...that cover some of the hottest CRISPR tools. Base Edit: Base editing plasmids allow you to make precise ...causing double-strand breaks. Our new page shows you base editing plasmids for mammalian systems, bacteria...bacteria, plants, and yeast. Popular cytosine base editor pCMV-BE3 has been requested nearly 400 times, and ... has already earned a blue flame for its adenine base editing capabilities. Epigenetics: CRISPR has made...for Gene Editing Day 9: Precision editing: Single Base Editing with CRISPR and Enhancing CRISPR Targeting...

...plasmids Cytosine base editors with minimized off-target DNA editing The original cytosine base editor, BE3,...research! This time: Optogenetic repression in yeast Base editing CRISPRi in Caulobacter crescentus Multiplexed...Multiplexed gRNA for human pluripotent stem cells CRISPR-based DNA demethylation Optogenetic repressors of gene... Read the paper in Nature Biotechnology Find the base editor plasmids CRISPRi in Caulobacter crescentus...Scientific Reports Find the plasmids Enhanced CRISPR-based DNA demethylation Figure 2: The components...

...IBEX Knowledge-Base, with every contributor listed as an author.  The IBEX Knowledge-Base is an invaluable...amplified staining methods, and oligonucleotide-based staining. Of course, if you’re interested in using... Ziv Yaniv, PhD, have created the IBEX Knowledge-Base, a community repository and resource for spatial...open-software community when designing the IBEX Knowledge-Base, seeking to create an iterative process that is .... Everyone who contributes to the IBEX Knowledge-Base is named on the Zenodo release in keeping with their...components of building both the community and the database. They particularly encourage people to upload ...

... on base editing from the first generation of base editors to the eighth generation (and dual base editors...Addgene gave to me: SHERLOCK and DETECTR, two CRISPR-based methods to detect nucleic acids.  On the third day...

...in the region around the cut site. It shows, base-by-base, the average amount of signal that disagrees...Editing Results with ICE on Synthego’s blog. CRISPR-based genome engineering has revolutionized the gene editing...Sanger sequencing data alongside the analysis of NGS-based amplicon sequencing data and found that the accuracy...enabling a ~100-fold reduction in cost relative to NGS-based amplicon sequencing. To use the tool, you start ...Analyze Complex Edits   Traditional Sanger sequencing-based analysis tools are unable to detect or analyze complex...region around the guide binding site(s). The sequence base calls from the .ab1 file are also shown above each...error-prone repair typically result in mixed sequencing bases downstream of the cut. Summary ICE generates NGS-quality...

... Course-based Undergraduate Research Experience (CURE). Having incorporated a discovery-based lab on planarian...and research-based learning in a year-long biochemistry laboratory : Part II – research-based laboratory...thinking skills (Knutson et al., 2010a,b). Classroom-based research projects have also been proposed as a mechanism...References Bangera, G., Brownell, S. 2014. Course-based undergraduate research experiences can make scientific... E., Kowalko, J., Keene, A. et al. 2022. Course-based undergraduate research experiences (CUREs) as a ...research to students; Using inquiry and research-based learning in a year-long biochemistry laboratory:...Implementation of a collaborative series of classroom-based undergraduate research experiences spanning chemical...

...positions to the base expression. When compared to the control — which should be base expression — a peak...with the negative control population (Figure 1). Based on where the GFP- population ends, you can draw ...cells and might be upregulated or downregulated based on your experiment. You can see from Figure 2 that...that determining where to place your gate based on the dot plot would be complicated.  Figure 2...   In such cases, instead of trying to quantify based on percentage positive or negative in your dot plot...populations of interest with normal gates, just based on a histogram plot instead of a dot plot. However...overlaps with the baseline suggesting this sample has a comparable expression to the baseline/negative control...

...of cancer through different mechanisms. New FRET based tools to study epigenetics Epigenetic research uses... Alice Ting’s lab has developed a new, plasmid-based biosensor to visualise changes in histone methylation...

...of fluorescence resonance energy transfer (FRET)-based calcium biosensors. Perhaps most notably, the Twitch.... “It gives you more information than intensity-based readouts.” Griesbeck also points out that the approach...now be applied to dozens of other published FRET-based sensors. For example, his team is now applying it...Universiteit Eindhoven has similar interest in FRET-based sensor proteins, particularly for the intracellular...

...your next experiment. Nucleic acid based machines Recombinase-based State Machines Enable Order-dependent...Roquet from the Lu Lab at MIT developed plasmid based systems that report the order in which specific ...received by a cell. These so-called recombinase-based state machines could potentially be used to report... Read this post to learn more about recombinase-based state machines and how they function. Adapting Toehold... Toehold Switches to Detect Zika Virus The RNA-based toehold switches described in this post are engineered...

...under non-denaturing conditions separate proteins based on size. Whether higher molecular weight complexes...tendency. Another technique that separates molecules based on size is ultracentrifugation. Sedimentation equilibrium...protein forms dimers. Finally, spectroscopic methods (based on fluorescence polarization) can be used to detect...EGFP. Constantini et al, (2012) developed a cell-based assay that does a better job of detecting homodimerization...whereas it was previously concluded to be monomeric based on gel filtration (Chudakov et al, 2007). We have... and negative controls for proper ER structure. Based on these data, we conclude that mKOk has dimerization...Amsterdam) where she develops genetically encoded FRET based biosensors.     Additional Resources on the Addgene...

... in AAV. A dCasMini-mediated adenine base editor allows for base editing, while gene editing can be done...perturbation by: Gabrielle Clouse While many CRISPR-based genetic screening methods have primarily used Cas9...genetic screening toolkit by optimizing a Cas12a-based method for screens. Cas12a’s ability to perform ...multiple genes in parallel. Lastly, this Cas12a-based screening method was used to create a genome-wide... Rachel Leeson This simple, inexpensive plasmid-based ladder system is a cost-effective alternative to...efficacy to dCas12a systems in both gene activation and base editing applications.  Figure 4: Overview...

...insert genes into plasmid backbones, base pairing between these ~4 base overhangs isn’t strong enough to ...recombination, it also doesn’t require a ligation step. Based on the robust system of homologous recombination...insert are (separately) digested to create 10–12 base “chewed back” overhangs, they can be annealed, forming...preventing T4 from chewing back the entire 10–12 bases. As such, the use of LIC is often limited to specially...overhangs must be accessible to allow for complementary base pairing, so SLIC can’t be used if the overhangs ...compared to Gibson assembly, a cloning method also based on homologous recombination. The higher temperature...

...Potential advantages of Cpf1 Type II CRISPR systems based on Cas9 were thought to be the simplest CRISPR systems...fashion, creating a 5 nucleotide 5’ overhang 18-23 bases away from the PAM. Cpf1’s staggered cleavage pattern...potential to eclipse any of the CRISPR nucleases in AAV-based applications. Get More Cpf1 Background Here THE ... Figure 1: Effects of (A) double and (B) single base pair gRNA-target mismatches on modification by AsCpf1...with single and double mismatches throughout the 23-base sequence. Double mismatches ablated Cpf1 activity...were present in the 3’ end of the target sequence (bases 19-23). Cpf1 is also sensitive to single mismatches...et al. saw no decrease in Cpf1 activity with 4-6 base deletions at the 3’ end of the target sequence. ...

...score match. This tool’s algorithm can be adjusted based on query, reference, and shared allele search preferences...called Cellosaurus, a growing cell line information database describing nearly 145,000 cell lines. Cellosaurus...researcher, Cellosaurus was able to both update their database and sent the updated information to the cell line...

...     A new positive selection-based RNAi method Until now, all DNA-based RNAi experiments typically involved...pervasive off-target effects. This conclusion was based upon numerous phenotype discrepancies between gene...considerations present at the first stage of an RNAi-based investigation. Creation of the RNAi trigger requires...considerations, such as the starting material (DNA-based vector or purified RNA) and the initial state of...allows easy creation of long hpRNA with Gateway-based cloning and straightforward positive selection of...occurrence of off-target effects. Nevertheless, amiRNA-based RNAi experiments routinely display highly specific...#137884). Both vectors allow the creation of amiRNAs based upon miRNA precursors of rice and Arabidopsis species...

... created a codon adaptation index (CAI) that is based on the codon usage frequency in a reference of highly...improve gene expression by changing synonymous codons based on an organism's codon bias. The idea is that scientists...can make mutations throughout a gene of interest based on an organisms codon usage bias to increase translational...code (Gustafsson et al., 2004), such as Hoogsten base pairs and wobble pairs.  Figure 2: (A) The...different tRNAs, including those that exhibit wobble base-pairing (a tRNA that can recognize multiple synonymous...Chappell SA, Zhou W, Mauro VP (2005) An mRNA-rRNA base-pairing mechanism for translation initiation in ...-0270(02)00362-x Stadler M, Fire A (2011) Wobble base-pairing slows in vivo translation elongation in ...

...a 30 nt guide when C or U base-flips are present across from the target base. However, they saw that in...Cas13 enzymes prefer targets with a given single base protospacer flanking site (PFS) sequence, but orthologs...so they cannot directly edit the genome. A Cas13-based RNA editing system would likely be reversible and...it is sensitive to mismatches in target RNA from bases 12-26 of a 30 nt target sequence. For the editing...targeting work with Cas13, REPAIR is the first CRISPR-based system to enable precise RNA editing. The addition...al. “A cytosine deaminase for programmable single-base RNA editing.” Science 2019 Jul 26;365(6451):382-...RNA Targeting with Cas13a Learn about CRISPR DNA Base Editing Resources on Addgene.org Browse All CRISPR...

... pegRNAs. Chromatin-based prime editing efficiency prediction While sequence-based prediction models from...building a chromatin-based prediction tool (ePRIDICT). In summary, machine learning-based prime editing ...way to predict the efficiencies of pegRNA designs based on patterns observed from high-throughput experiments...(used for PE3 and PE5 prime editing strategies) based on a DeepSpCas9 score ranking (Kim et al., 2019)... predicts how well a genomic site can be edited based on its chromatin environment. After selecting the...activity prediction by DeepSpCas9, a deep learning–based model with high generalization performance. Science...

...Class 2 CRISPR systems, including the type II Cas9-based system, require a single-component nuclease to mediate...fashion, creating a 5 nucleotide 5’ overhang 18-23 bases away from the PAM. With this information, they turned...advantages of Cpf1 over Cas9 Type II CRISPR systems based on Cas9 were thought to be the simplest CRISPR systems...with single and double mismatches throughout the 23-base sequence. Double mismatches ablated Cpf1 activity...were present in the 3’ end of the target sequence (bases 19-23). Cpf1 is also sensitive to single mismatches...et al. saw no decrease in Cpf1 activity with 4-6 base deletions at the 3’ end of the target sequence. ... Figure 2: Effects of (A) double and (B) single base pair gRNA-target mismatches on modification by AsCpf1...