...Fab Fragment? Consider the classic IgG antibody (Figure 1): it is composed of four peptide chains (two ...two identical heavy chains and two identical light chains) each with a constant region and a variable region...chains are covalently linked to one another by disulfide bonds between the two heavy chains and between...four chains make a Y-shaped protein that can be divided into the Fragment Crystallizable region (or the... means that it is relatively easy to physically divide up the different regions, both through genetic ...proteolytic digestion that retains an antibody’s disulfide bonds within the hinge region. The resulting product...')2 fragments, which reduces the hinge region disulfide bonds, resulting in two independent Fab fragments...

...amino acids or peptide tags. 5) Strong protein-protein interactions with other peptides. Created with ...Addgene’s antibodies are provided in a buffer with the antimicrobial agent sodium azide, which can interfere...spectral properties of the fluorophores you are considering to make sure that they’ll be compatible with ...antibody conjugates (especially if you start to consider clinical applications). But there are a few other...we would be remiss not to at least mention. Oligonucleotides allow for sensitive detection of low abundance...multiplexing potential. You will find antibody-oligonucleotide conjugates used in assays like proximity ligation...strong protein-protein interactions with other peptides, like an Fc-binding domain (Figure 1).  ...

... Other useful backbone modifications to consider include amide linkages, unlocked nucleic acids, and a...to simply swap out several of the ribonucleotides for deoxynucleotides. A partially DNA gRNA is surprisingly...perfect guide; now check out our blog posts on choosing the best Cas9 enzyme to pair with your guide and ...larger CRISPR RNA (crRNA). In addition to the 20 bp guide sequence, the crRNA also contains a ~20 bp repeat... a constrained ethyl.  Can’t decide which RNA backbone modification is best? Studies suggest coupling ...use in applications like siRNA and antisense oligonucleotides. Some of these synthetic modifications, specifically...photoactivatable and photocleavable gRNAs. Photocleavable guides are generated by introduction of a single photocleavable...

...in the face of evidence” (4). Frustration among scientists has been rising as evidence of climate change...not in the job of having evidence”, scientists want to make sure having evidence is the job of everyone ...marching for every cause. The references provided also only provide a snapshot into the opinions or research...Health endanger scientists’ ability to uphold their side of the bargain and do the research required to inform...while alienating the other While scientists do identify as Democrats more often than Republicans, the ...Republicans (16). This is a Pro-Science and Pro-Evidence march. Who is marching and why? Who? All sorts...include science in decision making, it will be considered a success. Below are resources for the march ...

...previously induced gene knockout. Besides amine- and DNA-binding dyes, nucleoside analogs like bromodeoxyuridine... useful for classifying and sorting cells by identifying lineage markers, like many of the cluster of ...one or several of those techniques can be used alongside standard surface labels. Here, I will introduce...streptavidin conjugates are commercially available for a wide range of fluorophores, allowing for great flexibility... you to include further (non-lineage) markers alongside, e.g., for cellular activation. In addition, FACS...interest is not located on the cell membrane but inside the cell. Due to their chemical nature, antibodies...antibodies to enter the cell (Figure 3). A useful side effect of the fixing/permeabilization (fix/perm)...

...mutagenesis of Spinach. In doing so, they identified the nucleotides that contribute to the instability of...describes the enrichment process used to evaluate oligonucleotides for binding affinity to a specific target,...molecule derivative of GFP called 3,5-difluoro-4-hydroxybenzylidene imidazolinone (DFHBI). DFHBI is membrane ...studies. Additionally, Spinach, the first aptamer identified to bind DFHBI, is highly unstable in vivo due...Spinach. It was developed in a study of artificial guide RNAs that can be used to simultaneously target cellular...Broccoli, the other green fluorescent aptamer, was identified through directed evolution. First, a pool of ... that activate the fluorescence of DFHBI were identified using systematic evolution of ligands by exponential...

...attribution: Zetsche, Bernd, et al. "Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system." Cell... cleaves in a staggered fashion, creating a 5 nucleotide 5’ overhang 18-23 bases away from the PAM. Cpf1...through homology-directed repair. Both Cpf1 and its guide RNAs are smaller than their SpCas9 counterparts,...smaller than any of these options (2.7 kb with 24 nt guides), so it has the potential to eclipse any of the... al. and Kleinstiver et al. characterized genome-wide editing efficiency of two Cpf1 orthologs known to...orthologs was only slightly lower than that of the widely used SpCas9 and comparable to SaCas9. As seen with... with Cas9 orthologs, Cpf1 efficiency varies widely with gRNA sequence. Both groups used multiple methods...

... dedicated summary page with PubMed identifiers and links provided, in addition to a list of individual...read-outs other than cell viability. The following video provides an in-depth walk through of how to use ORCS...and other species. By accurately compiling genome-wide datasets, which are often obscured in large supplementary...different datasets that might otherwise be hard to identify. Figure 2: Users can cross-interrogate ...linked to the original publication via its PubMed identifier, which can also be used to search the entire ...as the original quantitative gene-level scores provided by authors rather than sgRNA-level sequence read...tables available with the publication are also provided for the user. This approach serves to maintain...

...and are inconsistent between universities!) This guide tells you how to use online platforms, such as Zoom...recognizing the importance and benefit of remote and video communication. But first, take a deep breath; at...navigate a global pandemic can also be the thing that provides a new perspective that helps to clarify the harder...before selecting an advisor, it is always a good idea to introduce yourself to a few members of your desired...incredibly useful, as it gives a more comprehensive idea of that advisor’s research interests which can be...further discuss aspects of the lab, research, and provide them with a CV or transcripts if requested Thank...Thank them, and eagerly await a reply. It’s a good idea to email around three professors per program. Most...

...they can’t be avoided. What are off-target effects? The CRISPR-Cas system is an RNA-guided nuclease that... gene expression by various mechanisms. The RNA guide is typically 20 nts and the genomic locus must often... concern, or nothing to worry about all. Let’s consider several scenarios below for a CRISPR experiment...An off-target rate of 5% is far too high when considering human patients and the diseases associated with...off-target measurements at those sites to get an idea of how high the off-target frequency is (more on...many more. While such software typically only considers DNA sequence and doesn’t take into account chromatin...the genome to reduce off-targets, it is time to consider your Cas enzyme. Even if there are similar sequences...

...donor oligonucleotide can be electroporated into the mouse zygote (Qin et al., 2015).  When inside the ...However, if repair material is provided (in the form of an oligonucleotide or plasmid), precise changes ...genome, the mouse genome is made up of 3 x 109 nucleotides (nt), and encodes 23,000 or so genes. It would...for genome editing in eukaryotes, with the most widely used CRISPR genome editing system derived from ... CRISPR/Cas system makes use of 3 components, a guide RNA (gRNA) of about 125 nt that specifies the target...break (DSB) at the target site, and a donor oligonucleotide or plasmid as the repair material if needed...create a mouse model, the gRNA, Cas9, and donor oligonucleotide or plasmid components are brought together ...

... around the targeted nucleotide. Introduction of guanine-mismatches in the guide across from these off-target...CRISPR field. Just a year after Feng Zhang’s lab identified Cas13a (C2c2) (Abudayyeh et al., 2016) as a RNA-targeting...E488Q). They also adjusted the structure of the guide RNA by introducing a C opposite the target A to ...editing with various spacer lengths from 30-84 nucleotides, and designated this system REPAIRv1. Using next-generation... see if they could shrink the construct. They identified ADAR2DD(delta984-1090), which decreases the REPAIRv1... off-target editing. In one direct transcriptome-wide comparison, REPAIRv2 reduced off-target sites from...They note that RESCUE is most active in a 30 nt guide when C or U base-flips are present across from the...

... targeted locus on either side of the new sequence. This homology will guide the cell to use the donor...the first questions are where, which, and what. Identify where in the genome you would like to introduce...exogenous DNA can be used as well. Below are some considerations for designing donor DNA for successful HDR ...on the size of the knock-in. Single strand oligonucleotide donors (ssODNs) with homology arms as short...need to be used due to synthesis limits of oligonucleotides. These donors traditionally have larger homology... site until either the PAM site or part of the guide’s recognition sequence is destroyed via mutation....introducing a mutation in either the PAM or the guide recognition sequence in the donor DNA, you can guarantee...

...relative proportion of guide RNAs (gRNAs) across the population. Recall that guide RNAs are the targeting...’ll need something like Google Maps to help us identify and visualize addresses. That’s where enhancers...three-step approach for designing an experiment to identify the critical NANOG binding sites: 1. Find Enhancers... 1: (H3K4me1 + H3K27ac) – H3K4me3 The following video demonstrates how anyone can find enhancers with ...genetically programmed to CRISPR out the enhancers we identified in step 2. The result is a heterogenous population...to create targeted disease therapies with minimal side-effects on healthy cells and maximal effect on unhealthy... combination with the techniques from Table 1, provides us with powerful GPS-like techniques to explore...

...innovative ideas in all facets of the entrepreneurial process in order to develop those ideas into commercial...excellent people with mediocre ideas than mediocre people with excellent ideas. I am lucky to have launched...self-knowledge and an unwavering devotion to seeing your ideas become something tangible.  When you think of entrepreneurship...company, you need to determine whether or not your idea is viable.  Go to experts you trust and pitch what... received from our network.  We started with the idea of a biotechnology investment fund that would apply...our early stage.  After each pitch we made, we considered the feedback and adjusted our business model ...You’ll find that no matter how well you know your idea, you will rarely succeed in launching a company ...

...Trends: Most tools provide tabular HTML formatted output. Surprisingly only 2 tools provide FASTA output. ...success to technical expertise, thoughtful single guide RNA (sgRNA) design, and the abnormally low GC content...OMICtools. It must be confusing for a newcomer to decide on the right software to use. With so much choice...sgRNA design tools The goal of this post is to provide some insight into available CRISPR software tools...qualities of what determines good sgRNA design. Incidentally, the first software for sgRNA design (by Hsu...scoring algorithms and parameters. This tapestry of ideas is what makes choosing the right tool confusing ...functions are not entirely necessary but should be considered extremely useful, depending on design goals. ...

...resistance to pyrazinamide (PZA) (a drug commonly used to treat tuberculosis) and in identifying new drug ...wild lettuce. Aside from their use as food, infusions of L. taraxacifolia leaves are widely used to treat...Ersing Seeding Labs and Addgene collaborate to provide scientific resources, equipment, and plasmids to...specifically, the Addgene-Seeding Labs plasmid grant provide plasmids to these researchers. In the third round...control. In her current research, Sheen aims to identify new genes related to PZA-resistance by using different...using CRISPR on our blog). Her research should provide us with a better understanding of PZA action and...treat several diseases. Her goal is to identify and isolate pure plant compounds with antidiabetic properties...

...few minutes. The host could also set aside Breakout Rooms to divide the audience into small sessions and...To date, aside from doing science, he has assisted many researchers in gaining the confidence to launch... at the lab meeting, the team can discuss those ideas and build from those.   Figure 1: Virtual...focus on. Use collaborative cloud apps It is a good idea to use collaborative cloud tools such as Google ... and Sheets during Zoom lab meetings to chip in ideas and take minutes that can be accessed by anyone ..., where everyone stands up, disappears from the video, and prepares a coffee, or simply moves away from...I believe this is a great functionality that is widely used and could bring back a taste of that casual...

...Cas9, for which efficiency varies widely across guide RNAs, each guide tested with CasRx yielded >80% knockdown...Cas13d proteins they identified have little sequence similarity to previously identified Cas13a-c orthologs... to tap the wealth of CRISPR possibilities. To identify Cas13d, both groups used very general bioinformatic...independently process their own CRISPR arrays into guide RNAs. crRNA cleavage is retained in dCas13d and ...its low immunogenicity. But these studies also identified other advantages, including Cas13d-specific regulatory...produced very similar cleavage products even when guides were tiled across a target RNA, indicating that...Previous CRISPR exon-skipping approaches used two guide RNAs to remove a given exon from the genome, and...

...ongoing Plasmids 101 series. Plasmids 101 will provide you with an overview of general molecular biology...plasmid with an insert? Blue-white selection is a widely used method to do just that! (To read about other...lactose analogue, IPTG (isopropyl β-D-1-thiogalactopyranoside). (To be completely accurate, IPTG binds...x-gal (5-bromo-4-chloro-3-indolyl-β-D-galacto-pyranoside) into galactose and an insoluble blue pigment...encoding this section of amino acids (called the α-peptide) to a lacZΔM15-mutant bacterial cell in trans complements...engineered a multiple cloning site (MCS) into the α-peptide (represented as an orange wedge in the figure on... your DNA (red) is cloned into this MCS, the α-peptide gets interrupted as shown in cell A, and thus will...