...we'll cover Cas9 multiplexing options, but also check out our blog post about multiplexing with Cpf1.   ...BsaI-based E. coli multiplexing plasmid is available from the Koffas lab. Scaling up your multiplexed gRNAs with...Yamamoto Lab Multiplex CRISPR/Cas9 Assembly Kit: This kit is built for serious multiplexing and enables...with the MuLE system. Multiplexing from a single transcript You can also multiplex gRNAs via a polycistronic...PTG cassette.    Find gRNA multiplexing vectors at Addgene! Multiplexing in plants Qi-Jun Chen Lab Golden...this blog post.  Multiplexing in zebrafish Wenbiao Chen Lab Golden Gate Assembly Multiplex Plasmids: These...supplied on a separate plasmid. Multiplexing in Drosophila Bullock Lab Multiplex Plasmid: Two gRNAs can be ...

...protospacer Multiplexing CRISPR-Cas9 options before Cpf1 crRNA array Prior to this new Cpf1 multiplexing method...Cpf1 counterparts. Table 2. Cas9 Multiplexing Options  Multiplexing Method Delivery Method Advantages...Xie et al  Multiplexing crRNA expression with Cpf1  The key advantage of multiplexing crRNA expression... chart below for a summary of multiplexing efficiency in vivo. Multiplex gene editing with CRISPR-Cpf1...interested in using Cpf1 multiplexing in your research? Check out the Cpf1 multiplexing plasmids available ...Feng Zhang's lab describes how to use Cpf1 for multiplex genome editing.  For a few reasons, Cpf1 is a ...compared to Cas9.  Read on to learn more about Cpf1 multiplexing.  For an in-depth review of Cpf1, check out ...

...scalability and efficiency of multiplex gene editing. Moreover, existing multiplex strategies have been facing...developed DAP arrays for efficient multiplex base editing (MBE) and multiplex prime editing (MPE) with only...Schematic of multiplex gene editing with DAP array   Use DAP arrays can enable efficient multiplex gene ...leveraged such host-defense mechanisms to enable multiplex gene editing in a variety of cells and organisms...from a single array, has been a top choice for multiplex gene knockout or regulations, it lacks proper ...contains a double-win for both efficiency and multiplexity, with these constraints? The Gao lab first ...living organisms and has previously been used for multiplex Cas9-nuclease editing. Typically, a single array...

...Figure 1: Schematic of multiplex immunofluorescence approaches. A) Sequential Multiplex IF, where one target...just want to make some really beautiful images? Multiplex IF may be just what you need! How does it work...work? There are several ways to achieve multiplex IF. One of the most common approaches is to use sequential...before labeling the next target. B) Simultaneous Multiplex IF, where all the primary antibodies are combined...   Figure 2: Examples of simultaneous multiplex IF labeling of rat brain samples from the James...your antibodies When selecting antibodies for multiplexed IF, many of the same considerations apply as ... to avoid secondary antibodies altogether and multiplex with direct IF, using primary antibodies that ...

... construction system for CRISPR/Cas9-mediated multiplex genome engineering is now available thanks to ...new tool in Scientific Reports in June. “The multiplexity is one of the most advantageous properties of...established way of making an all-in-one vector for multiplex genome engineering.” Now, they’ve filled that... also be expanded to include even more loci. Multiplex genome engineering demonstrated in human cells...be applied to efficient viral vector-mediated multiplex genome engineering. The system now provides an...an efficient targeting strategy for multiplex editing of genomes. It will also allow simultaneous activation...see Yamamoto and Sakuma’s protocol. Reference: Multiplex genome engineering in human cells using all-in-one...

...will be necessary to improve its multiplexing capacity. Multiplexing is key due to the natural redundancy... Addgene Other Techniques for gRNA Multiplexing Multiplexed and programmable regulation of gene networks...editing efficiency. One previous strategy for multiplexing gRNAs used the RNA nuclease Csy4 from Pseudomonas... test the application of this system to gRNA multiplexing, Xie et al. constructed PTGs containing 8 gRNAs...mutations at all five loci. This highly efficient multiplexing further illustrates the potential of PTGs. Applications... III-gRNA, which will facilitate CRISPR/Cas9 multiplexing in systems with limited packaging capacity. ...including some simple comparisons. The easy multiplexing afforded by PTGs will improve the utility of...

... and simple to multiplex. Problems with FRET kinase reporters Difficult to Multiplex: FRET reporters...easy to multiplex. Their design allows flexible swapping of fluorescent proteins and multiplexing is simple...reporters are challenging to design and difficult to multiplex. The Covert Lab provides an alternative tool with...properties that limit which fluorophores can be multiplexed together. Challenging  to Design: FRET reporter...

...barcodes for live cell clonal multiplexing by: Rachel Leeson Multiplexed images in live cells remains ...knock-ins Visual barcodes for live cell clonal multiplexing Improving flexibility for in vivo proximity...Viviana Gradinaru Lab has previously developed the multiplexed version of the Cre-mediated AAV Targeted Evolution...lab has developed visual barcodes for clonal multiplexing of cells in live imaging application. Each visual...subclone, they were able to develop a 12-plex multiplex imaging system. Mixing the subclones within a ...researchers to avoid the off-target effects of hi-multiplexing within in a cell, while potentially allowing...

...Digital PCR for AAV Quantification.”   Multiplex PCR Multiplex PCR, as the name implies, is a method in...laboratory.  There are two main categories of multiplex PCR:  Single template PCR reaction - one template... of each template are used in one reaction. Multiplex PCR is used commonly in disease or pathogen identification... time and effort. Scientists can also utilize multiplex qPCR to quantify the concentrations of multiple...sample. It is important to note however, that multiplex PCR is more complicated to develop and is often...single pair of primers, like those stated above. Multiplex PCR is also used in high throughput SNP genotyping...Monitoring subtle changes in target DNA levels Multiplex PCR Amplify multiple targets from one sample ...

...conversion, gives users the flexibility needed for multiplexing. In the case above, you can’t costain with your...antibodies with the same isotype can not be used for multiplexed indirect staining methods since the secondary...the new Fc region. In addition to increasing multiplexing capabilities this process is frequently used...single-plexed experiments, it needs to be addressed when multiplexing. It is imperative that you use narrowly selective...selective isotype-specific secondary antibodies when multiplexing with chimeric antibodies. In the case above ...and mouse 1gG2a primary antibodies used in a multiplexed imaging experiment   We hope that this blog...

...IBEX is a highly multiplexed tissue imaging method that uses iterative staining and chemical bleaching...Radtke et al., 2020, 2022). I do mean highly multiplexed: you can image over 65 different parameters using...an invaluable resource for anyone performing multiplexed imaging, not just IBEX. The IBEX Imaging Community...., & Germain, R. N. (2020). IBEX: A versatile multiplex optical imaging approach for deep phenotyping ...resources on the Addgene blog Antibodies 101: Multiplex Immunofluorescence Negative Can Be Positive: Open...

...lab presented three ways to use CAPTURE 2.0: 1. Multiplex capture of chromatin interactions The Xu lab performed...Long-range chromatin interactions identified with the multiplexed CAPTURE 2.0 largely replicated interactions previously... a large cluster of enhancers. CAPTURE 2.0’s multiplexing and high resolution allowed the Xu Lab to simultaneously...role in gene regulation The high resolution and multiplexed chromatin ‘photos’ generated with CAPTURE 2.0..., Orkin, S.H., Zhang, M.Q., & Xu, J. (2020). Multiplexed capture of spatial configuration and temporal...

...optimizing for LightCycler machines.) Multiplexing It is possible to multiplex qPCR when using fluorescent probe-based...change, the multiplexed protocol may have to be re-optimized.  While troublesome, multiplexing your reference...the Lightcycler while being relatively easy to multiplex (Thellwell, 2000). See Table 1 for a comparison...noise Probe Allelic specificity Can be multiplexed Single amplicon validation technique Hydrolysis... reagents in your chemistry, unless running a multiplex reaction or on a rapid cycle machine. Melt Curves...possible (Vandesompele, 2002), and it's possible to multiplex your reference genes to save on plate space. For...six reactions in a single well. In practice, multiplexing is quite difficult and requires significant ...

...us about a new tool for efficient multiplex base editing and multiplex prime editing (requiring minimal...editing tool for large inserts.  DAP arrays for multiplex gene editing: Guest blogger Qichen Yuan tells ...

...Base editing CRISPRi in Caulobacter crescentus Multiplexed gRNA for human pluripotent stem cells CRISPR-...the paper in mBio Find the CRISPRi plasmids A multiplexed gRNA piggyBac transposon system for human pluripotent... deliver uniform and sustained expression of multiplexed gRNAs in human pluripotent stem cells. Previously... construct and the fluorescently-labeled gRNA multiplex. Read the paper in Scientific Reports Find the...

...as those generated by multiplexed delivery of multiple sgRNAs. Since multiplexing is commonly used to create...edits is critical to the CRISPR workflow. For a multiplex sample, ICE includes visual representations of...visualization helps the researcher see which of the multiplexed sgRNA was involved in a particular edit, and ...can analyze indels that result from single or multiplex CRISPR-Cas9 double-strand DNA breaks using SpCas9...the wild type sequence.  If you are viewing a multiplex sample, the cut site will be aligned to the most...

...Cis/trans activity, PAM requirement, gRNA, and multiplex ability Experimental guidelines and applications...you need more nitty-gritty details — how can I multiplex this enzyme? What other RNA components are required.... For each enzyme, it will also show you how multiplexed arrays would be designed, if that’s your end ...details to refine your search (trans activity, multiplex ability, gRNA design, etc.). With this feature...

...it except in the most dire of circumstances.) Multiplex assays Mo’ proteins, mo’ problems may not be a...common saying, but it probably should be. In a multiplex assay - one with multiple proteins of interest...different species and/or isotypes. For highly multiplexed assays, using secondary antibodies which recognize...of the number of protein targets, simplifying multiplex assays.  A brief note Since the indirect method...

... Depositor Collections MORF Feng Zhang - Multiplexed Overexpression of Regulatory Factors (MORF) Collection...ordering. ID Plasmid Gene/Insert Pooled Library The Multiplexed Overexpression of Regulatory Factors (MORF) pooled...