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We narrowed to 609 results for: CaS;

Showing: 101 - 150 of 609 results

  1. Hot Plasmids and Viral Preps - January 2021

    Type
    Blog Post
    Published
    Jan. 5, 2021, 2:15 p.m.
    ...Hypercompact genome editor CRISPR-Casɸ By Melina Fan The Doudna Lab adds to the CRISPR-Cas toolbox with their discovery...well-characterized Cas enzymes, which rely on multiple active sites. In addition, Casɸ has a minimal PAM...three Casɸ orthologs, Casɸ-1, Casɸ-2, and Casɸ-3. This system is notable for several reasons. Casɸ is only...70kDa, with a molecular weight roughly half of Cas9 or Cas12a. This is in part due to the fact that it only...demonstrate that CRISPR-Casɸ is active in vitro as well as in human and plant cells. Casɸ from Biggiephage...Blue-light inducible Cre for use in bacteria CRISPR-Casɸ mGold, a new yellow fluorescent protein New CRISPR...excision or insertion, inversion, translocation or cassette exchange. By utilizing recombinases to manipulate...

  2. Making CRISPR Plasmids Using Fragmid

    Type
    Blog Post
    Published
    Aug. 29, 2024, 1:15 p.m.
    ... it’s easy to switch out reporters, Cas proteins, guide cassettes, promoters, N’ and/or C’ terminus, and...specific to the type of module it is (Pol 2 protomer, Cas protein, ect...). Whatever individual components ...above selections.) CRISPR enzyme (Cas9 [sp], Cas9 [other], Cas12a, RNA-targeting) CRISPR mechanism ...destination vectors contain an ampicillin resistance cassette and an origin of replication, which are not schematized...using more than six or seven components — in most cases, that's making two or more plasmids — it may be ...

  3. Live and Let Dye: Self-Labeling Protein Tags

    Type
    Blog Post
    Published
    April 16, 2024, 1:15 p.m.
    ... dehalogenase that binds a synthetic chloralkane (CA) ligand. The current standard version known as HaloTag...TMP-tag) Few Easy HaloTag 295 Chloralkane (CA) Many Easy CLIP-tag 182 Benzylcytosine...) COS-7 cell ER labeled with Halo-Sec61β with SiR-CA (magenta) and outer mitochondrial membrane labeled...that your fusion protein is functional. In some cases, a small peptide tag like tetracysteine might be...

  4. A New Generation of Adenine Base Editors Improves Editing in Primary Human Cells

    Type
    Blog Post
    Published
    May 7, 2020, 1:15 p.m.
    ...impaired Cas protein (dCas or Cas nickase)  a guide RNA that targets the complex of TadA and dCas to the.... pyogenes Cas9 in the ABE complex with PAM-variant Cas9 proteins: the S. pyogenes NG-Cas9 (PAM: NG) to...constructs using a catalytically “dead” S. pyogenes Cas9 (dC9-ABE8.x-m/d). The dC9-ABE8.x-m/d constructs ...frequency more than 90% compared to ABE7.10. Choosing Cas9 proteins to increase the targeting scope of adenine... base editors The most commonly used S. pyogenes Cas9 requires the target site in the genomic DNA to contain... to create NG-ABE8.xm/d and the S. aureus Cas9 (PAM: NNGRRT) to create Sa-ABE8.x-m/d. With these variants...frequency over ABE7.10. The ability to use non-standard Cas9 in the ABE8 complexes enables a broader targeting...

  5. CRISPR Cheat Sheet

    Type
    Blog Post
    Published
    Jan. 27, 2021, 2 p.m.
    ...However, prime editing and the identification of other Cas proteins continue to expand the targeting range. ...the gRNAs to cut particular DNA or RNA sequences. Cas9 is a very commonly used CRISPR nuclease. Note:...versions of CRISPR nucleases that can’t cut DNA (dCas9, for example) that are used to increase or decrease...

  6. Hot Plasmids - August 2020

    Type
    Blog Post
    Published
    Aug. 18, 2020, 1:15 p.m.
    ...The MoClo CRISPR/Cas Toolkit for plants includes 95 plasmids consisting of CRISPR/Cas nucleases, base ...multiplexed gRNAs and of dCas9 fusions. The dCas-KRAB activator and the dCas-VPR repressor are introduced...an EGFP reporter. Expression of the dCas9-KRAB activator or dCas9-VPR repressor in hPSCs is also driven...piggyBac gRNA delivery and the all-in-one dCas9-KRAB and dCas9-VPR targeting plasmids can be found at Addgene...approaches rely on continual expression of both dCas9 and the gRNA. This expression can vary significantly... could quantify and track cells expressing both dCas9 and gRNAs. The multi-gRNA piggyBac vector ...plasmids for piggyBac gRNA delivery and the all-in-one dCas9 targeting plasmids! Hazelbaker et al., Sci Reports...

  7. Addgene’s Top 10 Blog Posts from 2014

    Type
    Blog Post
    Published
    Dec. 16, 2014, 8:01 p.m.
    ...by our guest bloggers, the experts in the CRISPR/Cas field, have been extremely helpful for scientists...Plasmids 101: Yeast Vectors By Marcy Patrick CRISPR-Cas9 FAQs Answered! By Caroline LaManna Plasmids 101...

  8. 22 Hot Plasmid Technologies from 2014

    Type
    Blog Post
    Published
    Jan. 6, 2015, 5:21 p.m.
    ...with Addgene which allow scientists to use CRISPR-Cas technology to perform genome-wide knockout screens...these new CRISPR screening tools, see our CRISPR/Cas Plasmids: Pooled Libraries webpage or read our blog...Knockout Screening. Also, check out our updated CRISPR-Cas resources at www.addgene.org/CRISPR/ to browse CRISPR...information on CRISPR technology? See our improved CRISPR-Cas Guide. Wang et al., Science. 2014. Jan 3;343(6166...Guilinger et al., Nat Biotechnol. 2014..) Our CRISPR-Cas collection of plasmids updates frequently, so visit...plasmids available! Interested in purifying Cas9? Check out pET-28b-Cas9-His from Alex Schier's lab, designed...the lab of Yonglong Chen, pCS2-3xFLAG-NLS-SpCas9-NLS is a Cas9 expression plasmid that was used by Chen...

  9. The CRISPR Software Matchmaker: A New Tool for Choosing the Best CRISPR Software for Your Needs

    Type
    Blog Post
    Published
    Nov. 3, 2015, 3:30 p.m.
    ...For this, some online tools such as Cas-OFFinder will suffice. Cas-OFFinder provides a bare-bones data...prove somewhat useful for the latest NTT targeting Cas protein, Cpf1. Separately, on-target efficacy is ... tools yet. As a genome editing tool, the CRISPR/Cas9 technology has been surrounded by a whirlwind of...only one, or a subset of organisms. In only a few cases the software is organism agnostic. Most tools are...will find that some tools cater to specific use-cases through the way their algorithms were developed ...with R. For the Java enthusiasts, take a look at sgRNAcas9. Top tools for transferring CRISPR technology ... parasite Plasmodium falciparum using the CRISPR-Cas9 system." Nature biotechnology(2014). PubMed PMID...

  10. Controlling for Off-target Effects with a New Genome-wide CRISPR Screen Design

    Type
    Blog Post
    Published
    Sept. 13, 2018, 1:55 p.m.
    ...-wide profiling of off-target cleavage by CRISPR-Cas nucleases." Nature biotechnology 33.2 (2015): 187...PMC4320685. 3. Fu, Yanfang, et al. "Improving CRISPR-Cas nuclease specificity using truncated guide RNAs."...effects of gRNA and Cas9 expression, they fail to account for the effects of Cas9-induced dsDNA breaks... either stably expressed Cas9 or that had been lentiviral infected with Cas9. Validating the Library The...Genome-wide CRISPR/Cas9 screens are a high-throughput systematic approach for identifying genes involved.... Unanswered questions about genome-wide CRISPR/Cas9 screen design Are non-targeting guides an appropriate...single-guide studies? Many strategies exist for reducing Cas9 off-target effects, but most studies have focused...

  11. Fueled by Coffee at #SfN14

    Type
    Blog Post
    Published
    Nov. 18, 2014, 7:15 p.m.
    ...I’ve seen evidence of the infiltration of CRISPR-Cas tools into neuroscience experiments. Speaking of...

  12. Optogenetics + CRISPR, Using Light to Control Genome Editing

    Type
    Blog Post
    Published
    Sept. 3, 2020, 12:15 p.m.
    ...Proteins Switch Off CRISPR-Cas Systems.  Listen to the anti-CRISPR podcast segment Simply expressing anti-CRISPR... group of proteins that are able to block CRISPR-Cas systems. They have been used to block genome editing....2014.12.011 Polstein LR, Gersbach CA (2015) A light-inducible CRISPR-Cas9 system for control of endogenous...applied to create photoswitchable spCas9, dCas9, and S. aureus Cas9 (saCas9) proteins, showing the versatility...specificity as full-length Cas9 (flCas9). When triggered by blue light (470nm), paCas9-1 induced indel mutations...proteins from N. crassa Split Cas9, Split Cas9 nickase, Split dCas9 Genome editing by NHEJ and HDR, repression...Photoswitchable Cas9 Richter et al., 2016 tsRC9 RsLOV from R. sphaeroides Cas9, dCas9 Gene editing ...

  13. Designer PUF Proteins for Any RNA Target

    Type
    Blog Post
    Published
    June 17, 2014, 7:57 p.m.
    ...updated versions of the kit.” Reference Abil Z, Denard CA, Zhao H.  Modular assembly of designer PUF proteins...

  14. Addgene's Top Blog Posts from 2020

    Type
    Blog Post
    Published
    Dec. 29, 2020, 2:15 p.m.
    ...SARS-CoV-2/COVID-19 Detection Methods Based on CRISPR/Cas by Shravanti Suresh Guest blogger Shravanti Suresh...

  15. What's New in CRISPR - March 2020

    Type
    Blog Post
    Published
    March 10, 2020, 1:15 p.m.
    ...with inactive Cas enzymes can alter gene expression via acetylation or demethylation. Casilio DNA Methylation...fluorescent dCas9-KRAB and dCas9-VPR for quantifying and tracking cells that express both the dCas9 construct... lower average Cas9-independent off-target DNA editing, ~5–50-fold lower levels of Cas9-dependent off-...Caulobacter genes using a catalytically dead Cas9 (dCas9) derived from Streptococcus pasteurianus  or ...Saccharomyces cerevisiae. The system consists of dCas9, the mammalian transcriptional repressor Mxi1, and...accessible and the complex enters the nucleus where dCas9-Mxi1 can repress gene expression. In the dark, the...s lab identified and validated CBEs with reduced Cas9-independent deamination. They generated these editors...

  16. CRISPR Protocol for Genomic Deletions in Mammalian Cell Lines [Video]

    Type
    Blog Post
    Published
    Feb. 18, 2015, 3:09 p.m.
    ...Plasmids CRISPR Information at Addgene More CRISPR-Cas Blog Posts    ...Genomic Deletions in Mammalian Cell Lines via CRISPR/Cas9" from the Journal of Visualized Experiments (JOVE...and Daniel Bauer's labs details the use of CRISPR/Cas9 to create genomic deletions in mammalian cell lines...motivations behind this research.   Using CRISPR/Cas9 for targeted genomic deletions We were inspired ...with the Porteus lab [2]. With the advent of CRISPR/Cas9, we began to explore the paired double-strand break...disrupted cells can be easily identified. Even in the case of clones with monoallelic deletion, the nondeleted...regularly interspaced palindromic repeats (CRISPR)/Cas9 nuclease system in mammalian cells. J.Biol.Chem....

  17. Sequencing Options for CRISPR Genotyping

    Type
    Blog Post
    Published
    Oct. 4, 2016, 2:30 p.m.
    ...High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells." Nature biotechnology 31.9...-wide profiling of off-target cleavage by CRISPR-Cas nucleases." Nature biotechnology 33.2 (2015): 187...Use Cas9 Activators Perform Genome-Wide Screens with CRISPR Generate Mouse Models with CRISPR/Cas9 Additional...techniques, please see “Methods for Optimizing CRISPR-Cas9 Genome Editing Specificity” by Tycko et al. 2016...improving the genome-wide specificities of CRISPR–Cas9 nucleases”
by Tsai and Joung 2016. Many thanks ...high-throughput screening strategy for detecting CRISPR-Cas9 induced mutations using next-generation sequencing...of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity." Nature biotechnology 31.9...

  23. Antibodies 101: Polyclonal Antibodies

    Type
    Blog Post
    Published
    July 14, 2021, 1:15 p.m.
    ...     References and Resources References Ascoli CA, Aggeler B (2018) Overlooked benefits of using polyclonal...their ability to bind the target antigen. In this case, the serum is run over a column containing the target...

  24. Back to Bacteria: CRISPR gRNA Multiplexing Using tRNAs

    Type
    Blog Post
    Published
    June 2, 2015, 6:06 p.m.
    ... gene networks with an integrated RNA and CRISPR/Cas toolkit in human cells. Nissim L, Perli SD, Fridkin...In the short time since its development, CRISPR/Cas9 genome editing has been used to study the effect...recombination. To further increase the utility of CRISPR/Cas9, it will be necessary to improve its multiplexing...RNAs (gRNAs) are commonly packaged in 400-500 bp cassettes containing the RNA pol III promoter, gRNA and ...and pol III terminator. These relatively large cassettes (considering the gRNA itself is ~100 bases) limit...construct size, it requires coexpression of Csy4 with Cas9 and the polycistronic transcript. As nucleases may... (15% and 9% of target sites.) How many loci can Cas9 modify? To test the application of this system to...

  25. Plasmids 101: FLEx Vectors

    Type
    Blog Post
    Published
    April 28, 2016, 2:30 p.m.
    ...Central PMCID: PMC2593125. 4. Saunders A, Johnson CA, Sabatini BL. 2012. Novel recombinant adeno-associated... switch conditional knock-in cassette. As shown in Figure 2, the cassette should contain a reporter DNA...For this FLEx switch to successfully work, the cassette would need to contain the BFP coding sequence ...antisense orientation (Figure 1, top). The entire DNA cassette would be flanked by two pairs of target sites-...in the opposing orientation, you have created a cassette that will express GeneR only in the presence of...recombination event would excise the reporter from the cassette, allowing for expression of GeneR. This allows...

  26. Multiplex Genome Editing with CRISPR-Cpf1

    Type
    Blog Post
    Published
    May 9, 2017, 2:12 p.m.
    ...Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas System. Cell,163(3), 759-771. PubMed PMID: 26422227...Comparing the Cas9 and Cpf1 CRISPR Nucleases   Cas9 Cpf1 Components crRNAtracrRNACas9 crRNACpf1 ... guide page for a review of Cas9.  For a brief comparison of Cpf1 vs. Cas9, see the table below. Table... Endonuclease size spCas9: ~4 kbsaCas9: ~3 kb ~3.9kb crRNA/gRNA length gRNA: ~100 nt crNA: ~42...promoters).  spCas9 and its gRNAs are also larger than their Cpf1 counterparts. Table 2. Cas9 Multiplexing...Lentiviral Expression Cassettes lentiviral transduction One vector expresses Cas9, eGFP, and up to 4 ... Requires co-transfection of Cas9 Nissim et alTsai et al PTG Cassettes transfection gRNAs expressed ...

  27. CRISPRainbow and Genome Visualization

    Type
    Blog Post
    Published
    Feb. 28, 2017, 3:30 p.m.
    ...Loci in Living Human Cells by an Optimized CRISPR/Cas System.” Cell 155 (2013): 1379-1491. Pubmed PMID:... a colorful Cas9 toolbox, Ma et al. turned to SpCas9 and its orthologs NmCas9 and St1Cas9. Each ortholog...locus, and to do so in vivo. Catalytically dead Cas9 (dCas9) cannot induce gene editing, but it retains its...bright-field image. Image from Ma et al, 2016. Using dCas9 to visualize genomic loci in vivo The flexibility...By using a gRNA to target fluorescently labeled dCas9 to a given genomic locus, you can track the localization...to noise ratio of the system, they modified GFP-dCas9 and the gRNA scaffold to enhance complex assembly...amount of background fluorescence from unbound GFP-dCas9. With these improvements, labeling efficiency was...

  28. Antibodies 101: Fab Fragments

    Type
    Blog Post
    Published
    Sept. 5, 2024, 1:15 p.m.
    ...References and resources References Bates A, Power CA (2019) David vs. Goliath: The Structure, Function...anti-IgG Fab fragments make good secondaries for cases where you need to pre-form primary-secondary antibody...antibody recognizes endogenous antigens. In these cases, even though one full antibody may be able to get...

  29. Antibodies 101: Affinity Reagents

    Type
    Blog Post
    Published
    April 14, 2022, 1:15 p.m.
    ...Elsevier Science & Technology Books. Bates A, Power CA. David vs. Goliath: The Structure, Function, and ...

  30. Plasmids 101: Modular Cloning

    Type
    Blog Post
    Published
    March 12, 2024, 1:15 p.m.
    ...libraries depend on many gRNAs delivered with a relevant Cas nuclease. MoClo can be used to assemble multiplexed... part, will behave in a cell. For unpredictable cases, MoClo provides two answers: first, sets of parts...

  31. Hot Plasmids and Viral Preps - March 2021

    Type
    Blog Post
    Published
    March 23, 2021, 1:15 p.m.
    ...They're used to determine the PAM requirements of Cas enzymes. CRISPR-ARE is a new system from Jiazhang...and editing with a single Cas9-VPR in Saccharomyces cerevisiae. To control Cas9 activity and influence DNA... editor targets DNA (via catalytically inactive dCas9) to modify chromatin. A fluorescent protein can ...CRISPR system developed by Taekjip Ha’s lab where Cas9-gRNA complex binds the target DNA without cleavage...cleavage until it's activated by light. Once activated, Cas9 cleavage can induce DSBs within minutes. New CRISPR...Jacobs-Wagner’s lab produce different levels of dCas9 expression and contain different antibiotic resistance... lab developed a chemogenetic system that brings Cas9 to ubiquitin ligase for degradation.      New...

  32. Experimenting in an Open Source lab: from CRISPR to Cats

    Type
    Blog Post
    Published
    July 23, 2015, 2:30 p.m.
    ...contributed by Derek Jacoby from Makerspace Victoria, CA Over the decades science has become increasingly ...with  Addgene. We now have the incredible CRISPR/cas9 tools at our disposal to to modify the worm genome...be useful to our model  so that we can use CRISPR/cas9 to build the mutants, analyse their motor behaviour...project, but the primary goal is learning in this case.   Funding Makerspaces One of the main challenges...- everyone contributes each month - with only occasional supplementation with grant funds, money is always...

  33. Addgene's Cancer Collection Pages Connect the Research Community

    Type
    Blog Post
    Published
    Sept. 6, 2016, 2:30 p.m.
    ...Gateway Cloning Platform (Life Technologies, Carlsbad, CA) that represents 180 genes from the Ras signaling...work. Both cases yield the same result – uncontrolled movement of the vehicle or, in the case of cancer... a global scale, tens of millions of new cancer cases arise and millions of people die from cancer-related...and brake pedal of a vehicle, respectively. In the case of a mutation that turns a proto-oncogene permanently... pathway and be eliminated from the body. In the case of cancer, however, the apoptotic pathway is circumvented...

  34. Capturing a Moment with Marc Zimmer’s “The State of Science”

    Type
    Blog Post
    Published
    July 30, 2020, 1:15 p.m.
    ...fast, and cheap distribution of the newest CRISPR-Cas plasmids, has been a key driver of the technology...complete without the inclusion of some interesting case studies, and Zimmer provides several. In Part Three...winners for developing the technology. Though these case studies are “old,” they lead Zimmer to one of his...research, how they conduct their research, and in some cases, if they conduct their research. Even if Zimmer ...

  36. Hot Plasmids and Viral Preps - May 2021

    Type
    Blog Post
    Published
    May 18, 2021, 1:15 p.m.
    ...anti-CRISPRs were identified that inhibit the S. aureus Cas9, a Cas protein that is smaller and easier to deliver...CRISPR/Cas9 constructs. zCas9i is a high efficiency intron-optimized SpCas9-coding gene. SlugCas9 is a ...promoters driving expression of Cas9 were used in this study.  The zCas9i cloning kit is a plant cloning... of the population.  Because indel generation by Cas9 can occur at a steady rate, scientists found a way...a compact Cas9 ortholog from Staphylococcus lugdunensis that recognizes a NNGG PAM and has comparable ...comparable activity to SaCas9. A new yeast inducible CRISPRi library - Ampl133 targets S. cerevisiae genes with...

  37. Uncovering Ancestral Bias in CRISPR Research

    Type
    Blog Post
    Published
    Nov. 29, 2022, 2:15 p.m.
    ...pieces of RNA that bind to a cell’s DNA, telling the Cas machinery where to cut.      Fig. 2: Heatmap... the guide from binding to the genome or prevent Cas9 from cutting, both of which can introduce significant... are undercounted by approximately 20%. In some cases, this may lead researchers to deprioritize genes...

  38. CRISPR Antimicrobials

    Type
    Blog Post
    Published
    May 3, 2016, 2:30 p.m.
    ...Addgene. 2. Bikard, David, et al. “Exploiting CRISPR-Cas nucleases to produce sequence-specific antimicrobials...systems (Find the Citorik Plasmids here). In both cases, they used 1-2 CRISPR crRNAs targeting sequences...gene being encoded by a high-copy plasmid. In the case of the genomically encoded quinolone resistance,...

  39. MXS Chaining

    Type
    Blog Post
    Published
    Feb. 7, 2017, 3:30 p.m.
    ...Gibson DG, Young L, Chuang RY, Venter JC, Hutchison CA, Smith HO.Enzymatic assembly of DNA molecules up ...sequences. Intermediate chaining products (called “cassettes”) can be readily reused in the assembly of any...any later construct (i.e.,functional cassettes are recyclable) Can only work if the restriction sites used...

  40. Viral Vectors 101: The Retroviral Lifecycle

    Type
    Blog Post
    Published
    July 27, 2023, 1:15 p.m.
    ...Delivery of Gene-Editing Systems Based on CRISPR/Cas: Current State and Perspectives. Viruses, 13(7), ...molecular biologists working with vectors are, in most cases, interested in transferring genes to mammalian cells...viral protein reverse transcriptase (RT). In the case of a natural virus, both the protein and the genetic...particle, along with its RT-deficient genome. In both cases, the lipid membrane is the only component that is...whatever other tool you chose to express. In the case of a natural virus, this new chromosomal locus is...

  41. Viral Vectors 101: Preparing Pooled Libraries

    Type
    Blog Post
    Published
    July 13, 2023, 1:15 p.m.
    ...CRISPR libraries. The Genome Engineering using CRISPR/Cas Systems Forum- Moderated by the Feng Zhang lab, this...Resources and references References Genome-scale CRISPR-Cas9 knockout and transcriptional activation screening...Brigham MD, Sanjana NE, Zhang F. Genome-scale CRISPR-Cas9 knockout and transcriptional activation screening...

  42. CRISPR Activation: A Practical Guide

    Type
    Blog Post
    Published
    Aug. 18, 2016, 2:30 p.m.
    ....2018.04.011 Polstein LR, Gersbach CA (2015) A light-inducible CRISPR-Cas9 system for control of endogenous...CRISPR/Cas9 is an enormously plastic tool and has taken the scientific world by storm. While Cas9 has been...required for Cas9-based activation. CRISPRa for genome-wide studies One of the best uses for Cas9 activators...context of the gene, Cas9 activators efficiently solve these problems.   Which Cas9 activator should I ...Best Cas9 Variant for Your Next Experiment Find out More about Genome-wide Screens with CRISPR/Cas9 Learn...with AAV Resources on Addgene.org Find Cas9 Activators Find Cas9 Repressors Catch Up on Your CRISPR Background...there has also been significant work on constructing Cas9 transcriptional activators. These constructs allow...

  43. What's Your Organism? Expanding Genomic Tools via the NSF EDGE Program

    Type
    Blog Post
    Published
    May 2, 2019, 12:41 p.m.
    ...reptiles at all. But new success is coming with CRISPR/Cas in lizards. The Menke Lab was able to make an albino...   It’s not an overstatement to say that CRISPR/Cas9 editing is one of the key tools now providing access... be immortal. Manihot esculenta (Blake Meyers) Cassava is the fifth most grown crop in the world so it...-Mediated Ovary Transduction of Cargo)  delivers Cas9 RNP to the arthropod germline by injection into ...

  44. Four Base Editing Reporters to Monitor and Enrich Editing in Real-time

    Type
    Blog Post
    Published
    July 7, 2020, 1:15 p.m.
    ...monitor APOBEC-Cas-9 mediated editing. eGFP reporters for single base editing by APOBEC-Cas9 Though the ACE... a APOBEC-Cas9n-UGI complex. So how exactly does this work? Figure 1: APOBEC- and Cas9-mediated ...and enrichment of DNA editing by APOBEC–Cas9 or cleavage by Cas9 in living cells. Nucleic Acids Research...genome, but they’re inefficient compared to CRISPR/Cas9 edits that rely on double strand DNA breaks. Due...can help you do just that. Monitoring APOBEC and Cas9-mediated editing in real time The Harris lab created...APOBEC (a family of cytidine deaminase proteins) and Cas9-mediated editing in real time (St. Martin et al..... Image from St. Martin et al., 2018. APOBEC-Cas9n-UGI targets its preferred trinucleotide motifs (...

  45. Hot Plasmids: Fall 2024

    Type
    Blog Post
    Published
    Nov. 21, 2024, 1:30 p.m.
    ...its high resistance to antibiotics. Several CRISPR/Cas-based systems have been developed for other mycobacteria...Overview of the dual-plasmid CRISPR/Cas9 workflow in M. abscessus. A) Sth1Cas9 mediates double strand break ...mScarlet-based tags for live imaging in C. elegans CRISPR/Cas9 for gene disruption in M. abscessus  Bioluminescent...Mycobacterium abscessus using a dual-plasmid CRISPR/Cas9 system By Aryana Khosravani Deborah Hung’s lab has... has developed a novel dual-plasmid CRISPR/Cas9 system for gene disruption in Mycobacterium abscessus,...resistance interfering with selection. Using an inducible Cas9 expression and removing the sgRNA plasmid after ...Figure 4). This work marks the first use of CRISPR/Cas9 in M. abscessus. The simplicity and efficiency of...

  46. Plasmids 101: Gibson Assembly and Other Long-Homology Based Cloning Methods

    Type
    Blog Post
    Published
    March 1, 2016, 3:30 p.m.
    ...Gibson DG, Young L, Chuang RY, Venter JC, Hutchison CA 3rd, Smith HO. Enzymatic assembly of DNA molecules...modularity to overlap-directed methods (Casini 2014). In this case linker sequences are added before the...In this case, rather than using a restriction enzyme or PCR to make linearized vector, the Cas9 enzyme ...standard sequences with overlap regions, such as in the case of MODAL (modular overlap-directed assembly with...restriction site for linearization. This is a perfect case for the use of Gibson assembly combined with the...assembled into a vector using Gibson assembly. In this case the vector was PCR amplified to contain regions ...Li K, Wang B, Jin S, Reiser M, Lockey RF. CRISPR/Cas9 nuclease cleavage combined with Gibson assembly ...

  47. Fluorescent Proteins 101: When GFP lets you down

    Type
    Blog Post
    Published
    Aug. 23, 2018, 12:05 p.m.
    ...loci in living human cells by an optimized CRISPR/Cas system." Cell 155.7 (2013): 1479-1491. PubMed PMID...anaerobic conditions needs to be examined on a case-by-case basis. GFP is sensitive to acid The chromophore...nuclease-deficient Cas9 can bind loci in the genome that are defined by a gRNA. Since Cas9 is a protein, a...the localization of the fusion protein. Also, in cases where the protein of interest is relatively small...the function of the protein. This is clearly the case when proteins undergo post-translational modification...

  48. Harnessing Bacterial Toxins for Allelic Exchange

    Type
    Blog Post
    Published
    Aug. 15, 2019, 12:30 p.m.
    ...RNA-guided editing of bacterial genomes using CRISPR-Cas systems." Nature biotechnology31.3 (2013): 233. PubMed...expression of a gene with an antibiotic resistance cassette. This leaves a “scar” in the chromosome, sometimes...(Thomason et al., 2014). In recent years, CRISPR-Cas9 technology has also aided in the creation of scarless...vector contains sacB, an chloramphenicol resistance cassette, and the R6K origin of replication to allow replication...amount of user-to-user variability, and in some cases, counter-selection escape can occur: single crossover...pDS132 (Philippe et al., 2004): Replaced the sacB cassette with one of three toxins from the Khetrapal paper...15109831. Pyne, Michael E., et al. "Coupling the CRISPR/Cas9 system with lambda red recombineering enables simplified...

  50. CRISPR-mediated Plant Base Editors

    Type
    Blog Post
    Published
    Jan. 3, 2019, 1:35 p.m.
    ...editing in rice. In Genome Engineering: The CRISPR-Cas Revolution, Cold Spring Harbor Laboratory (2018)....by tethering cytosine deaminase with Cas9 nickase (nCas9). nCas9 creates a nick in the DNA by cutting ...base editors replacing the SpCas9 with the mutant versions of SpCas9 and SaCas9, which recognize PAM sequences...Nickase Cas9 (D10A) Deaminase PAM requirements Reference CBE SpCas9 rAPOBEC1 NGG 6 SpCas9 Target-AID...PmCDA1) NGG 5 SpCas9-VQR rAPOBEC1 NGA 8 SaCas9-KKH rAPOBEC1 NNNRRT 8 ABE SpCas9 TadA-TadA* NGG 9... 9-12 SpCas9-VQR TadA-TadA* NGA 8 SaCas9 TadA-TadA* NNGRRT 9 SaCas9-KKH TadA-TadA* NNNRRT 8 SpCas9...tRNA adenosine deaminases (ecTadA), fused it with dCas9, and transformed it into E. coli harboring the defective...
Showing: 101 - 150 of 609 results