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We narrowed to 609 results for: CaS;

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  1. Hot Plasmids Podcast Episode 2: New RFPs, AAVs, & More

    Type
    Blog Post
    Published
    Sept. 8, 2017, 7:52 p.m.
    ... the Addgene Podcast.     Additional Resources on the Addgene Blog Listen to Our Podcast Interview with...In the second episode of our Hot Plasmids podcast series, you'll learn about new red fluorescent proteins...plasmids webpage. Listen to the entire Hot Plasmids podcast! Hot plasmids covered in this segment Hot Plasmid...

  2. Even more elegant: Single injection CRISPR/Cas9 in C. elegans

    Type
    Blog Post
    Published
    July 7, 2015, 3:36 p.m.
    ...for CRISPR/Cas9 editing in C. elegans: Engineering the Caenorhabditis elegans genome using Cas9-triggered...2013, a remarkable nine papers describing CRISPR/Cas9 genome engineering methods for C. elegans were released...Multiple features of C. elegans have made CRISPR/Cas9 so successful in this species, namely, the robust...create a new and efficient selection-based CRISPR/Cas9 method to generate knock-ins on any genetic background...background. Their new method uses a selection cassette containing genes for hygromycin resistance and the...heat-shock inducible Cre to generate a self-excising cassette (SEC). To make it possible fluorescently label...into the genome. Figure 1: Self-excising cassette schematic from Dickinson et al. (2015). Used with...

  3. Simple CRISPR-based Epigenetic Editing: dCas9-directed DNA Demethylation

    Type
    Blog Post
    Published
    Dec. 14, 2021, 2:15 p.m.
    ...Enzymatic domains tethered to dCas9 often interact with DNA independently of dCas9 targeting (Galonska 2018...binding is crucial for dCas9 to interfere with DNMT1 even through replication. The dCas9 also needs to be removeable...has several great options: pLV hUbC-dCas9-T2A-GFP for removable dCas9 expression and pLM-CMV-R-Cre. Selection... Some enzymatic fusions are godsends. Tethering dCas9 to the histone acetyltransferase p300 or the DNA...absent before. But other enzymatic fusions, such as dCas9-TET, can, like the iron ball-and-chain that they...-based epigenetic approach (spoiler alert: it’s dCas9… with no modifications). Ready to get started?   ...dioxygenase  When I started to work on building a CRISPR/dCas9-based tool for DNA demethylation in 2015, none existed...

  4. Hassle-free 96-well Format Epitope Tagging Using Cas9 Ribonucleoprotein

    Type
    Blog Post
    Published
    June 28, 2018, 3:01 p.m.
    ...PubMed Central PMCID: PMC5947990. 3. Jarvik JW, Telmer CA. 1998. Epitope tagging. Annu Rev Genet 32: 601-618... Figure 2: Cas9 RNP complexed with 2-part cr:tracrRNA tags genes more effectively than Cas9 complexed ...Enhanced CRISPR/Cas9-mediated precise genome editing by improved design and delivery of gRNA, Cas9 nuclease,...target site. With the widespread adoption of CRISPR/Cas9 technology and rapid improvements in the field, ...set out to develop a hassle-free, highly efficient Cas9 protein-based method for epitope tagging in mammalian...genes at a time using Custom Synthetic cr:tracrRNA/Cas9 Ribonucleoprotein (csRNP) delivery Epitope tagging...delivery of three reagents to cells: a guide RNA, Cas9 protein, and a donor DNA that contains the tag sequence...

  5. Behind-the-scenes of the Isolation of the Thermostable IgnaviCas9 From a Yellowstone Hot Spring

    Type
    Blog Post
    Published
    Nov. 12, 2019, 2 p.m.
    ...other Cas proteins Read all of our CRISPR blog posts Read about GeoCas9, another thermostable Cas9 Resources...indicates that like GeoCas9 and ThermoCas9, IgnaviCas9 is a type II-C Cas9. IgnaviCas9 is most similar to...thermostable Cas9 enzymes have previously been characterized: GeoCas9 from the Doudna lab and ThermoCas9 from ...Find IgnaviCas9 here! What can you do with IgnaviCas9? Why would a thermostable Cas9 be useful? It can enable...launched in the Quake Lab. Identifying IgnaviCas9, a hyperthermostable Cas9 This year it was wonderful to learn... Ignavibacteriae phylum genome. IgnaviCas9 is a hyperthermostable Cas9 CRISPR nuclease that is active ...assembled genomes, but also led to the discovery of IgnaviCas9, a new CRISPR enzyme from a sample from Mound...

  6. Celebrating Outstanding CRISPR/Cas9 Achievements at the Dr. Paul Janssen Award Dinner

    Type
    Blog Post
    Published
    Sept. 15, 2014, 6:05 p.m.
    ... to CRISPR/Cas9 Technology? Learn More: Addgene's CRISPR/Cas9 Guide History of CRISPR Cas - A tale of... use CRISPR/Cas9 to generate mice with genes knocked out in the insulin signaling cascades in only 4 months...awards that will be bestowed upon discovers of CRISPR/Cas9 genome engineering, Dr. Jennifer Doudna and Dr. ...offered the great analogy of comparing the CRISPR/Cas9 system to Norton Antivirus. Antivirus programs work... crispr/cas9...

  7. Adenoviral Delivery of CRISPR/Cas9 Aims to Expand Genome Editing to Primary Cells

    Type
    Blog Post
    Published
    Dec. 10, 2020, 1 p.m.
    ...advanced genome editing machinery such as CRISPR-Cas. Figure 1: Schematic of targeted gene knock-out...explorations of the use of AdV for in vivo CRISPR-Cas delivery utilized the Non Homologous End Joining ..., Cowan CA, Rader DJ, Musunuru K (2014) Permanent Alteration of PCSK9 With In Vivo CRISPR-Cas9 Genome ...capacity is large enough to carry the Cas9 gene and a gRNA expression cassette in one single viral particle. ...DNA Adenoviral CRISPR/Cas9 genome editing tools can also be used to deliver Cas9 and DNA templates in ... CRISPR/Cas9 research! To find more information about the adenoviral delivery of CRISPRS/Cas9 using the... out the plasmids at Addgene: pAdSh.PGK.Cas9 (expresses S. pyogenes Cas9 from the PGK promoter) and U6...

  8. Introducing an All-in-One CRISPR/Cas9 Vector System for Multiplex Genome Engineering

    Type
    Blog Post
    Published
    Nov. 12, 2014, 3:54 p.m.
    ...versions of Cas9, nuclease and nickase, but we are now adopting dCas9, dCas9-VP64, and dCas9-KRAB in our...They went on to show that their CRISPR/Cas9-nuclease and CRISPR/Cas9-nickase vectors work successfully to...all-in-one vector construction system for CRISPR/Cas9-mediated multiplex genome engineering is now available...one of the most advantageous properties of CRISPR/Cas9 compared to ZFNs and TALENs,” said Tetsushi Sakuma...offers ease and efficiency for all-in-one CRISPR/Cas9 vectors expressing up to seven guide RNAs (gRNAs...Figure 1: Schematic overview of the all-in-one CRISPR/Cas9 vector construction system for multiplex genome ...Golden Gate cloning method to ligate expression cassettes of the gRNAs into a single vector in tandem. They...

  9. CRISPR Methods for Bacteria: Genome Engineering, CRISPRa, CRISPRi, Base Editing, and More

    Type
    Blog Post
    Published
    Sept. 28, 2020, noon
    ... screened 11 other Cas9 orthologs to identify a more efficient, well tolerated, Cas enzyme. They identified...binding the DNA or extending the transcript. Different Cas orthologues and subtypes function somewhat differently...from the Lu lab found early success using CRISPR-Cas to target subsets of bacteria in population based...that leverage the remarkable properties of CRISPR-Cas systems is base editing. This process is generally...Fischetti VA, Marraffini LA (2014) Exploiting CRISPR-Cas nucleases to produce sequence-specific antimicrobials...bacterial gene expression using an engineered CRISPR-Cas system. Nucleic Acids Research 41:7429–7437 . https... Streptomyces Species Using an Engineered CRISPR/Cas System. ACS Synth Biol 4:723–728 . https://doi.org...

  11. Hot Plasmids April 2018 - Protein Degradation, Nanoscopy, FIRE-Cas9, and Yeast Expression Tools

    Type
    Blog Post
    Published
    April 23, 2018, 2 p.m.
    ...Listen to the Fire-Cas9 Podcast Segment Jerry Crabtree’s lab has developed a new CRISPR/Cas9-based system to...epigenetic modulation. While catalytically inactive cas9 (e.g. dCas9) has been fused to a variety of protein modules...Inducible Recruitment for Epigenome editing by Cas9 (FIRE–Cas9), combines these approaches to enable rapid...here. No time to read? Listen to our Hot Plasmids Podcast! TRIM-Away: Targeted endogenous protein degradation...contributed by Mary Gearing Listen to the TRIM-Away podcast segment CRISPR and RNAi have helped researchers...Eric J. Perkins Listen to Endosomal pH Sensors podcast segment The first Addgene plasmid deposit from ...Communications. 2017. PubMed PMID: 28924139. FIRE-Cas9 for inducible interrogation of the epigenome Article...

  12. Hot Plasmids - June 2019 - Optogenetics, Acoustic Reporter Genes, microRNAs, and the CRISPR-Cas9 system CHIME

    Type
    Blog Post
    Published
    June 6, 2019, 12:41 p.m.
    ...CRISPR-Cas9 system CHIME Article contributed by Aliyah Weinstein  Listen to the CRISPR podcast segment...    Listen to this episode of the Hot Plasmids podcast Light-mediated block of synaptic transmission ...contributed by Klaus Wanisch  Listen to the optogenetics podcast segment An optogenetic tool that allows light-mediated...Lukas Morgan  Listen to the acoustic reporter gene podcast segment In recent years, scientists are beginning... Michael Lemieux  Listen to the miRNA reporter podcast segment Discovered in the early 1990’s, microRNAs...custom nucleases for each gene of interest. CRISPR-Cas9 tools have been used in immunology for in vitro ...Medical School developed a solution: CHIME, a CRISPR-Cas9 system that can edit genes in any immune lineage...

  13. Hot Plasmids February 2024

    Type
    Blog Post
    Published
    Feb. 20, 2024, 2:45 p.m.
    ...optimizing the Cas protein delivery with Peptide-Assisted Genome Editing (PAGE). They fused the Cas enzyme to...). C) Workflow for Cas-RNP-PAGE editing of primary T cells, by co-incubation of Cas-RNP and assist peptide...from Zhang et al. 2023 with permission.   Find Cas-PAGE plasmids here!   Zhang, Z., et al. (2023). ...delivery was much better with Cas12, which uses a much shorter crRNA. Cas12-RNP-PAGE achieved high editing...peptide” that facilitates endosomal escape. Combining Cas9-PAGE with lentiviral sgRNA expression yielded editing...lines and primary cells. Although cell entry of a Cas9 ribonucleoprotein (RNP) was limited (likely due ...editing frequency (nearly 100% in some cases) and minimal toxicity, including in clinically-relevant lines...

  14. Finding nucleic acids with SHERLOCK and DETECTR

    Type
    Blog Post
    Published
    April 16, 2020, 1 p.m.
    ...targets in one reaction, Cas enzymes, such as variations of Cas13 and Cas12a, are combined with different... Reporter Cas12a (Cpf1) is a CRISPR Cas variant that can also cleave dsDNA similarly to Cas9 (Zetsche ...Functional Characterization of Diverse Class 2 CRISPR-Cas Systems. Molecular Cell 60:385–397 . https://doi....Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas System. Cell 163:759–771 . https://doi.org/10.1016...Joung J, Verdine V, Donghia N, Daringer NM, Freije CA, Myhrvold C, Bhattacharyya RP, Livny J, Regev A, ...doi.org/10.1126/science.aaq0179 Myhrvold C, Freije CA, Gootenberg JS, Abudayyeh OO, Metsky HC, Durbin AF...scientists have utilized CRISPR-Cas9 protein variants, Cas13, and Cas12a, to develop simple, portable, ...

  15. Overcoming the AAV Size Limitation for CRISPR Delivery

    Type
    Blog Post
    Published
    Sept. 16, 2020, 1 p.m.
    ... mouse liver.  Two other smaller Cas proteins include Cas12b and CasX. While there are no papers yet published...toxicity. More AAV-based CRISPR systems Smaller Cas proteins SaCas9 isn't the only CRISPR enzyme that’s small... published that deliver these Cas proteins with AAV, at just 1,108 and 986 amino acids, respectively, ...(2013) Multiplex Genome Engineering Using CRISPR/Cas Systems. Science 339:819–823 .https://doi.org/10.1126...Horvath P, Magadán AH, Moineau S (2010) The CRISPR/Cas bacterial immune system cleaves bacteriophage and...one AAV, Cas9 must be made smaller. Previous attempts to “shrink” Cas9 include the use of St1Cas9 (~3.3 ... aureus Cas9 (SaCas9, 1053 amino acids) displayed cleavage activity in mammalian cells. SaCas9 produced...

  16. CRISPR-based Genome Editing Systems For Editing in E. coli

    Type
    Blog Post
    Published
    April 13, 2021, 1:15 p.m.
    ...TnsB, and TnsC pQCascade expresses the transposase TniQ, the Cas proteins Cas6, Cas7, Cas8, and the crRNA... her Ph.D. from the Chinese Academy of Sciences (CAS) Center for Excellence in Molecular Plant Sciences...California. He is also an intern at the Yang Lab of the CAS Center for Excellence in Molecular Plant Sciences...focuses on the efficiency of transposon-encoded CRISPR/Cas systems in bacteria, specifically on optimizing strains...   Yiwen Zhang entered the Sheng Yang Lab of the CAS Center for Excellence in Molecular Plant Sciences...DE3)/pCas strain. In response, we modified the pCas/pTargetF system and updated it into a pEcCas/pEcgRNA...these CRISPR systems. The original pCas/pTargetF system Our original pCas/pTargetF system consists of one...

  17. Harnessing TnpB for Genome Editing: A Compact and Versatile Tool for Your Research

    Type
    Blog Post
    Published
    July 29, 2025, 1:15 p.m.
    ...just one to cleave both strands. Similar to how Cas proteins require a PAM (protospacer adjacent motif) ...protein, much like how PAM sites change for different Cas species. For instance, the TnpB from Deinococcus ...offering access to regions previously unreachable with Cas-based editors. TnpBs have a familiar targeting mechanism..., much like the single guide RNA (sgRNA) used in Cas systems. The structure is also reminiscent of sgRNAs...editing (Molla et al., 2021), which require fusing Cas proteins with additional enzymes or domains (increasing...well-known CRISPR-associated nucleases are Cas9 and Cas12a, the heavyweights in the genome editing toolbox...acids in size. To give a visual analogy: if Cas9 or Cas12a is a soccer ball, TnpB is a baseball — much...

  18. 3 Tips to Improve HDR Efficiency for CRISPR Editing in Human Cells

    Type
    Blog Post
    Published
    Sept. 5, 2017, 1:58 p.m.
    ...accuracy with CRISPR/Cas-blocking mutations After genome editing with CRISPR/Cas9 in human cells was described...strongly encourage any researcher to incorporate CRISPR/Cas-blocking mutations at both targeted alleles when ...another ssODN template that contains the same CRISPR/Cas-blocking mutation but not the intended mutation. ... RNA should be minimized by incorporating CRISPR/Cas-blocking mutations into the HDR repair template. ...et al. "Multiplex genome engineering using CRISPR/Cas systems." Science 339.6121 (2013): 819-823. PubMed...at the Rockefeller University in NYC. The CRISPR/Cas9 system is a versatile tool for precise gene editing...organisms and model systems. We have used CRISPR/Cas9 extensively for the purpose of making sequence-specific...

  19. Lentiviral CRISPR Libraries Enable Genome-Scale, Knockout Screening

    Type
    Blog Post
    Published
    Feb. 20, 2014, 4:57 p.m.
    ...CRISPRs: Using CRISPR/Cas to Edit Disease Out of the Genome History of CRISPR Cas - A tale of survival ... crispr cas...It’s clear that CRISPR-Cas9 technology has really changed the game for anyone looking to quickly and ...Lander library uses sequential infection first of Cas9, then with the sgRNAs, and can result in a higher...that have previously been stably transfected for Cas9 expression. The Yusa lab has used their library ...application offer proof-of-principle that CRISPR-Cas9 can be used to target any sequence in the genome... Genetic Screens in Human Cells Using the CRISPR/Cas9 System. Wang T, Wei JJ, Sabatini DM, Lander ES. ...

  20. CRISPR in the Clinic

    Type
    Blog Post
    Published
    Nov. 2, 2023, 1:15 p.m.
    ...currently in clinical trials includes Cas3 (the shredder member of the Cas family) as part of a cocktail therapy...CRISPR therapies. Systemic therapies involve CRISPR-Cas delivery directly to the patient. Patient-derived...systemic CRISPR therapy. They engineered a CRISPR-Cas construct targeting the diseased allele specifically...Molina MA, Berkhout B, Herrera-Carrillo E. A CRISPR-Cas Cure for HIV/AIDS. Int J Mol Sci. 2023 Jan 13;24(...There can be no doubt that CRISPR/Cas9 technology has been a breakthrough for the genome-editing field...phenotype could be rescued by zygote co-injection of Cas9 and a single-guide RNA targeting the mutant allele... patient. The first human trial involving CRISPR Cas9 for these diseases was sponsored by Vertex Pharmaceuticals...

  21. The AAVantages of AAV in CRISPR Screens

    Type
    Blog Post
    Published
    Feb. 4, 2025, 2:15 p.m.
    ...stably express Cas effectors in specific tissues, eliminating the need to deliver the Cas protein altogether...from the comfort of Cas9, and opt for a completely different Cas effector, like Cas12a (Cpf1). Even more...of genes with minimal effort. The versatility of Cas effectors allows for a wide variety of screens, including... to use variants of Cas9, like split Cas9, or a smaller Cas9 ortholog like SaCas9. You could even venture...experiments, particularly in vivo ones. In those cases, AAV vector screens may be AAVantageous!       In...advantage of AAV vectors is how the cargo — in this case, the CRISPR components — are expressed. Unlike lentivirus...at double-stranded break points caused by CRISPR-Cas9 editing. The good news is that this integration ...

  22. CRISPR 101: Making a Knock-In Cell Line

    Type
    Blog Post
    Published
    Dec. 22, 2022, 2:15 p.m.
    ...can be! Cas12a is a Cas family member which generates sticky ends upon cutting. Cas12a may promote HDR by...! Use a Cas that facilitates HR What if promoting HR was as simple as swapping out your Cas9? It can ...prime a Cas break for HR is to fuse an HR protein to the nuclease. Early HR factors fused to Cas9 have been...like to introduce your knock-in. Then choose which Cas enzyme to use and design a gRNA to where you would...editing job to get done. However, this is not the case for knock-ins. Knock-in mutations typically require...Nat Chem Biol). Mutate the PAM or sgRNA sequences Cas9 likely goes through multiple rounds of cleavage ...guarantee there won’t be re-targeting after HDR. If your Cas9 cuts in the coding region of a gene, make sure that...

  23. Cpf1: A New Tool for CRISPR Genome Editing

    Type
    Blog Post
    Published
    Oct. 14, 2015, 2:30 p.m.
    ...CRISPR/Cas9.  Figure 1: Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system...single RNA-guided endonuclease of a class 2 CRISPR-Cas system." Cell (2015). PubMed PMID: 26422227. Find... P., et al. "Genome-wide specificities of CRISPR-Cas Cpf1 nucleases in human cells." Nat Biotechnol. (...An updated evolutionary classification of CRISPR-Cas systems." Nat Rev Microbiol. (2015). PubMed PMID:...needed by Cas9 for cleavage. In certain cases, Cpf1 may be better suited for genome editing than Cas9 - read...than that of the widely used SpCas9 and comparable to SaCas9. As seen with Cas9 orthologs, Cpf1 efficiency...rate is similar to that of high fidelity Cas9s eSpCas9 and SpCas9-HF1. Both AsCpf1 and LbCpf1 ribonucleoproteins...

  24. How to Design Your gRNA for CRISPR Genome Editing

    Type
    Blog Post
    Published
    Sept. 24, 2020, 1:15 p.m.
    ...active, not only for SpCas9 but also for some other Cas enzymes. It seems to be the case that there is no ...potential gRNAs. While SpCas9, with a PAM preference of NGG, is still the most widely-used Cas enzyme, the development...Koblan LW, Liu DR (2020) Genome editing with CRISPR–Cas nucleases, base editors, transposases and prime editors..., Doench JG (2020) Design and analysis of CRISPR–Cas experiments. Nat Biotechnol 38:813–823 . https://...Collins R, Ragavendran A, Brand H, Erdin S, Cowan CA, Talkowski ME, Musunuru K (2014) Low Incidence of...development of SaCas9, NmeCas9, Cas12a enzymes, and engineered variants thereof offers additional PAM options... capacity of a locus. When using S. pyogenes Cas9 (SpCas9), potential target sites are both [5’-20nt-NGG...

  25. Addgene Depositors Get More Citations

    Type
    Blog Post
    Published
    April 20, 2017, 2:30 p.m.
    ...Zhang’s original 2013 gene editing paper on CRISPR/Cas amassed nearly 2,400 citations in its first four ...you can see the 2013 ‘bump’ related to the CRISPR/Cas papers and Addgene deposits, but papers published...et al. "Multiplex genome engineering using CRISPR/Cas systems." Science 339.6121 (2013): 819-823. PubMed...

  26. Synthetic Photobiology: Optogenetics for E. coli

    Type
    Blog Post
    Published
    Sept. 8, 2015, 2:30 p.m.
    ...Davidson EA, Scouras A, Ellington AD, Marcotte EM, Voigt CA. Nature. 2005 Nov 24;438(7067):441-2. PubMed. Find... ZB, Chevalier AA, Levskaya A, Marcotte EM, Voigt CA, Ellington AD. Cell. 2009 Jun 26;137(7):1272-81. ... in Escherichia coli. Tabor JJ, Levskaya A, Voigt CA. J Mol Biol. 2011 Jan 14;405(2):315-24. doi: 10.1016...used with many types of proteins, including Cas9 and dCas9-transcription factors, for which low expression...

  27. CRISPR 101: Homology Directed Repair

    Type
    Blog Post
    Published
    Jan. 26, 2023, 2:15 p.m.
    ...researchers for many applications. Your favorite Cas enzyme, a guide RNA to your site of interest, and... efficiency. One important point to note is that Cas enzymes can continue to cleave DNA if the gRNA/PAM... Repair (HDR) for Increased Efficiency of CRISPR/Cas-mediated Transgenesis (2018). Pubmed. The Road Less... Repair (HDR) for Increased Efficiency of CRISPR/Cas-mediated Transgenesis (2018). Pubmed. CRISPR References...Homology directed repair and genome engineering CRISPR-Cas9 has revolutionized the genome engineering world ...PAM site remains intact. In other words, Cas9 will keep cutting and repairing your target DNA until a ...insertions such as fluorescent proteins or selection cassettes. This class of template should have homology arms...

  28. CRISPR Meets Synthetic Biology: A Conversation with MIT’s Christopher Voigt

    Type
    Blog Post
    Published
    April 22, 2015, 2:06 p.m.
    ...multi-input CRISPR/Cas9 genetic circuits References: Nielsen AA, Voigt CA. Multi-input CRISPR/Cas genetic circuits...describe what’s possible when multi-input CRISPR/Cas genetic circuits are linked to the regulatory networks...almost limitless number of DNA binding proteins with Cas9 guided by different promoters. The challenge in ... that stop it from being used. One thing is that Cas9 is toxic. When cells carry CRISPR circuitry, it ...orthogonal variation. The first one worked in each case. Sometimes in CRISPR people talk about off-target...

  29. Viral Vectors 101: Calcium Sensors

    Type
    Blog Post
    Published
    Aug. 10, 2023, 1 p.m.
    ... or intracellular landscape, remember: Keep Ca+2lm and Ca+2rry on! References and Resources References...GECO sensor (GEX), where one wavelength changes with Ca while another wavelength is insensitive. Additional...

  30. Hot Plasmids - October 2020

    Type
    Blog Post
    Published
    Oct. 13, 2020, 1:15 p.m.
    ...our CRISPR Plasmids and Resources page.  CasPhi is 70 kDa Cas protein identified exclusively in huge bacteriophage...glycosylation. Get the library at Addgene CasRx is a RNA targeting Cas system that can be used in Drosophila... melanogaster. Get the plasmids at Addgene The Cas Hybrid for Multiplexed Editing and screening Applications... (CHyMErA) Knockout Libraries express hybrid Cas9-Cas12a gRNAs under a single U6 promoter. The paralog... codon-optimized, engineered Streptococcus canis Cas9 nuclease that has a broad 5′-NNG-3′ PAM. Get the... hybrid gRNA library deletes human alternative cassette exons and disrupts genes with alternative exons...

  31. Reaching out to China: Canton Nucleic Acids Forum (CNAF) 2015

    Type
    Blog Post
    Published
    Dec. 1, 2015, 3:30 p.m.
    ... of the University of Georgia studies the CRISPR/Cas system as it relates to its natural role in prokaryotic...systems to defend against both DNA and RNA viruses - 2 cas systems regulate at the DNA level and 1 at the RNA...Tel Aviv University then spoke about using CRISPR/cas to address the growing clinical problem of antibiotic...bacterial phage vectors with CRISPR/cas9 sensitizing gene cassettes. Thse vectors are engineered to replace... tetracycline cas9 inducible knock-out system. His lab is also exploring the use of cas9 proteins from... I could tell that they were mushrooms. In some cases I couldn’t tell if the bags contained flora or fauna...There are many clever ways labs are using CRISPR/cas9 and more are being reported. Zhou Songyang from ...

  32. CRISPR 101: Epigenetics and Editing the Epigenome

    Type
    Blog Post
    Published
    June 24, 2020, 5:45 p.m.
    ...Staphylococcus aureus Cas9 (Sa-Cas) and deposited plasmid IF311: pMAGIC (R4-R3) NLS-Sa dCas9-NLS-LSD1. LSD1 ...easier! With the help of guide RNAs, fusions between Cas proteins and the epigenetic modifier could be targeted...Crawford GE, Reddy TE, Gersbach CA (2015) Epigenome editing by a CRISPR-Cas9-based acetyltransferase activates...Hilton IB, Crawford GE, Reddy TE, Gersbach CA (2017) CRISPR–Cas9 epigenome editing enables high-throughput...including pcDNA-dCas9-p300 Core and pcDNA3.3-Nm-dCas9-p300 Core, as well as pLV-dCas9-p300-P2A-PuroR for...pCMV-dCas9-D3A) and Tet-dependent (TetO-dCas9-D3A) constructs. For lentiviral expression, Fuw-dCas9-Dnmt3a...types. They have also fused LSD1 to x-Cas9(3.7). x-Cas9(3.7) is a SpCas9 mutant that exhibits increased PAM...

  33. What's New in CRISPR - September 2019

    Type
    Blog Post
    Published
    Sept. 10, 2019, 12:56 p.m.
    ...in bioRxiv Find the GeneWeld vector kit CasX, a small Cas protein, is active in E. coli and human cells...ago, Jennifer Doudna’s lab discovered a new Cas protein, CasX, from metagenomic analysis of microbial DNA...GeneWeld vectors to create knock-ins CasX Drug inducible CRISPR/Cas9 activation E. coli genome-wide inhibition...demonstrating that CasX is active in both E. coli and human cells. Using the structure of CasX at eight different...and inactive CasX for E. coli and human expression. Read the paper in Nature Find the CasX plasmids ...inducible CRISPR/Cas9 systems for transcriptional activation and genome editing by fusing Cas9 to a mutated...vectors GeneWeld from Jeffrey Essner’s lab uses CRISPR/Cas9 and a donor vector to create knock-ins of fluorescent...

  34. Fluorescent Tagging of Endogenous Genes with SapTrap

    Type
    Blog Post
    Published
    Oct. 12, 2017, 2:26 p.m.
    ...other CRISPR/Cas systems used for endogenous protein tagging, please visit our website CRISPR/Cas Plasmids...example, Masato Kanemaki's lab has developed a CRISPR/Cas-based system for tagging endogenous proteins with...this system lies in the ability of the endonuclease Cas9 to create a DNA double stranded break (DSB) at a..., and co-injection of the targeting vector and a Cas9 expression plasmid will insert the desired genetic...reactions are then mixed and transfected along with a Cas9 expression vector. They can also be introduced directly...into a Drosophila S2 cell line stably expressing Cas9. This PCR toolkit offers C- and N-terminal tagging... promote homology directed repair after a CRISPR/Cas9 induced break. Learn more about these constructs...

  35. A History of Genome Engineering in Popular Culture

    Type
    Blog Post
    Published
    Feb. 25, 2020, 2:15 p.m.
    ...to target new genomic sites. The discovery of the Cas proteins in 2005 was a major breakthrough in the ...the world of genetic engineering as CRISPR-Cas editing soon replaced the protein-based DNA-binding domain...domain with a single-guide RNA sequence.  The Cas proteins, just like its predecessor induce a double-strand...editors (meganucleases, ZFNs, TALENs, and CRISPR-Cas) is that they induce double-strand breaks (DSBs) ... the genome. They fused a catalytically impaired Cas9 protein to a DNA-editing enzyme to generate the ...based on a prime editor which is a fusion between Cas9 and reverse transcriptase. Using an elongated template...The new frontier of genome engineering with CRISPR-Cas9. Science 346:1258096 . https://doi.org/10.1126/science...

  36. CRISPR 101: Drug discovery and CRISPR

    Type
    Blog Post
    Published
    May 4, 2023, 1 p.m.
    ...for drug discovery, there is more on the horizon. Cas enzymes have been engineered to have many unique ...References Fellmann, C., et al., Cornerstones of CRISPR-Cas in drug development and therapy. Nat Rev Drug Discov...characterized or have a simple pathology. In these cases, preliminary identification of potential targets...odds of finding a quality drug downstream. In some cases, there may already exist a drug which targets some... of genome edits accessible, fast, and easy. In cases where the mutational basis of a disease is known...CRISPR 101: Making a Knock-In Cell Line Using CRISPR-Cas9 to Edit Disease Out of the genome Tips for a first...

  37. Addgene @ Keystone: Thoughts on Precision Genome Engineering and Synbio

    Type
    Blog Post
    Published
    Jan. 15, 2015, 1:50 p.m.
    ... engineering from ZFNs through TALENs and CRISPR-Cas nucleases, provided important context for the rest...to plan your CRISPR experiment for our new CRISPR/Cas users) and updated Synbio page to people who were...the CRISPR-Cas9 system. Mehmet Fatih Bolukbasi from Scot Wolfe's lab described a chimeric Cas9 that is improving...followed by three of the biggest names in the CRISPR-Cas9 field – Jennifer Doudna, Feng Zhang, and Keith Joung...

  38. Tips for CRISPR Gene Editing in Mice

    Type
    Blog Post
    Published
    June 28, 2016, 10:59 a.m.
    ...et al. "Analysis of off-target effects of CRISPR/Cas-derived RNA-guided endonucleases and nickases." Genome...large scale mouse mutagenesis by injecting CRISPR/Cas plasmid into zygotes." Development, growth & differentiation...mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering." Cell 153.4 (2013): ...plasmid contains humanized Cas9 expression cassette and sgRNA expression cassette. The target sgRNA can be...expression cassette reconstitution. When the target sequence was digested by sgRNA guided Cas9 endonuclease... encoding Cas9 into the oocyte. This original method was developed as the use of CRISPR/Cas9 in mammalian... guest blogger Samantha Young. The use of CRISPR/Cas9 for gene editing has expanded since its adaptation...

  39. CRISPR 101: Cytosine and Adenine Base Editors

    Type
    Blog Post
    Published
    Feb. 13, 2025, 2:15 p.m.
    ...as a template. To do so, they used a Cas nickase, instead of dCas9. The resulting editor, BE3, nicks the...editing requires three elements. They are, broadly: A Cas protein (catalytically dead or nickase) fused to ...a deaminase that makes the edit A gRNA targeting Cas to a specific locus A target base for editing within...within the editing window specified by the Cas protein These elements were the starting point towards ...evolution of an adenine base editor with improved Cas domain compatibility and activity. Nature Biotechnology...individual Cas9. They also mutagenized the cytidine deaminase portion of the base editor to create SpCas9 base..., a base editor containing high fidelity Cas9 variant HF-Cas9 (Rees et al., 2017). HF-BE3 showed 37-fold...

  40. CRISPR Guide Update

    Type
    Blog Post
    Published
    Oct. 10, 2024, 1:15 p.m.
    ... Addgene blog PRIME Editing CRISPR 101: Cas9 vs the Other Cas(s) Progress Towards a PAM-free CRISPR... transposases or the Cascade-Cas3 system. We also describe newly engineered Cas9s with more flexible requirements...and enhanced editing, as well as alternatives to Cas9. All of this is accompanied by new or refreshed ...

  41. Plasmids 101: Modular Cloning Applications and Kits

    Type
    Blog Post
    Published
    May 14, 2024, 1:15 p.m.
    ...these kits, as there are many options! CRISPR CRISPR-Cas can harness the power of MoClo to expand both targetability...sgRNAs, MoClo can also be used to test a variety of Cas enzymes with different PAM requirements and editing...optimization, and more. While there are many CRISPR-Cas libraries and tools available for certain model organisms...elements impact viral titer and to compare different Cas enzymes in all-in-one AAV delivery. While Fragmid... chromosomal integration of larger expression cassettes (Shaw et al., 2023). Those looking to express ...unique sgRNAs into a multi-TU targeting construct (Cas9, sgRNA, reporter, etc.). This allows for the multiplexing...repurposed for CRISPR applications (gene insert = Cas9 + gRNA), Addgene distributes specialized CRISPR ...

  42. Fluorescent CRISPR Reporters: SRIRACCHA and GEmCherry2

    Type
    Blog Post
    Published
    May 26, 2020, 1:15 p.m.
    ...reversible, integrated reporter for assaying CRISPR/Cas-stimulated HDR activity) The Madison lab created ...fcell.2018.00054 Sander JD, Joung JK (2014) CRISPR-Cas systems for editing, regulating and targeting genomes...contains Cas9. The Madison lab also created an inducible SRIRACCHA system (iSRIRACCHA) where Cas9 expression... labor intensive and quite time consuming.   As Cas9/gRNA activity is crucial for successful CRISPR editing...the scientific community for efficient real-time Cas9 activity assays. Recently scientists have created...fluorescent reporter assays to quantify targeted Cas9 cleavage, and repair through homology directed repair...containing the reporter gene that was cleaved by Cas9 and repaired via HDR using the supplied donor plasmid...

  43. Developing a cold tolerant E. coli using specialized chaperones

    Type
    Blog Post
    Published
    Jan. 28, 2020, 2:35 p.m.
    ... stable Cas expression under low temperature conditions, thereby enabling efficient CRISPR-Cas mediated... our strategy using the pMBP-LbCas12a and pC013-Twinstrep-SUMO-huLwCas13a plasmids from Addgene, which...

  44. Antibodies 101: Isotypes

    Type
    Blog Post
    Published
    Oct. 19, 2021, 1:15 p.m.
    ...    References and resources References Janeway CA Jr, Travers P, Walport M, et al. Immunobiology: The...https://www.ncbi.nlm.nih.gov/books/NBK27106/  Janeway CA Jr, Travers P, Walport M, et al. Immunobiology: The...complement system proteins, which can initiate a cascade that helps attack extracellular pathogens.  Transportation...encode those proteins are designated by the lower-case Greek letters μ, δ, γ, ε, and α. Some isotypes have...

  45. CRISPR 101: Non-Homologous End Joining

    Type
    Blog Post
    Published
    April 16, 2015, 3:45 p.m.
    ...advantage to using the CRISPR/Cas system for genome engineering is the fact that Cas9 can be easily programmed...https://doi.org/10.1016/j.cell.2013.08.021 Waters CA, Strande NT, Pryor JM, Strom CN, Mieczkowski P, Burkhalter.... Repair of Cas9-induced breaks by NHEJ As illustrated below, NHEJ-mediated repair of Cas9-generated breaks...discuss NHEJ, and how it impacts what happens to Cas9-mediated DSBs in the genome. Non-homologous end...consequently the principal means by which CRISPR/Cas9-introduced breaks are repaired. The following factors...introduce indels. Given the end structure of the Cas9 DSB (blunt or near-blunt ends without nucleotide...population of cells constitutively expressing a targeted Cas9 should possess indels in the majority of their chromosomes...

  46. The 12 Days of CRISPR: 2021

    Type
    Blog Post
    Published
    Dec. 28, 2021, 2:15 p.m.
    ...of CRISPR, Addgene gave to me: a miniature CRISPR-Cas system for mammalian use. On the second day of CRISPR...: efficient CRISPR editing with a hypercompact Cas12f1 and engineered guide RNAs. On the sixth day of ...programmed genome editing by a miniature CRISPR-Cas12f nuclease. On the eighth day of CRISPR, Addgene ...the eleventh day of CRISPR, Addgene gave to me: a Cas9 nickase 101 blog post.  On the twelfth day of CRISPR...

  47. Technologies Enabled by NanoLuc® Luciferase

    Type
    Blog Post
    Published
    Feb. 8, 2018, 12:17 p.m.
    ...Yang, J. et al. (7) wished to trigger intracellular Ca++ release with an optogenetic receptor, melanopsin...composed of Venus fluorescent protein, the Troponin C Ca++ binding domain, and NanoLuc® Luciferase. CalFlux...stimulation with NanoLuc-based luminescence (BRET) Ca++ sensing. Nature Comm. 7, 13268. PubMed PMID: 27786307...endogenous tagged genes generated through CRISPR/Cas9 knock-in. Finally, NLuc is very inviting for endogenous...of photobleaching, autofluorescence, and, in the case of exciting cyan-excitable donors, phototoxicity...

  48. Treating Muscular Dystrophy with CRISPR Gene Editing

    Type
    Blog Post
    Published
    Jan. 26, 2016, 3:30 p.m.
    ...Thakore PI, Majoros WH, Reddy TE, Gersbach CA. Multiplex CRISPR/Cas9-based genome editing for correction of...Ran FA, Yan WX, Asokan A, Zhang F, Duan D, Gersbach CA. In vivo genome editing improves muscle function ...Perez-Pinera P, Brown MT, Majoros WH, Reddy TE, Gersbach CA. Correction of dystrophin expression in cells from...long period of time. High fidelity Cas9s such as eSpCas9 and SpCas9-HF should be explored to reduce off-target...mutation in dystrophin exon 23. Long et al. used SpCas9 in conjunction with AAV9, whereas Nelson et al....al. and Tabebordbar et al. used the shorter SaCas9 with AAV8 and AAV9, respectively. Although each study... it’s important to realize that DMD is a simpler case than other genetic diseases we’d like to treat with...

  49. The 12 Days of CRISPR

    Type
    Blog Post
    Published
    Jan. 3, 2023, 2:15 p.m.
    ... 11th day of CRISPR, Addgene gave to me: Cas-E-le-ven! (Cas11)🎵 On the 10th day of CRISPR, Addgene gave...🎵 On the 3rd day of CRISPR, Addgene gave to me: dCas9-based gene editing for cleavage-free genomic knock-in...day of CRISPR, Addgene gave to me: High-fidelity Cas13 variants for targeted RNA degradation with minimal...

  50. Giving gRNAs a Facelift - Synthetic and Beyond

    Type
    Blog Post
    Published
    Oct. 20, 2022, 1:15 p.m.
    ...al. Chemically modified guide RNAs enhance CRISPR-Cas genome editing in human primary cells. Nat Biotechnol...Photoswitchable gRNAs for Spatiotemporally Controlled CRSIPR-Cas-based Genomic Regulation. ACS 6, 5, 695-703 (2020...surprise then that stability of the RNA used in CRISPR-Cas9 experiments – the “gRNA” – suffers from the same...still effectively serving their function of guiding Cas9 to generate a targeted break. These advances in ...modifications The recognition sequence for CRISPR-Cas9 targeting is led by a ~20 bp gRNA which is part ...trans-activating crRNA (tracrRNA) which interacts with the Cas9 protein. These RNA molecules are vulnerable to attack...partially DNA gRNA is surprisingly well-tolerated by the Cas9 protein and can increase on-target efficiency. In...
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