细菌CRISPR/Cas9载体质粒列表-BioVector NTCC保藏中心2014 updated

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The following Cas9 and gRNA expression plasmids have been designed for use in bacteria.

Cut

Fully functional Cas9 enzymes designed to introduce a double-strand break (DSBs) at a specific location based on a co-expressed gRNA-defined target sequence. DSBs are preferentially repaired in the cell by non-homologous end joining (NHEJ); a mechanism which frequently causes insertions or deletions (InDels) in the DNA, possibly resulting in frameshifts. If a repair template with high homology to the DNA surrounding the DSB is introduced along with the Cas9 and gRNA plasmids, the cell may instead repair the break using homology-directed repair (HDR). HDR is much less error-prone than NHEJ and can be used to faithfully introduce specific genomic changes.

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pCas9	tracr/Cas9	Bacterial Expression, CRISPR ; E.coli		Marraffini	RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nat Biotechnol. 2013 Jan 29. doi: 10.1038/nbt.2508.	Add to Cart
pET-28b-Cas9-His	Cas9 (Other)	Bacterial Expression, CRISPR		Schier	Efficient mutagenesis by Cas9 protein-mediated oligonucleotide insertion and large-scale assessment of single-guide RNAs. PLoS One. 2014 May 29;9(5):e98186. doi: 10.1371/journal.pone.0098186. eCollection 2014.	Add to Cart
pMJ806	Cas9	Bacterial Expression, CRISPR	T7	Doudna	A Programmable Dual-RNA-Guided DNA Endonuclease in Adaptive Bacterial Immunity. Science. 2012 Jun 28.	Add to Cart
pwtCas9-bacteria	wild-type Cas9	Bacterial Expression, CRISPR	pLtetO-1	Qi	Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression. Cell. 2013 Feb 28;152(5):1173-83. doi: 10.1016/j.cell.2013.02.022.	Add to Cart
pCRISPomyces-2	sSpCas9 (Synthetic), gRNA cassette (Synthetic)	Bacterial Expression, CRISPR	rpsL(XC)-BbsI, gapdhp(EL)	Zhao	High-Efficiency Multiplex Genome Editing of Streptomyces Species Using an Engineered CRISPR/Cas System. ACS Synth Biol. 2014 Dec 8.	Add to Cart
pET28a/Cas9-Cys	Cas9-Cys (Other)	Bacterial Expression	T7 Promoter	Kim	Gene disruption by cell-penetrating peptide-mediated delivery of Cas9 protein and guide RNA. Genome Res. 2014 Apr 2.	Add to Cart
DS-SPcas	Cas9 (Other), tracrRNA precursor (Other)	CRISPR	proC, tracrRNA promoter	Church	Orthogonal Cas9 proteins for RNA-guided gene regulation and editing. Nat Methods. 2013 Sep 29. doi: 10.1038/nmeth.2681.	Add to Cart
pCas	cas9 (Other)	Bacterial Expression, CRISPR ; Lambda Red recombinase	native cas9 promoter	Yang	Multigene editing in the Escherichia coli genome using the CRISPR-Cas9 system.Appl Environ Microbiol. 2015 Jan 30. pii: AEM.04023-14.	Add to Cart
pCRISPomyces-1	sSpCas9 (Synthetic), tracrRNA (Other), crRNA cassette (Synthetic)	Bacterial Expression, CRISPR	rpsLp(XC)-BbsI, rpsLp(CF), gapdhp(EL)	Zhao	High-Efficiency Multiplex Genome Editing of Streptomyces Species Using an Engineered CRISPR/Cas System. ACS Synth Biol. 2014 Dec 8.	Add to Cart
pET-Cas9-6xHis	Cas9_6xHis (Other)	Bacterial Expression	T7	Liu	Cationic lipid-mediated delivery of proteins enables efficient protein-based genome editing in vitro and in vivo. Nat Biotechnol. 2015 Jan;33(1):73-80. doi: 10.1038/nbt.3081. Epub 2014 Oct 30.	Add to Cart
pET-Cas9-NLS-6xHis	Cas9-NLS-6xHis (Other)	Bacterial Expression	T7	Liu	Cationic lipid-mediated delivery of proteins enables efficient protein-based genome editing in vitro and in vivo. Nat Biotechnol. 2015 Jan;33(1):73-80. doi: 10.1038/nbt.3081. Epub 2014 Oct 30.	Add to Cart
pET-NLS-Cas9-6xHis	NLS-Cas9-6xHis (Other)	Bacterial Expression	T7	Liu	Cationic lipid-mediated delivery of proteins enables efficient protein-based genome editing in vitro and in vivo. Nat Biotechnol. 2015 Jan;33(1):73-80. doi: 10.1038/nbt.3081. Epub 2014 Oct 30.	Add to Cart
pTrex-b-NLS-hSpCas9	NLS-hSpCas9	CRISPR ; Trypanosoma cruzi expression	Ribo-HX1	Tarleton	CRISPR-Cas9-Mediated Single-Gene and Gene Family Disruption in Trypanosoma cruzi. MBio. 2014 Dec 30;6(1). pii: e02097-14. doi: 10.1128/mBio.02097-14.	Add to Cart
SP-Cas9	SP-CAS9 (Other)	Bacterial Expression, CRISPR	T7	Geijsen	Efficient Intracellular Delivery of Native Proteins. Cell. 2015 Apr 23;161(3):674-690. doi: 10.1016/j.cell.2015.03.028.	Add to Cart

Interfere

A catalytically inactive Cas9 (dCas9) or dCas9-repressor peptide fusion can be used to knock-down gene expression by interfering with transcription of the gene. Design your gRNA sequence to direct the dCas9 repressor to a specific genomic sequence. Potential target locations can include promoter regions, regulatory regions, and early coding regions. If the plasmid that you choose does not also express a gRNA, you will need to use a separate gRNA expression plasmid to target the dCas9 repressor.

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pdCas9	tracrRNA (Other), dcas9 (Other), CRISPR array	Bacterial Expression, CRISPR		Marraffini	Programmable repression and activation of bacterial gene expression using an engineered CRISPR-Cas system. Nucleic Acids Res. 2013 Jun 12.	Add to Cart
pdCas9-bacteria	dCas9 (bacteria) (Other)	Bacterial Expression, CRISPR	pLtetO-1	Qi	Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression. Cell. 2013 Feb 28;152(5):1173-83. doi: 10.1016/j.cell.2013.02.022.	Add to Cart
pMJ841	Cas9 (Other)	Bacterial Expression, CRISPR	T7	Doudna	A Programmable Dual-RNA-Guided DNA Endonuclease in Adaptive Bacterial Immunity. Science. 2012 Jun 28.	Add to Cart
10xHis-MBP-TEV-S. pyogenes dCas9 M1C D10A C80S H840A C574S	dCas9 M1C D10A C80S H840A C574S (Other)	Bacterial Expression		Doudna	Programmable RNA recognition and cleavage by CRISPR/Cas9. Nature. 2014 Sep 28. doi: 10.1038/nature13769.	Add to Cart
DS-SPcasN-	Cas9, nuclease-null (Other), tracrRNA precursor (Other)	Bacterial Expression, CRISPR	proC, tracdrRNA promoter	Church	Orthogonal Cas9 proteins for RNA-guided gene regulation and editing. Nat Methods. 2013 Sep 29. doi: 10.1038/nmeth.2681.	Add to Cart

Activate

A catalytically inactive Cas9 (dCas9) fused to a transcription activator peptide can increase transcription of a gene. Design your gRNA sequence to direct the dCas9-activator to a specific genomic sequence. Potential target locations can include promoter regions and regulatory regions. If the plasmid that you choose does not also express a gRNA, you will need to use a separate gRNA expression plasmid to target the dCas9-activator.

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	pWJ66	tracrRNA (Other), dcas9-w (Other), CRISPR array	Bacterial Expression, CRISPR		Marraffini	Programmable repression and activation of bacterial gene expression using an engineered CRISPR-Cas system. Nucleic Acids Res. 2013 Jun 12.	Add to Cart
	pWJ68	tracrRNA (Other), w-dcas9 (Other), CRISPR array	Bacterial Expression, CRISPR		Marraffini	Programmable repression and activation of bacterial gene expression using an engineered CRISPR-Cas system. Nucleic Acids Res. 2013 Jun 12.	Add to Cart

Empty gRNA Expression Vectors

Select a gRNA expression plasmid based on factors such as selectable marker or cloning method. When using the CRISPR/Cas system, you will need to express both a Cas9 protein and a target-specific gRNA in the same cell at the same time. Single plasmids containing both the gRNA and Cas9 act as an all-in-one vector, but their function (cut, nick, activate, interfere...) is limited to that of the Cas9 present on the plasmid. gRNA plasmids that do not co-express Cas9 require a separate plasmid that does so; however, these independent gRNA plasmids can be paired with a wide variety of Cas9 plasmids and therefore are not limited to a single Cas9 function.

	gRNA Plasmid	Promoter	Cloning Enzyme(s)	Validated In	Resistance	Co-expressed Cas9	Depositing lab
Cas9 species = S. pyogenes (PAM = NGG)
	pCRISPR		BsaI	E. coli, S. pneumoniae	Kanamycin	none, need Cas9 plasmid	Marraffini
	pCas9		BsaI	E. coli, S. pneumoniae	Chloramphenicol	yes, cut	Marraffini
	pgRNA-bacteria	BBa_J23119	SpeI + HindIII		Ampicillin	none, need Cas9 plasmid	Qi