...relies on two distinct Cas9 variants—nuclease-competent or nuclease-null—in order to effect different perturbations...imagine transfecting cells with both the nuclease-positive and nuclease-null variants of Cas9 along with the... Cas9 has two catalytic domains, each of which cleaves one strand of DNA upon binding the protospacer....the protein’s nucleolytic capability, and this nuclease-null or “dead” Cas9 (dCas9) variant can then be... many cases this is due both to lower relative nuclease efficiencies as well as to more stringent targeting... An alternative approach is to modulate Cas9’s nuclease activity by modifying the gRNA with which it is...functional changes to Cas9. For instance, when native (nuclease-competent) Cas9 interacts with a ≤15nt gRNA that...

... the task. Do your homework. Learn what your future potential colleagues study. Think about how you might...  Erik Snapp just released version 3.4 of his eBook, How to Get a Faculty Position. This edition has ...you have any questions, comments or suggestions, please do not hesitate to email me at [email protected]... interviews but no job offers, is a failure to clearly articulate one's talents and ideas. Some key points...in your specialty area, it's a safe bet that at least some committee members will NOT be in your area....impressive to answer an expert's question than to leave everyone confused about your research because you...markers force people to be more succinct. Slide decks leads to the opposite and can suffocate an exciting idea...

...functional Cas9 nuclease, so there’s no potential for aberrant DNA cleavage. Direct HIV-1 cleavage allows T-...the HIV-1 genome from infected cells. Read on to learn how CRISPR can take on HIV-1 in vitro, and what ...immune system, notably CD4+ T-cells, and eventually leads to acquired immunodeficiency syndrome (AIDS) in ...that most of the HIV-1 genome had been excised, leaving only a small portion of the LTRs joined together...expression. A pooled analysis found no evidence of Cas9 cleavage at predicted off-target sites with up to 7 mismatches...which could result in higher rates of off-target cleavage. In both cases, translating the in vitro work ...1 is limited to a subset of T-cells, but it is unclear if enough T-cells could be reached to ablate a ...

... by specific sites that allow them to be cleaved and released. These constructs tend to be smaller than... Csy4 RNA nuclease from Pseudomonas aeruginosa. When overexpressed, Csy4 efficiently cleaves gRNAs sandwiched...above, except that they use an endogenous nuclease system to cleave the gRNAs. gRNAs are flanked by glycine... P and Z recognize the tRNA sequences, cleave them, and release the gRNAs. PTGs are assembled into a wt...your potential options and cloning methods, but please scroll down for plasmids suitable for other model...Assembly uses Type IIS restriction enzymes, which cleave outside of their recognition sequence, creating...transcriptional repression), and Fok1-dCas9 (dimeric nuclease). Gateway assembly method Frew Lab Multiple Lentiviral...

...synthetic modular version of the COX8 leader sequence called Leader Of Cox8 Repeated (LOC8R) to more accurately...DNA recognition domain and a mostly c-terminal nuclease domain. Feng Zhang's group utilized the bi-lobed...Cas9(C)-2xNLS) fragments facilitates target DNA cleavage upon co-expression and spontaneous self-assembly...actively shuttled out of the nucleus due to the nuclear export sequence. Treatment with rapamycin induces...nucleus, where the functional Cas9 molecule can cleave the target DNA. The inducible split cas9 approach... systems such as TAL effectors or Zinc Finger Nucleases, the Gersbach lab has modified the RNA-guided ...by the use of sequence-specific enzymes (e.g. nucleases, recombinases) which irreversibly excise or invert...

... Facebook, etc.) to learn about available jobs, fields, and trends “Never Leave an Empty Seat” at conferences...Let us know what topic you'd like to learn more about next. Leave your suggestions in the comments section...: “Academia or industry?” Once academia was the clear answer with a well-defined path to professorship...government. This great resource allows the undecided to learn about a variety of science professions based on ...the lab highlight “transferable” skills such as leadership, management, and communication. A great approach...diverse audiences Organization Mentorship Leadership Science writing competitions (e.g. Access...internships Communication Teamwork Management Leadership Networking University technology transfer...

...webinars, and learn more about the field.  Read the CCST Science Policy Career Guide to learn more about ...field from a more traditional policy background, learning the science part on the job. Each path to science...policy professional over Twitter or LinkedIn to learn more about their career trajectory. If there’s one...one role can encompass many of these activities leading to a rich day-to-day work life.  Action Items:  ... profession. Figure 1: Science policy can lead you to many paths! For a more details, check out...The big secret about science policy that I didn’t learn until arriving here in DC, is that so much policy...little P” policy, the possibilities are endless leading to many ways for you to find your own niche.  Action...

...These very cute birds learn their songs like humans learn speech. Finches must learn early, use auditory...branches of the evolutionary tree at least 6-7 times and on at least 2 separate continents. Mimulus (Andrea...and I met scientists from 21 different labs who clearly also share this wonder of the organism.   It’s...beyond obtaining “omic” data and had to include clear plans to connect Genotype to Phenotype thus making...) -  perhaps you’ll choose to study Crepidula atrasolea or Prochlorococcus with your next project. Gymnotiformes... and if she decides to raise their offspring or leave them to their own devices. Chytrid fungi (Lillian... aspects of cognition. Spiralians - Crepidula atrasolea (Jonathan Henry) This black footed slipper snail...

...field, requiring people who are willing to quickly learn, and apply their knowledge to a variety of problems...problems. In this post, I’ll share what I’ve learned as a healthcare consultant and give you an idea of what...activities depending on the expertise of the senior leadership and members of their consulting staff. Who would...The consultants who enjoy their jobs most, get pleasure from taking up such complex challenges, quickly...ameliorated by thoughtful planning and engagement, leaving mental energy to enjoy the more rewarding and educational...in Bioengineering, and one of the things that pleasantly surprised me in my first few months as a healthcare...molecular behaviors of chemicals and proteins to learn how a particular drug or medical device works. This...

...been used since - including: mCerulean/mVenus, mCerulean/Amber, mCerulean/SYFP2A, mTurquoise/mVenus and... fused to the N-terminus of monomeric Cerulean mCerulean C1 Cyan Mammalian Express a gene of interest...microscopy (FLIM) method. Acceptor photobleaching method The acceptor photobleaching method is simple but limited... quenched when FRET occurs. By photobleaching the acceptor, you release the donor's quenching and the ...also been used to measure conformational changes, cleavage activity using FRET-based biosensors, and interactions...Mammalian Expresses mammalian optimized Aquamarine mCerulean N1 Cyan Mammalian Express a gene of interest fused...interest fused to the C-terminus of monomeric Cerulean mTurquoise2 Cyan Mammalian Constructs to target mTurquoise2...

...specific DNA sequences and companion endonucleases that recognizes and cleave the same DNA sequence if not methylated... questions is the same--methylation! Read on to learn about how DNA methylation may affect your restriction...restriction digests. Why methylate? Restriction enoducleases are so ubiquitous in the lab that it is easy...prevent them from being degraded by restriction endonucleases. A given prokaryote typically has genes encoding...whether certain restriction enzymes can effectively cleave DNA. Common lab E. coli K12 strains such as DH5alpha... activating enzymes such as DpnI. For example, cleavage by XbaI may be blocked due to methylation if the...results that would either be difficult to interpret or lead you to conclude your plasmid is not correct! Conversely...

...mentees efficiently Allows all group members to lead and learn at the same time Broadens the network of the...).  Assign each meeting a leader from the group in a rotation. The leader for that meeting is responsible...choosing a topic and leading the discussion. This is a chance to practice leadership and communication skills...Harvard Medical School researcher Connie Cepko to learn about her mentoring style. While working on this...intrinsic to their success. Their stories made it clear to me that, executed correctly, the group mentoring...a similar development stage, they have a lot to learn from each other. Some have taken my advice and I...forming your own peer mentoring group Find 5-8 colleagues that you think might be interested and willing...

... of CRISPR/Cas9 by altering the nuclease activity of the Cas9 nuclease itself. Design and testing of eSpCas9...improved the on-target specificity of the Cas9 nuclease with engineered variants: eSpCas9, SpCas9-HF1,... eSpCas9 Looking at the structure of the Cas9 nuclease (PDB ID: 4OO8 and 4UN3), Slaymaker et al. hypothesized...charged groove formed by the Cas9 HNH and RuvC nuclease domains 2. Interactions between the target strand... separation and target DNA binding to the Cas9 nuclease. Stable strand separation is maintained through...genomic sites. Knowledge of Cas9’s dynamic structure leads to the development of HypaCas9 Researchers in the... which portions of Cas9 might be important for nuclease specificity. They found that eSpCas9(1.1) and ...

...you can learn more about possible strengths and weaknesses, and view everything as a learning experience...project she needs one of you to lead. At first you might think, “I’m not a leader,” because you don’t see yourself...develop your nascent leadership skills. You might just find that you’re more of a leader than you thought!... portion of the interview out of the park. It’s clear that you know how to do the job. You may think this...Stop! Take a step back and think of a time you’ve lead your team in a defined direction in the past. Maybe...opportunity much different? Might you already have leadership skills that are just not being tapped at the ...time to write down all the skills that you have learned and that you frequently have to exercise in order...

...the learning of many facts but the training of the mind to think something that cannot be learned from...research, they gain confidence and learn to work independently, which can lead to greater success in meeting...take a different approach. His own most impactful learning experiences happened in the lab, and he wanted...settled on a two-semester model, in which students learn to express, purify, and analyze MDH in semester ... work and become more actively involved in the learning process. As trainees become more comfortable with...trading structure and predictability for improved learning outcomes and the opportunity to make new discoveries...department. So in 2016, Provost partnered with colleagues at other institutions to form a support network...

...college, and science, are places for learning and growth! As you learn more about yourself and the world ...This post was contributed by guest blogger Eleanor Wang, a Research Specialist in Patrick Hsu's lab at...science, experimental design, and execution. Can lead to an independent project. Lab technician / ...Talk to your professors about their research, and learn about what makes them excited to do what they do...what you’re working on is important! Better than leaving yourself in the dark, but if you never ask, you...also more than eager to help you along your path. Learning about their career paths and goals helped me reflect...concept to me when I was in high school, but I’ve learned the value in finding mentors wherever I go. This...

...gaga over the newest gene-editing protein - a DNA-cleaving Argonaute from Natronobacterium gregoryi, or NgAgo... CRISPR proteins Cas9 or Cpf1 - keep reading to learn more! Background: Argonaute and NgAgo In eukaryotes...role in RNA interference, binding RNA guides to cleave foreign RNA. Argonaute proteins are also present...thermophilus (TtAgo), showing that this protein could cleave DNA in vitro, directed by phosphorylated DNA guides...Swarts et al. were unable to show TtAgo-mediated cleavage in mammalian cells, likely due to high temperature...Argonaute. They identified NgAgo and showed that it can cleave plasmid and genomic DNA in a variety of mammalian... 10 genes, they did not observe differences in cleavage efficiency. If this finding is replicated in larger...

... is a strict requirement for Cas9-mediated DNA cleavage. The need for more PAM sequences While PAM sequences...identify mutants of S. pyogenes Cas9 that were able to cleave target DNA sequences located upstream of either... to wild-type SpCas9, which also displays some cleavage with an NGA PAM. This variant may increase the...VQR, EQR, and VRER Cas9 variants are capable of cleaving genomic DNA in mammalian cells and zebrafish embryos...using wild-type SpCas9. The number of off-target cleavage events for the VQR and VRER variants is similar...genome engineering was Cas12a (formerly Cpf1), a nuclease that generates double strand breaks in target ...formerly C2c2) has been adapted for targeted RNA cleavage in mammalian cells. The REPAIR (RNA Editing for...

...further improved, by adding a nuclear localization sequence (SV40 Large T nuclear localization sequence) and...step, the two compatible target sites are cleaved out, leaving behind a vector with the inverted DNA flanked...Additional Resources on the Addgene Blog Learn More about FLEX-vectors Learn More about Cre-Lox Check out Retrograde... Cre expression have been developed. Read on to learn more about FLP-FRT, Cre-lox, and how combinations...asymmetric core. Recombination between these sites can lead to excision, inversion, and translocation of DNA...Continuous inversion of the DNA sequence of interest can lead to poor expression of genes within that sequence...between the two target sites with identical sequence leads to inversion of the flanked DNA sequence and the...

...example, learn about how researchers are using “big data” to look at Lyme Disease in new ways. Or learn about...otherwise never learn from the valuable research we are doing. Browse the site, learn from others, share... fails to reach our primary target audience – colleagues in our field. Secondary audiences that may also...therefore must discover new ways to reach our colleagues and other interested audiences quickly and concisely...articles as the dominant tool for sharing ideas. This leads to everyday obstacles for many of us, such as finding...can select the disciplines and the years. Which leads to the question of how we can support researchers... disciplines. But there remains a great deal to learn from the science of others. Improving science communication...