...10.1146/annurev-biophys-062215-010822 Nishimasu, H., Ran, F. A., Hsu, P. D., Konermann, S., Shehata, S. I.... NUC HNH Cut DNA target strand RuvC Cut DNA non-target strand CTD (or PI) Recognize PAM...information on these regions.   Cas9’s transition from weak, random binding to precision gene editing requires...left) and transparent (right). The RNA guide sequence is extended as a single unpaired strand in the center...10 of the guide RNA, prearranged for base pairing with a complementary DNA strand. These 10 nucleotides...the non-target strand for the PAM sequence and doesn’t mind if the PAM complementary strand is mismatched...of the PAM in the non-target DNA strand (dark gray). Target DNA strand, light gray.   Once it has found...

...Gileadi lab Xenopus SP6 for transcription and injection pCS2P+ - Transcription from the SP6... elegans Transgenic Expression in organisms pBigT - For making transgenic mice. Contains...Tools for nematode transgenesis Rosenberg Lab Plasmids - Transgenic single-copy gene ...expression and packaging Reporters, shRNA expression, transgenics and genome modification Species-Specific...Mammalian CMV, SV40, EF1a, CAG pSG5L Flag HA - Transient expression pWPXL - Lentiviral ...expression pBI-MCS-EGFP - Tet-inducible Transient expression - Gradia Lab plasmids... high expression of UAS-driven transgenes Worm unc-54, variety of worm gene promoters C...

...reprogramming factors Efficient germ-line transmission obtained with transgene-free induced pluripotent stem cells...Reprogramming Plasmids Differentiation and Transdifferentiation Plasmids Stem Cell Research Plasmids Stem...differentiated somatic cell into another via transdifferentiation. This method of cell lineage conversion ...technology. Browse Addgene's collection of Transdifferentiation Plasmids below. Many of the techniques for... for reprogramming, differentiation, and transdifferentiation utilize plasmids to increase expression ...human Klf4, Oct4, c-Myc, and Sox2 as VP-16 transcriptional activating fusions in single lentiviral vectors...human iPSC generation OCT4 and SOX2 Work as Transcriptional Activators in Reprogramming Human Fibroblasts...

...For easy-to-transfect cell types (e.g. HEK293 cells), transfection with standard transfection reagents may...mammalian cell line that can be transfected at high efficiency Lentiviral transduction Cas9 and gRNA can be present...present in a single lentiviral transfer vector or separate transfer vectors May contain reporter gene ...screens using CRISPR AAV transduction CRISPR elements are inserted into an AAV transfer vector and used to ...ribonucleoprotein (RNP) complexes are advantageous due to the transient Cas9 expression. The table below summarizes the...details, see Plasmids 101: Mammalian Vectors ) Transient or stable expression of Cas9 and/or gRNA in a ... mammalian cell lines Useful for difficult to transfect cell types, can also be used in vivo A common ...

...introduce gRNA-targeted single-strand breaks in DNA instead of the double-strand breaks created by wild type... opposite strands of your DNA in close proximity. These double nicks create a double-strand break (DSB...dead dCas9 fused to a transcriptional activator peptide can increase transcription of a specific gene. ... BspQI Transfection Puromycin yes, cut Ji-Long Liu 45946 pU6-BbsI-chiRNA dU6 BbsI Transfection none, need...Genome Editing Cut Base Edit Nick Prime Edit Transcriptional Regulation Activate Interfere Epigenetics RNA...functional CRISPR/Cas enzymes will introduce a double-strand break (DSB) at a specific location based on a gRNA-defined... Edit Cas9 H840A nickase fused to a reverse transcriptase (RT) is capable of installing targeted insertions...

...testing transcription factor binding to reporter plasmid pH3U3-zif268 (omega) . Transcription factors ... answer many interesting questions. Does my transcription factor bind to a particular sequence? Do these...wild-type Cas9 from S. pyogenes for inducing double stranded breaks. For a more comprehensive list of our bacterial...see here . pGRG Series Various Plasmids Tn7 Transposition Nancy Craig A gene of interest cloned into one... one of these plasmids can be transposed into the attTn7 attachment site found in many enterobacterial...enterobacterial genomes. The bacteria of interest are transformed with the appropriate construct, grown at the permissive...permissive temperature (32°C) to allow transposition, and the construct is then cured by growing at the ...

...levels of transgene expression. Additionally, these viruses are able to target a broad range of dividing...genome. Disadvantages of adenoviruses include transient transgene expression, as well as the substantial immune...a class of medium-sized, non-enveloped, double-stranded DNA viruses known to cause mild to severe respiratory...both plasmids. This DNA molecule can then be transfected into mammalian packaging cell lines to generate...of interest? Search our database for adenoviral transfer plasmids that have inserts. ID Plasmid Type Description... Shuttle For production of viruses containing transgene under a chosen promoter Vogelstein 16403 pShuttle-CMV... Shuttle For production of viruses containing transgene under CMV promoter Vogelstein 16404 pAdTrack Shuttle...

...mutation, the best HR template for transfection is probably a single stranded DNA (ssDNA) oligo which usually...positively transfected. Alternatively, you can use a selection marker to select transfected cells (for...nickase doesn't have the ability to induce double-stranded breaks like the wildtype Cas9 does. There are ... The human U6 promoter prefers a 'G' at the transcription start site to have high expression, so adding...need to add an additional 'G'. Where are double strand breaks (DSBs) induced, compared to where the target..., the most efficient and easiest way is to co-transfect several plasmid together, with each plasmid having...two spacers for each locus into PX330, then co-transfect or delivery the two plasmids together into your...

...Plant transcription factors for Agrobacterium-mediated transient expression or plant transformation - Silin...parts for assembling plant transformation vectors - Daniel Voytas. Transcription Factors The following collections...expression in plants: Maize Transcription Factor ORFeome - John Gray. Rice Transcription Factor ORFeome - John...Plant Base Editors Save Time with Transient Plant Leaf Transformations RNA Interference in Plant Biology... Addgene Blog: Plant Biology CRISPR Cloning Transcription Factors RNAi Empty Backbones Fluorescent Proteins...Engineered biocontainable RNA virus vectors for non-transgenic genome editing across crop species and genotypes...several cassettes and generate multi-construct transgenic plants - Jan Lohmann. MoClo Plant Parts II and...

...introduce gRNA-targeted single-strand breaks in DNA instead of the double-strand breaks created by wild type... opposite strands of your DNA in close proximity. These double nicks create a double-strand break (DSB...dead dCas9 fused to a transcriptional activator peptide can increase transcription of a specific gene. ...functional CRISPR/Cas enzymes will introduce a double-strand break (DSB) at a specific location based on a gRNA-defined...Genome Editing Cut Base Edit Nick Prime Edit Transcriptional Regulation Activate Interfere Epigenetics RNA...base editors convert C->T (or G->A on the opposite strand) within a small editing window specified by the...guanosine to create A->G (or T->C on the opposite strand) mutations. ID Plasmid Promoter PI Publication ...

...introduce gRNA-targeted single-strand breaks in DNA instead of the double-strand breaks created by wild type... opposite strands of your DNA in close proximity. These double nicks create a double-strand break (DSB...dead dCas9 fused to a transcriptional activator peptide can increase transcription of a specific gene. ...functional CRISPR/Cas enzymes will introduce a double-strand break (DSB) at a specific location based on a gRNA-defined...Genome Editing Cut Base Edit Nick Prime Edit Transcriptional Regulation Activate Interfere Epigenetics RNA...Base editors convert C->T (or G->A on the opposite strand) within a small editing window specified by the...guanosine to create A->G (or T->C on the opposite strand) mutations. CRISPR-X, a base-editing system from...

...reverse transcriptase and integrase. Once in a target cell, the virus uses the reverse transcriptase in tow...long-term transgene expression. Some disadvantages of γ-retroviruses are that they can only transduce dividing...Kasparov Popular γ-Retroviral Transfer Plasmids Common γ-retroviral transfer plasmids. Want a retroviral...Plasmid for transgene expression; also expresses luciferase Lowe 9044 pMIG MSCV Plasmid for transgene expression...Addgene, including popular packaging, envelope, and transfer plasmids. Viral...envelope). Additionally, γ-retroviruses integrate randomly into the host's genome, which can lead to oncogenesis...of interest? Search our database for retroviral transfer plasmids that have inserts. ID Plasmid 5'LTR Description...

...material will be rearranged. The schematic below shows the three types of rearrangements: inversion, deletion...deletion and translocation. Inversion: If the loxP sites are on the same DNA strand and are in opposite... maintained). Translocation: If the sites are on separate DNA molecules, a translocation event is generated...evaluating infection/transfection efficiency, selecting desired clones, and making transgenic mice. ID Plasmid...Cre-Lox system and how it can be used for genetic rearrangements and recombination. Use Addgene's curated tables... Mammalian Luo 47117 pT3TS-Cre for in vitro transcription of Cre lac Bacterial Ekker 47551 pDD104 (Peft...pTC-CMV-Tet CreER expression and tetracyclin-dependent transgene/shRNA expression ApoE.HCR.hAAT Mammalian Ehmer...

...introduce gRNA-targeted single-strand breaks in DNA instead of the double-strand breaks created by wild type... opposite strands of your DNA in close proximity. These double nicks create a double-strand break (DSB...dead dCas9 fused to a transcriptional activator peptide can increase transcription of a specific gene. ...Genome Editing Cut Base Edit Nick Prime Edit Transcriptional Regulation Activate Interfere Epigenetics RNA...functional CRISPR/Cas enzymes will introduce a double-strand break (DSB) at a specific location based on a gRNA-defined...base editors convert C->T (or G->A on the opposite strand) within a small editing window specified by the...guanosine to create A->G (or T->C on the opposite strand) mutations. ID Plasmid Gene/Insert Promoter Selectable...

...interfering RNA) via transfection, or shRNA (short hairpin RNA) via transfection or viral delivery of ...silencing is achieved through the delivery of a double-stranded RNA (dsRNA) that matches the mRNA target sequence...expression; Designed for inducing RNAi in a wide range of cell types, tissues and organisms Luk Parijs ...or off PI 11643 pLVCT-tTR-KRAB 2nd generation; Transgene (CAG promoter) ‐ OR ‐ shRNA (H1 promoter when ...Trono 11779 pLVCT-rtTR-KRAB-2SM2 2nd generation; Transgene (CAG promoter) ‐ OR ‐ shRNA (H1 promoter when ...Didier Trono 11644 pLVET-tTR-KRAB 2nd generation; Transgene (hEF-1alpha promoter) ‐ OR ‐ shRNA (H1 promoter...Didier Trono 11642 pLVPT-tTR-KRAB 2nd generation; Transgene (hPGK promoter) ‐ OR ‐ shRNA (H1 promoter when...

...Adenoviruses have high transduction efficiency and direct high but transient transgene expression. γ-Retrovirus...virus for gene transfer in clinical trials. These viruses have a relatively high transduction efficiency,...Dividing/Non-dividing Yes Low AAV Transient or Stable* single stranded linear DNA ∼4.5 Kb 18-26 Dividing.../Non-dividing No* Very Low Adenovirus Transient double stranded linear DNA >8 Kb 105 Dividing/Non-dividing...are versatile tools because of their ability to transduce non-dividing cells. These viruses enable long-...immunogencity. These viruses can direct long-term transgene expression, but generally are not permanently ...permanently into the host genome, enabling long term transgene expression. Overview of Viral Plasmids While plasmids...

...introduce gRNA-targeted single-strand breaks in DNA instead of the double-strand breaks created by wild type... opposite strands of your DNA in close proximity. These double nicks create a double-strand break (DSB...dead dCas9 fused to a transcriptional activator peptide can increase transcription of a specific gene. ...Genome Editing Cut Base Edit Nick Prime Edit Transcriptional Regulation Activate Interfere Epigenetics RNA...functional CRISPR/Cas enzymes will introduce a double-strand break (DSB) at a specific location based on a gRNA-defined...base editors convert C->T (or G->A on the opposite strand) within a small editing window specified by the...guanosine to create A->G (or T->C on the opposite strand) mutations. ID Plasmid Gene/Insert Promoter Selectable...

...proteins by size. Transfer proteins from the gel to a membrane. Incubate the membrane with an unconjugated...block the membrane to remove excess antibody and prevent unwanted binding. Incubate the membrane with a ...Wash and block the membrane. Activate the HRP with a substrate kit. Image the membrane using either X-ray...hybridoma cells into plasmids. Plasmids are then transfected into cells and induced to express antibodies....variable regions, shown in light blue and light orange, that bind to a specific antigen, triggering an...constant region, and is shown in dark blue and dark orange. The constant region is a stable part of the antibody...responsible for the variable region goes through a randomized reorganization process, creating a unique binding...

...mEGFP Sialyltransferase 1 Golgi 109122 NPM1-mEGFP AICSDP-50 mEGFP Nucleophosmin Nucleolus (granular component...124606 SOX2-mEGFP AICSDP-60 mEGFP Transcription factor SOX-2 Transcription Factor 124607 ACTN2-mEGFP AICSDP...Nucleolus (granular component) 133963 UBTF-HaloTag AICSDP-80 HaloTag Nucleolar transcription factor UBF...101784 SLC25A17-mEGFP AICSDP-27 mEGFP Peroxisomal membrane protein PMP34 Peroxisomes 101785 TUBA1B-mTagRFP-T...AICSDP-42 mTagRFPT CAAX domain of K-Ras Plasma Membrane 114403 LMNB1-mTagRFP-T AICSDP-29 mTagRFPT Lamin...mEGFP RNA-binding protein FUS Paraspeckles/stress granules 114411 TNNI1-mEGFP AICSDP-34 mEGFP Troponin I,...159745 TFAM-mEGFP AICSDP-72 mEGFP Mitochondrial transcription factor A (TFAM) Mitochondrial nucleoids 164499...

...SFFV ALS Clifford Brangwynne 101224 pHR-FUSN-mCh-Cry2olig FUS SFFV ALS Clifford Brangwynne 101226 pHR-HNRNPA1C-mCh-Cry2WT...Clifford Brangwynne 122441 pHR-FUSN-miRFP670-Cry2WT FUS Cry2WT, miRFP670 SFFV ALS Clifford Brangwynne 122668...U6 Parkinson's Frank Soldner 180433 pU6-pegRNA-LRRK2-G2019S-3b LRRK2 U6 Parkinson's Frank Soldner 180434...U6 Parkinson's Frank Soldner 180435 pU6-pegRNA-LRRK2-G2019S-3d LRRK2 U6 Parkinson's Frank Soldner 180436... Parkinson's Frank Soldner 180437 Px330-EGFP-LRRK2-CRISPR/Cas9 LRRK2 U6 Parkinson's Frank Soldner 180439...Parkinson's Randy Schekman 205713 mNeonGreen-alpha-synuclein SNCA mNeonGreen CMV Parkinson's Randy Schekman... CMV Parkinson's Randy Schekman 205715 alpha-synuclein-3H SNCA CMV Parkinson's Randy Schekman 205822 CL20...