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  1. Four Factors that Differentiate the Stem Cell Field

    Type
    Blog Post
    ... of applications with the potential to save lives. While most scientists recognize and appreciate the ... Yamanaka himself, during his keynote address, appeared almost incredulous that the field has exploded...daunting because they have no clear cut answers, I applaud the stem cell community for addressing these important...but they are also the reason this research is happening. Stem cells scientists have certainly not lost...

  2. Optimizing Donor DNA for Enhanced CRISPR Genome Editing

    Type
    Blog Post
    ...altering its function). HDR is crucial for certain applications, for example, correcting the allele that causes...this question led us to develop a new, rational approach to improve the efficiency of sequence replacement...introducing a cut, with residence times on DNA of approximately 6 hours. To our surprise, we measured identical...region allowed sequence replacement rates of approximately 1%. This is by no means a large number, but ... order of magnitude greater than transfection approaches. Moreover, the cost of reagents is relatively...

  3. Resume Writing for Non-academic Science Careers

    Type
    Blog Post
    ...together a grant application? Are you familiar with the publishing or patent application processes? How ...How can you stand out among all the candidates applying for the position?   Here are a few things to consider...makes sense for your skills, and the job you are applying for. Also remember that a resume is typically ...have done is closely related to the job you are applying for, in which case talk about your research, as...

  4. Targeted Mutagenesis with EvolvR

    Type
    Blog Post
    ... by the Dueber Lab at Berkeley, provides a new approach for generating novel genetic variants in bacteria...the key components of EvolvR and its potential applications. Challenges of existing mutagenesis methods...exist, but there are a few drawbacks with these approaches that makes it challenging for users to achieve...is capable of mutagenesis in E. Coli, but this approach requires special bacterial growth conditions in...more than one mutation near the target site. Applications of EvolvR The Dueber Lab used EvolvR to discover...

  5. Antibodies 101: ChIP

    Type
    Blog Post
    ...next-gen sequencing. ChIP is an incredibly useful application for exploring these interactions, and can give...including epigenetic modifications. But it's not an approach for the faint of heart!  The Process ChIP can...preferred method, enzymatic digestion can be appropriate when your protein of interest is a low abundance... upfront investment than many other antibody applications, this investment can well be worth it once you... idea to add "patience and an extra-methodical approach" to the top of your protocols when embarking on...

  6. Luminescent Imaging with Nano-lanterns

    Type
    Blog Post
    ...multicolored nano-lanterns for use in various applications, including optogenetics, biosensors, and fusion...furimazine. In addition to the previously described applications, the enhanced signal of eNL allows their use...characterizing Nano-lanterns and eNL for many different applications. For long-term imaging, especially where photobleaching...concern, these constructs come in very handy. This approach is especially suited to complement optogenetic...Therefore, especially thick samples may not be appropriate for use with Nano-lantern technology. Multicolor...

  7. Sharing is Caring: But How to Distribute Open Hardware?

    Type
    Blog Post
    ...disciplines. In biology, innovative new imaging approaches are constantly being developed which combine ...biological protocols and revolutionary physical approaches. However, with each new technical achievement... greatly increases accessibility. Through this approach, the Planktoscope, a portable device for studying...on-going creativity with the construction plans. Appropriate open-source licenses for hardware (e.g. CERN ...optical setups. References Joel T. Collins, Joe Knapper, Julian Stirling, et. al, "Robotic microscopy for...

  8. Clear and Accessible Writing

    Type
    Blog Post
    ...principles we used in our daily content writing applied to alt text. We also were aware of the need for...they hoping to get from it? The image in Figure 1 appears on our protocol page for AAV Purification by Iodixanol...a lot of jargon and have a technical, detailed approach. Other times, it is more helpful to use plain ...start small with alt text or immediately dive into applying these four steps elsewhere, engaging in this practice...from posters to papers.  We hope this helps, and happy writing!   Resources Additional resources on the...

  9. Choosing a CRISPR Nuclease: Site Accessibility, Specificity, and Sensitivity

    Type
    Blog Post
    ...advances and how some of these variants may be appropriate for your specific research question. Considerations...off-target activity, which may be undesirable in many applications. At the time of our first blog post on choosing...Using a combined random and directed mutagenesis approach, the Anna Cereseto Lab created pX-evoCas9, which...enzymes may better suit specific experimental approaches. One example is CasX, characterized by the Oakes...While no enzyme is perfect for all experimental approaches, knowledge of both the advantages and limitations...

  10. RANbodies: Reporter Nanobody Fusions

    Type
    Blog Post
    ...some reporters are better suited for particular applications. Read on to learn more about each reporter or...sensitive than HRP RANbody detection, it is approximately as sensitive as detecting the same antigen with...less sensitive than HRP RANbody detection but approximately as sensitive as antigen detection with a standard...detected with a labeled anti-HA or -MYC antibody, approximately as sensitive as detecting antigen with a standard...Detection with an anti-Chicken IgY antibody is approximately as sensitive as detecting antigen with standard...

  11. Visualizing Protein Turnover In Situ

    Type
    Blog Post
    ...brains. However, it is not known why or how this happens. Therefore, I set out to study how protein turnover...protein turnover in situ in neurons. The classic approach for studying protein turnover is radioactive pulse-chase...immunocytochemistry is perhaps the most common approach. With a good and specific antibody and the proper...proteins with FUNCAT Professor Schuman’s technique applies metabolic labeling using a noncanonical amino acid... observed with fluorescence microscopy. This application of AHA labeling is known as fluorescence noncanonical...

  12. Antibodies 101: Fab Fragments

    Type
    Blog Post
    ...regions, both through genetic and proteolytic approaches. This comes in handy when, for example, you want... as secondary antibodies, have much broader applicability. It is fairly easy to find these in a Fab fragment...pre-form primary-secondary antibody complexes before applying them to samples, such as when performing IHC in...can be used as a block in similar scenarios. By applying unconjugated Fab fragments that are the same species...fragments described above can be used for similar applications and have similar advantages, though with some...

  13. Choosing the B(right)est Fluorescent Protein: Aggregation Tendency

    Type
    Blog Post
    ...two posts in this series described a practical approach to selecting a bright fluorescent protein and ...aggregates. This property can be tolerated in some applications (e.g. labeling of cells or tracking promotor...promotor activity), but it is problematic in applications in which the fluorescent protein is used as an inert...cells. To understand how well fluorescent proteins approach this ideal situation, cellular assays have been...can be used in the OSER assay including CytERM-mKOkappa, CytERM-mTurquoise2, and CytERM-dTomato. When...

  14. CRISPRainbow and Genome Visualization

    Type
    Blog Post
    ...DNA-binding proteins to label certain loci, but this approach is not scalable for every locus...unlike CRISPR... 9 and 13. They next turned their attention to mapping pairs of intrachromosomal loci. The technique successfully...authors’ knowledge, this work represents the first mapping of intrachromosomal loci, a major benchmark in ...-MS2-PP7 Despite the success of Ma et al.’s approach, fluorescently labeled Cas9’s come with a number...up fluorescent CRISPR labeling, they took a new approach: labeling the gRNA itself. In their CRISPRainbow...

  15. Plasmids 101: Golden Gate Cloning

    Type
    Blog Post
    ...engineering fields. We’ll walk you through how to apply this precise and easy-to-use system to your cloning...designed correctly, the recognition sites do not appear in the final construct, allowing for precise, scarless...are eliminated by digestion/ligation and do not appear in the final construct. The destination vector ...adapted Golden Gate cloning for inserting the appropriate oligonucleotides specifying a gRNA target sequence...different combinations, or other new and exciting applications, this system is an incredibly powerful tool ...

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

    Type
    Blog Post
    ... editing in primary human cells  Cell therapy approaches can use base editors to correct mutations in ... reduced off-target editing frequencies. For applications that require high DNA editing specificity, for... for example for potential therapeutic approaches, the authors recommend mRNA delivery, the V106W version...deamination, an important attribute for therapeutic application. ABE8s demonstrate an overall improved base editing...editors with increased activity and therapeutic application. Nature Biotechnology. https://doi.org/10.1038...

  17. Overcoming the Challenges of Lentiviral Production

    Type
    Blog Post
    ...end-users, size of the transgene, and downstream applications should all be considered when choosing the correct...example, in our hands we find that a double harvest approach at 48 and 72h produces high titer virus for transfer...procedure for a single 10 cm2 dish can be easily applied to multiple dishes, remember that handling multiple... as resources available, cost and downstream applications. For example, while the PEG precipitation method... When assessing lentiviral titer, don't compare apples to oranges After choosing a packaging system and...

  18. Design Tips for Prime Editing

    Type
    Blog Post
    ...When possible, the PE3b/PE5b approach is recommended over the PE3/PE5 approach. Figure 7: Nicking...its design changes for every new prime editing application. And optimizing its sequence is important, as...with BioRender.com. PE3b and PE5b: In this approach, the nicking sgRNA is designed to bind only after...BioRender.com. MMR inhibitors PE4 and PE5: These approaches inhibit MMR to improve editing efficiency, but...

  19. Hot Plasmids February 2024

    Type
    Blog Post
    ..., its dimeric nature can interfere with some applications. Several labs have been working to produce monomeric... for molecular fusion and membrane-targeting applications. Nature Methods, 1–9. https://doi.org/10.1038...successful in primates. The authors used a multispecies approach to generate and screen a library of AAV9 variants...proven itself a good option for essentially all applications. Addgene’s plasmid collection includes over ...clone (M2) has been widely used in a variety of applications. Find anti-DYKDDDDK recombinant antibodies here...

  20. Quickest Way to Deposit Plasmids: The Deposit Spreadsheet

    Type
    Blog Post
    ...information to Addgene Material Transfer Agreement (MTA) approval  Send plasmids to Addgene  Quality control  In...can also directly paste sequence data into the appropriate full and partial sequence cells. *Note: If you...descriptive plasmid name and match the name as it appears in its publication or as it is commonly known in...descriptive purpose focuses on the practical application of the plasmid and will help a scientist outside... account username. In order for the plasmids to appear in your Addgene account under My Plasmids, we need...

  21. High Complexity Golden Gate Assembly with NEB’s DAD

    Type
    Blog Post
    ...circular assembly, with the 52-piece approach yielding approximately ~800-fold fewer plaques than the 10...thought otherwise! They suspected a data-driven approach might allow for an increase in the complexity....to sets of up to 6-8 overhangs, the data-driven approach was needed to massively expand complexity. The...Overhang Ligation Fidelity by T4 DNA Ligase and Application to DNA Assembly. ACS Synthetic Biology. https...

  22. Viral Vectors 101: Virus Safety

    Type
    Blog Post
    ...to take, and if something does happen, ensure you can receive appropriate treatment and/or monitoring. ...protection are key to lab safety and the same principles apply to viral handling. Gloves are always required and...start an experiment, just in case something does happen.   Finally, the best way to be safe is to know ...valuable tools without endangering yourself or others. Happy transducing! Resources Resources on Addgene.org...

  23. Story of a SynBio Startup: RevBio's Epiphany (or Lack Thereof)

    Type
    Blog Post
    .... We both had an applied science bent, so the engineering aspect of this job appealed to us. However, ...possibilities in life for employment?  It never happened – there was never a “moment”. But there were a...about continuing with the business. We ended up applying to a business accelerator freely stating that ...your thoughts with us in the comments below. We’re happy to answer questions about this transition (so far...

  24. Viral Vectors 101: Producing Your rAAV

    Type
    Blog Post
    ...contain rAAV. To minimize contamination, use appropriate decontamination procedures (Korte et al., 2021...surfaces that come into contact with the rAAV and apply common cell culture practices diligently throughout... toxic transfection reagents and keep the cells happy. Harvesting virus Once you have transfected your...al., 2010). For certain in vitro and in vivo applications, you can consider using unpurified AAV from ..., 2017). You can learn more about common AAV applications by reading our Viral Vectors 101 series!  This...

  25. Cloning Mammalian Cells with the Agarose Method

    Type
    Blog Post
    ...carefully) holding plates up to the light, where they appear as opaque dots (colonies should contain at least...only 1.5 ml of each are used). This solution is capped, swirled and tested for the correct temperature...and not spread out onto the plate. We test the (capped) 1:1 agarose/DMEM solution against a part of the...densities, then dispose of the plates that do not appear to be yielding well-spaced clones. For rapidly ...population of transfected clones. The technique is applicable to a wide variety of cell lines, from HEK to ...

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

    Type
    Blog Post
    ...uncontrolled cellular proliferation. We have appreciated for some time the causes of this uncontrolled...cellular machinery necessary for division. This approach also kills dividing non-cancerous cells, such ... University of California, San Francisco, is approaching the Ras problem by exploiting the different mutant...genes from the Ras signaling pathway. Its major appeal is that it enables the simple construction of vectors...any of these genes through several different applications such as fluorescent protein fusion studies and...

  27. Google Forums Round Up: First Impressions of NgAgo

    Type
    Blog Post
    ...been excitedly testing NgAgo's genome editing applications. So, how does NgAgo stack up thus far? A few...tips. Based on the discussions in the forums, it appeared that many researchers were having difficulty achieving... target site. Overall, 64% of researchers were unhappy with the new method and 65% wanted to wait until... the comments section below. Keep pipetting and happy genome editing! Check Out Other Argonaute Plasmids... S. Lander, and Feng Zhang. "Development and applications of CRISPR-Cas9 for genome engineering." Cell...

  28. SciComm: Taking Science to Elementary and Middle Schools

    Type
    Blog Post
    ...lot of activities (try for a dozen!). The best approach may not be “let’s try to make this STEM activity...that would compliment the STEM curriculum. As it happened, I’d met some of the 8th grade science teachers...opinion, it was more important to communicate the applications of CRISPR over the details of how it worked....analogy that involves student participation, make it happen! How should we use CRISPR? How shouldn’t we use... I came out of the experience with a renewed appreciation of our science teachers’ difficult-but-rewarding...

  29. AAVs in Retinal Gene Therapy

    Type
    Blog Post
    ...in the limelight. On December 19, 2017 the FDA approved Luxturna™ the first gene therapy for the treatment...AAV-based gene therapy treatment for a genetic disease approved by the FDA is for a rare eye disease and not another...trial started, and 10 more years to obtain FDA approval and make history in the form of Luxturna™. A ...medicine. References and further reading 1. FDA approves gene therapy for blindness 2. Gene Therapy emerges...-1250015778 7. Yu W and Wu Z., 2018. In vivo Applications of CRISPR-Based Genome Editing in the Retina...

  30. Plasmids 101: Choosing an Antibiotic Resistance Gene

    Type
    Blog Post
    ...common antibiotic resistance genes and which applications are best (or worst) for each antibiotic. The... after it has been heat-shocked or electrically zapped. As a result, a shorter recovery period of 30 minutes...is expensive (relative to amp), and for some applications, the benefit it offers is marginal. Fortunately...antibiotic resistance ultimately comes down to your applications and needs. The more niche and specific your ...recap of the pros and cons of each antibiotic and happy selecting!   Antibiotic Resistance Stability...

  31. Getting to Know Expansion Microscopy

    Type
    Blog Post
    ...with an abundance of protocols and examples of applications from across disciplines now available. In this...Tissue expansion is not compatible with in vivo applications and there’s just not much to be done about that...and Stiles, 2022). Fourth, signal continuity can appear reduced in ExM, which could impact how you interpret...pre-expanded samples can sometimes take on a fragmented appearance post-expansion, but that doesn’t mean we should...especially when paired with complementary experimental approaches. Since it arrived on the scene, ExM has helped...

  32. Uncovering Ancestral Bias in CRISPR Research

    Type
    Blog Post
    ...variations in gene expression, they found that for approximately 40% of the identified genes, the genes were ...lines from individuals of African descent have approximately 20% more single nucleotide polymorphisms (SNPs...ancestry-associated cancer dependencies are undercounted by approximately 20%. In some cases, this may lead researchers...map. Now, any data that came from CRISPR guides mapped to a SNP would be ignored. Working with Lia Petronio...8 Lewis, Anna CF, Santiago J. Molina, Paul S. Appelbaum, Bege Dauda, Anna Di Rienzo, Agustin Fuentes, ...

  33. Lab to Office Culture Shock

    Type
    Blog Post
    ...move from lab to office will likely happen eventually. If you approach that transition dreading the change... scientist position at Addgene, the skeptical applicant asked if she would partake in “critical thinking...thinking” in the position to which she was applying. I probably did not respond to her question as well then...adjustment period will be that much longer. If you approach the transition embracing the opportunity for growth...

  34. Plasmids 101: Plasmid Incompatibility

    Type
    Blog Post
    ...In this Plasmids 101 post, we’ll cover why this happens, how it might affect your work, and how understanding...rely on chance (or random diffusion) to make this happen.  There are several different versions of the plasmid...way to bacterial cloning glory, scientists have applied these concepts to deal with the more sinister plasmids...pestis using plasmid incompatibility. Letters in Applied Microbiology 47:235–240 . https://doi.org/10.1111...Bacterial plasmid partition machinery: a minimalist approach to survival. Current Opinion in Structural Biology...

  35. Transferable Skills Guide: Career Planning Resources

    Type
    Blog Post
    ...connection you made at the job fair when you formally apply for the position. This post on Business Insider ...re likely to find current job openings. A good approach is to do this first for companies at which you...connection. That way, once you’ve identified an appropriate job opening, you can send your contact an email...the assistance of recruiters for identifying appropriate job candidates. If you do receive such a call...those relationships. Then when you are ready to apply for your dream job, you’ll be in the best possible...

  36. Twenty Years of Sharing: Addgene's Viral Vector Service

    Type
    Blog Post
    ...viral vector service in 2016. We realized we could apply the lessons learned from our plasmid lab to develop... tools we make available. This prioritization happens for good reasons. We began offering our viral vector...remains the more requested option, and jGCaMP8 appears to be growing in requests faster than jGCaMP7 (...viral vectors. This process builds on our former approach and involves reading primary literature, soliciting...redesigned Data Hub in 2022. It’s influenced the way we approach our viral vector educational resources, from eBooks...

  37. Transferable Skills Guide: Creativity

    Type
    Blog Post
    ... tangible and applicable to your situation.   Thinking Outside the Box: Team Approaches At some point ...required to develop new projects and test novel approaches, a necessary skill set for any scientist. In ...lab   Thinking Outside the Box: Individual Approaches Whether it’s finding the source of contamination...creative skills you have developed in the lab can be applied anywhere! References and Resources References ...

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

    Type
    Blog Post
    ...to generate new pEcgRNA. pEcCas/pEcgRNA can be applied not only in coli K-12 strains, but also in E. coli...genome editing in microorganisms which could be applied to optimization of genome editing tools.   Jieze...engineered cells for green chemistry and biomedical applications.   Yiwen Zhang entered the Sheng Yang Lab of...research focuses mainly on the development and application of a multiplex genome editing system based on...Escherichia coli Genome via the CRISPR-Cas9 System. Appl Environ Microbiol 81:2506–2514 . https://doi.org...

  39. Tips for CRISPR Gene Editing in Mice

    Type
    Blog Post
    ...contains overlapping 5′ and 3′ EGFP fragments under the ubiquitous CAG promoter. The approximately 500 bp...website invites you to input the target region (approximately 500bp is best) and select the organism. It will...different types of gRNA design software. When you are happy with your selection, ordering the sgRNA is simple...process, you should first develop primers to the approximately 500 base pair sequence surrounding your gRNA...

  40. Tips for Using FRET in Your Experiments

    Type
    Blog Post
    ...exist in the real world and corrective approaches and appropriate controls are required to make this method... this technique has revolutionised the way we apprehend molecular complexes and is still a very useful...experiment. You can then choose the most efficient approach for your own system. The simplest and the most...14. Zimmermann T,  Terjung S. FRET Basics and Applications: An EAMNET Teaching Module http://www.embl.de...

  41. Plasmids 101: Biotinylation

    Type
    Blog Post
    ...molecular biology applications for biotin and avidin. A short list of some other common applications and links...downstream application. Biotin labelled oligonucleotides can be used in a multitude of applications including... feng, Liu, N. hua, Shu, L. yuan, & Sun, H. “Application of avidin-biotin technology to improve cell adhesion...

  42. Transferable Skills Guide: Conflict Resolution

    Type
    Blog Post
    ...sell them. Types of Conflict Before explaining my approach to conflict resolution, it’s important to recognize...day-to-day operations and keeping our customers happy, but inevitably there will be gray areas that either...you can help everyone walk away from a conflict happy, feeling they each got enough of what they wanted...Thomas Kilmann Conflict Model describes 5 basic approaches to conflict resolution based on various levels...yourself gravitating most frequently towards these approaches, you may need to put more conscious effort into...

  43. CRISPR 101: Homology Directed Repair

    Type
    Blog Post
    ...one of two ways - each junction resolution could happen on the crossing strand (horizontally at the purple...strand. While this pathway may not be immediately applicable in DSB-induced genome engineering, it has biological...that can be harnessed by researchers for many applications. Your favorite Cas enzyme, a guide RNA to your...Mouse Geneticist's practical guide to CRISPR applications. Singh P, et al. Genetics 114.169771; (2014)... PubMed. Optimization of Genome Engineering Approaches with the CRISPR/Cas9 System. Li, K et al. PLoS...

  44. Hot Biosensors 2022: Year-End Roundup

    Type
    Blog Post
    ...getting dark and wintry with the end of year approaching. We’re always looking for something to brighten...They generated variants optimized for different applications (v82 has higher affinity for glutamate but slower... in the fluorescent properties, pYtags are an appealing tool for monitoring RTK signaling.  Figure 4:...lower binding affinities to broaden the range of applications. Image reproduced from Torres Cabán et al. 2022...screening platform developed here is also a promising approach for developing sensors for other targets. Figure...

  45. Viral Vectors 101: Integrase-Deficient Lentiviral Vectors

    Type
    Blog Post
    ...decades. Widely used across various research applications, they have become indispensable tools for manipulating...-lasting changes in cells. However, for some applications, integration might be considered a double-edged... certain types of clinical or basic research applications. Considerations for IDLV use If you’re reading...Integrase-defective lentiviral vectors: progress and applications. Gene Therapy, 17(2), 150–157. https://doi.org...lentiviral vector: prospects for safe clinical applications. PeerJ, 10, e13704. https://doi.org/10.7717/...

  46. 10 Steps to a Perfect Science Talk

    Type
    Blog Post
    ... talks and how to make them serve you well. The happy byproduct might just be a career opportunity. The...non-academic settings. Many of these guidelines will apply to any type of science presentation, but, for more...and venue before tailoring each presentation appropriately.  Find out who will be listening.  Are they ...goal is to get the listeners to be thinking “What happens next?”  Describe how you pushed to a solution or... use no notes and can concentrate on delivery, rapport with the audience, and their own excitement about...

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

    Type
    Blog Post
    ...ways to clone DNA fragments which have provided appealing alternatives to restriction enzyme cloning. These... amplification with primers that contain the appropriate homologous sequences. NEB recommends an overlap...sequences remaining in the final product. The long overlapping region between fragments also better ensures ... of the fragments as compared to the smaller overlapping sequences left with a restriction digest. A study...cloning kits available which are all based on long-overlapping regions, have no requirements for restriction...

  48. Introducing the Addgene Antibodies 101 eBook!

    Type
    Blog Post
    ...resource for anyone looking to use antibody-based applications in their research.  Like all our eBooks, the...through the process. If you’re confident in your application and protocols, but can’t quite seem to find an...for Your Experiment.” Whatever your antibody application question, the Antibody 101 eBook is a great place....org with any thoughts, questions, or feedback. Happy reading! ...

  49. How to Design Your gRNA for CRISPR Genome Editing

    Type
    Blog Post
    ...blog post will discuss differences between these approaches, and provide updates on how best to design gRNAs...capture the mRNA cap, provides the most accurate mapping (Radzisheuskaya et al., 2016). In this case, location...location and sequence are of approximately equal importance in design – an optimized sequence will do ... using knockout or transcriptional modulation approaches. Avoiding off-target effects The off-target ...fully predict which sites will and will not show appreciable levels of modification. Whole-genome sequencing...

  50. Generating Mouse Models Using CRISPR/Cas9

    Type
    Blog Post
    ...it got “dirty”, with multiple sequence traces overlapping each other! When the count was done, 75% of the...but not least, when using CRISPR, remember to appreciate the fact that it was first discovered in an obscure... with Biogen and is particularly interested in applying CRISPR for mouse genome editing.     Haoyi Wang...interested in using CRISPR to develop therapeutic applications that better human health.     References Cong...Stem Cell/Blastocyst and CRISPR/Zygote Injection Approaches. Sci Rep 8: . https://doi.org/10.1038/s41598-...
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