter (which drives sgRNA expression in pLentiCRISPR-E). Also note that the Kleinsteiver et al Cas9-HF1 construct (Nature 529:490) loses a significant amount of efficiency when using truncated guides, and this may be true for eSpCas9 also but hasn’t been formally tested at the time of plasmid submission.  For this reason, consider using guides that start with a native 5’G.

■ 質粒圖譜

■ 質粒序列

質粒序列請下載：ZK612pLentiCRISPR-E哺乳編輯質粒.txt