...Systemic capsids packaging Cre-dependent transgenes can also be paired with Cre-expressing transgenic mouse...with appropriate cell type-specific promoters or Cre lines. Spatial transcriptomics suggests that AAV-...Huber, N., Pasca, S. P., & Gradinaru, V. (2016). Cre-dependent selection yields AAV variants for widespread...Deverman, B. E., & Gradinaru, V. (2020). Multiplexed Cre-dependent selection yields systemic AAVs for targeting...have developed techniques like CREATE (Deverman et al., 2016), M-CREATE (Ravindra Kumar et al., 2020),...Research in Neurobiology, 4, 100086. https://doi.org/10.1016/j.crneur.2023.100086. Challis, R. C., Ravindra...

...and Genome Editing, 3–4, 100013. https://doi.org/10.1016/j.ggedit.2022.100013 O’Brien, R. E., Bravo, J...part of the Class 1 CRISPR family, which means it requires multiple Cas effectors to carry out genomic editing...different compositions of the Cascade complex. Created with BioRender.com.   The Cascade complex Cascade...Figure 2: Cascade complex from CRISPR type I-E. Created with BioRender.com. Thanks to this complex,...Tomoji Mashimo lab and the Tilmann Weber lab — to increase efficiency of these systems, though consistent...General overview of the Cascade-Cas3 mechanism. Created with BioRender.com.   The reeling mechanism...unwinding of dsDNA occur simultaneously, which creates a loop in the target strand that is cut occasionally...

...useful tool for marking and tracking cells is the the Cre/lox recombination system. A researcher can flank ...expression is turned on or off in cells expressing Cre. The result of a system like this is a mosaic of ...respectively. The permanent labelling of cells using cre-lox is advantageous when compared to photoactivable... In the past 15 years efforts have been made to create transgenic mice expressing fluorescent protein ...auto-fluorescence (Drobizhev et al. 2011). But this requires other lasers than the standard Ti:Sapphire laser...wavelength as it has been shown that even a small incremental change of the excitation wavelength can significantly...called Brainbow, the Ubow mouse strain has been created to fate map Langerhans cells and follicular dendritic...

... Zinc Finger Consortium: OPEN Reagents (Kit # 1000000013 ) Depositing Labs: Keith Joung This kit consists...17. PubMed . Description Use of the OPEN method requires OPEN pools (available by request from the Joung...paper . How to Cite this Kit These plasmids were created by your colleagues. Please acknowledge the Principal...Investigator, cite the article in which they were created, and include Addgene in the Materials and Methods...publication was a gift from Keith Joung (Addgene kit # 1000000013)" For your Reference section: Oligomerized pool...

...breaks caused by Cas12 create short 3’ overhangs, which leads to slightly increased efficiency when conducting...and Genome Editing, 3–4, 100013. https://doi.org/10.1016/j.ggedit.2022.100013 Additional resources on ...systems has led to extreme sequence divergence that requires a multi-pronged analysis to sort effectively. ...type to subtype to variant (shown in italics). Created with BioRender.com.   Class 1 versus class ...underutilized compared to Class 2. Class 1 Despite their incredible abundance, Class 1 CRISPR systems are less popular...Cascade. As you might imagine, this is handy for creating large genomic deletions. Type I systems are also...with Cas3 (pink). Complex shown is from type I-E. Created with BioRender.com.   Type III Type III is ...

...channel pipette 20–200 µL single channel pipette 200–1000 µL single channel pipette 1–10 µL multichannel pipette...enough master mix for all samples). Each sample requires 15 μL of master mix. Pro-Tips Use a "Universal...95 uL 20X 200X Dilution 3 20 uL Dil. 2 80 uL 5X 1000X Dilution 4 20 uL Dil. 3 80 uL 5X 5000X Dilution ...indicates the presence of primer dimers which can increase background signal and alter the Ct values of your...

... ligase The Roux Lab in 2016 optimized BioID to create BioID2, a smaller and improved promiscuous biotin..., BioID2 exhibits enhanced proximal labelling, requires less biotin, and allows scientists to more selectively...was its low sensitivity. Sensitivity could be increased with higher expression levels but for many fusion...until they identified a mutant with significantly increased sensitivity that did not affect the function or...miniTurbo were developed in the Ting Lab in 2018 to increase the speed of proximity labeling by promiscuous...APEX2, although it can rapidly label proteins, requires H2O2 which can be toxic to living cells. To circumvent... BASU exhibits 1,000 fold faster kinetics and increases the signal to noise ratio 30-fold compared to ...

...used for large scale edits. These proteins, like Cre recombinase or phage derived serine integrases, insert... GAT; increase nuclease fidelity SpCas9-NG - NG; increase in vitro activity SpG - NGN; increase nuclease...others work to increase Cas9’s proofreading capabilities. No matter the method, increased fidelity enzymes...backbone HypaCas9 - increase Cas9 proofreading and discrimination evoCas9 - decrease off-target effects...domains to expand PAM recognition, increase specificity, and decrease off-target effects Sniper-Cas9 - ...compatible with truncated gRNAs to increase specificity SuperFi-Cas9 - increases fidelity with reduced nuclease...integration of donor DNA. Genome-Wide Screens Using CRISPR In a genetic screen, a population of cells with mutations...

...C-terminal fluorescent protein tags that can be used to create fluorescent protein fusions with your protein of...expression of a recombinant protein with a 6x His tag. Requires a strain with T7 RNA polymerase. Amp resistant...in response to green light. BIOFAB plasmid set 1000000037 N/A N/A Drew Endy This kit contains plasmids ...expression from that element. When this activation requires a transcription factor to bind to a small molecule...heterodimer formation with dominant negative AraC decreases GFP expression. Zinc Finger Consortium: Modular...strain for performing a bacterial two-hybrid (B2H) screen of modularly assembled zinc fingers. Return to ...

...reactions. The BP reaction creates an attL-flanked entry clone. The LR reaction creates an expression clone ...entry clone and destination vector creates two products: the desires expression clone and a byproduct containing...reactions. Recombination of attP and attB sites creates attL and attR sites.   Gateway vectors contain...Gateway plasmids.    Figure 3: Method A to create an entry clone: recombination of an attB-flanked...attL-containing entry vector.   Figure 4: Method B to create an entry clone: TOPO cloning the insert into an...attL-containing entry vector.   Figure 5: Method C to create an entry clone: Restriction cloning the inset into...vector will recombine with the attL-entry clone to create the expression clone.    Figure 6: Generating...

...fluorescent signal. Sanger can sequence approximately 500-1000 bases downstream of the known primer region with...study DNA fragments of interest, such as genes. Creation of recombinant DNA Molecular Cloning Plasmids ... With current cloning technology, it is easy to create and modify plasmids containing the genetic element...vector can also include an enhancer sequence which increases the amount of protein or RNA produced. Expression...into target cells. You can use these plasmids to create viral particles, such as lentiviral, retroviral...used in place of ampicillin. Preparing Antibiotics Create a stock solution of your antibiotic. Unless otherwise... dissolved in dH 2 0. Addgene recommends making 1000X stock solutions and storing aliquots at -20°C. To...

...sonication to break DNA up into fragments of 300-1000 bps in length. Note: This sonication process may...cytometry requires a negative control, a small subset of the sample with no antibodies. It also requires single-antibody... cell cultures using hybridomas, cell cultures created by fusing B cells with myeloma cells (immortalized...immortalized B cells). The genetic reorganization that creates variety in the antibody allowing for their extreme...animals immunized as described above are used to create hybridomas, which produce large amounts of a single...recombinant antibodies. Recombinant antibodies are created by cloning antibody genes from specific hybridoma...goes through a randomized reorganization process, creating a unique binding site. This process, known as ...

...used for large scale edits. These proteins, like Cre recombinase or phage derived serine integrases, insert... GAT; increase nuclease fidelity SpCas9-NG - NG; increase in vitro activity SpG - NGN; increase nuclease...others work to increase Cas9’s proofreading capabilities. No matter the method, increased fidelity enzymes...backbone HypaCas9 - increase Cas9 proofreading and discrimination evoCas9 - decrease off-target effects...domains to expand PAM recognition, increase specificity, and decrease off-target effects Sniper-Cas9 - ...compatible with truncated gRNAs to increase specificity SuperFi-Cas9 - increases fidelity with reduced nuclease...integration of donor DNA. Genome-Wide Screens Using CRISPR In a genetic screen, a population of cells with mutations...