細(xì)菌CRISPR/Cas9載體質(zhì)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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| Plasmid | Gene/Insert | Vector Type | Promoter | PI | Publication |
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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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| Plasmid | Gene/Insert | Vector Type | Promoter | PI | Publication |
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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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| Plasmid | Gene/Insert | Vector Type | Promoter | PI | Publication |
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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 | |
|---|---|---|---|---|---|---|---|
| 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 | ||
Last Updated: February 28, 2014
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