...observed in the absence of genome editing (Fig. 2 and 3) (5, 3). Figure 2: Gene activation with nuclease-competent... Figure 3: Gene repression with nuclease-competent Cas9. When complexed with a truncated gRNA, Cas9...its nucleolytic activity is eliminated (Fig. 1) (1, 3). The gRNA can be further modified by incorporation...methods 12.9 (2015): 823-826. PubMed PMID: 26167643. 3. Kiani, Samira, et al. "Cas9 gRNA engineering for ...target site. Benefits of truncated gRNAs Importantly, the use of truncated guides has largely been demonstrated...protospacer, in the genome. Native Cas9 has two catalytic domains, each of which cleaves one strand of DNA...gRNA (20nt complementarity to target site) or truncated gRNA (≤15nt complementarity to target site). When...

...found climbing mountains or playing with his two cats. Keira Havens grew up in Hawaii where she was fascinated...genetic tools facilitates plant science. Challenge #3: Intellectual property There is another challenge ...biotechnologists face is public perception of genetic modification (GM) and synthetic biology. The mistrust and...cannot build what we do not understand, and the complicated and unique metabolic processes of plants are ...

...Forward Primer will use the sequence 5'-ATGTGGCATATCTCGAAGTAC-3' for the region that binds the ORF and ...primer, making our Forward Primer 5'-GAATTCATGTGGCATATCTCGAAGTAC-3'. Many restriction enzymes do not cut...final Forward Primer sequence of 5'-TAAGCAGAATTCATGTGGCATATCTCGAAGTAC-3'. For the Reverse Primer, the design... the ORF, including the stop codon (5'-TGGCATATCTCGAAGTACTGA-3'), then adding NotI (GCGGCCGC) and then... This gives us a sequence of 5'-TGGCATATCTCGAAGTACTGAGCGGCCGCTAAGCA-3' (30bp with 18bp of homology to ...chose for our reverse primer (5’-TGGCATATCTCGAAGTACTGAGCGGCCGCTAAGCA-3’) into this calculator we get a ...assist with restriction enzyme digestion (usually 3-6bp) Restriction Site: Your chosen restriction site...

... for the amplification step since Taq polymerase leaves a single deoxyadenosine (A) at the 3' ends of ...this enzyme recognizes the DNA sequence 5´-(C/T)CCTT-3' and digests double stranded DNA at this sequence.... is stored as a covalent bond between the cleaved 3′ DNA strand and a tyrosyl residue of topoisomerase... provide vectors or cloning arms with overhanging 3´ deoxythymidine (T) residues that are covalently linked...potential positive clones. Once you introduce your 3’-end “A” overhang insert, the magic of TOPO cloning...Reaction: Mix together the PCR product and TOPO Vector.  3. Incubate 5 Minutes at Room Temperature: You can place...proofreading polymerases will also remove all unpaired 3’ ends in your PCR product. If you need to decrease...

...high IL-3 showed mostly blue fluorescence, indicating a quick cell cycle. Cells that had IL-3 withdrawn...Figure 3: BaF3 cells were transduced with H2B-FT virus and grown in varying concentrations of IL-3. Cells..., which depend on IL-3 to proliferate. The cells cultured with a low dose of IL-3 showed reduced proliferation... concentrations of IL-3 show a high proportion of blue fluorescence, indicating cell proliferation. BaF3...BaF3 cells maintained in media without IL-3 show only red fluorescence. Image from Eastman et al., 2020...Streptococcus canis Cas9 nuclease that has a broad 5′-NNG-3′ PAM. Get the plasmids at Addgene The Human GlycoGene... labeling proteins with different subcellular locations. Image from Goudeau et al., 2020. Goudeau...

...ligase 25 °C 1 hour 3-base or 4-base T4 DNA ligase 25 °C 18 hours 3-base or 4-base T4 ...ligase 37 °C 1 hour 3-base or 4-base T4 DNA ligase 37 °C 18 hours 3-base or 4-base Bbsl-HF...-16 °C cycling 3-base Sapl, 1x NEBridge Ligase MM 37-16 °C cycling 3-base Table 1: Enzyme... the same overhang twice; (2) avoid palindromes; (3) no overhangs with the same three nucleotides in a...could be done using overhang sets that broke rules 3—5. Fidelity, it turns out, might depend more on the...must be used. Each of the tools works with either 3-base or 4-base overhangs, and with a variety of different...BspQI, 1x NEBridge Ligase MM 42-16 °C cycling 3-base  PaqCl®, 1x T4 DNA Ligase Buffer 37-16 ...

...proper insertion at the 5’ and 3’ junctions, respectively. These results indicate that PITCh is a robust method...PMID: 26456817. Find plasmids from this publication at Addgene. 3. Zhang, Chi, Xiuhua Meng, Xiaolei Wei...the PITCh vector should be constructed with 5’ and 3’ microhomology to the insertion locus flanking the...the GFP-Puro cassette and one in between the 5’ and 3’ microhomologies in the genomic locus. The first two...breaks allow for two sets of microhomologies (5’ and 3’) to anneal, knocking the GFP-Puro cassette into the...plasmid contains a GFP-Puro cassette flanked by 5' and 3' microhomology and PITCh-gRNA binding sites. This ...microhomologies in the PITCh vector ~20 bp 5’ and 3’ microhomologies are added to the GFP-Puro cassette...

...market; you need one with 5’->3’ polymerase activity (for amplification), 3’->5’ exonuclease activity (increases...increases amplification fidelity), and no 5’->3’ exonuclease activity (which could potentially truncate the ...on a high percentage agarose gel (~3%). Note that primer duplication will evade detection in the initial...PubMed PMID: 8470801. 3. Saiki, R. K., et al. "Primer-directed enzymatic amplification of DNA." Science 239...on either side of the desired mutation (usually 1-3 mismatched bases) is sufficient for your primers to...present, a minor extension can usually ensure that the 3’-base(s) do not form secondary structures. The introduction...template (e.g. 0.1-1.0 ng/μl). Smaller plasmids (~3 kb) are generally more efficiently amplified than ...

...bigger than ∼3 kb are packaged less efficiently. Subcomponents: U3; Unique 3'; region at the 3' end of viral...SIN γ-retrovirus lack most of the U3 region of the 3’ LTR and are primarily used in gene therapy research...viral genomic RNA (but found at both the 5' and 3' ends of the provirus). Contains sequences necessary...transcription. R; Repeat region found within both the 5' and 3' LTRs of retro/lentiviral plasmids. U5; Unique 5';...the viral genomic RNA (but found at both the 5' and 3' ends of the provirus). 5’ LTR Acts as an RNA...proceeds through U5 and the rest of the provirus. 3’ LTR Terminates transcription started by 5' LTR ... be used interchangeably. Are γ-retroviruses replication incompetent? Yes, as with lentiviruses, the genes...

...Teacher, Vol. 79 No. 3, March 2017; (pp. 208-223). Link: http://abt.ucpress.edu/content/79/3/208 2. Fire, S....Nature, 391 (1998), pp. 806-811. PubMed PMID: 9486653. 3. Newmark, Phillip A., et al. "Ingestion of bacterially...DE3. For Phagocata morgani and Phagocata gracillis, the process is a little more complicated. These strains...Girardia sp., Dugesia dorotocephala, Phagocata morgani, and Phagocata gracillis. There are 11 different ... coli. Thus, to get the dsRNA into Phagocata morgani and Phagocata gracillis, one has to purify the dsRNA... in situ hybridization experiments. A recent publication [1] from the Sánchez lab describes the plasmids...the deposit) are described in the Sánchez lab publication [1] so that the students performing these experiments...

...come in contact with target cells (Fig. 3).     Figure 3: The life cycle of a retrovirus. Image...protein (IN) during step 3 (King et al., 2008) (Hu & Temin, 1990). At step 3, the double stranded DNA ...Image courtesy of Jeremy Luban, MD. Figure 3 depicts the viral life cycle. One should ponder this figure...of DNA into the host cell’s genome (shown in step 3), but note also that it is worthwhile to generalize...complex retroviruses. Microbiological Reviews, 56(3), 375–394. https://doi.org/10.1128/MR.56.3.375-394.1992...produce infectious murine leukemia virus. Virology, 46(3), 866–876. https://doi.org/10.1016/0042-6822(71)90087...retroviruses ” (Cullen, 1992) because of their more complicated genomes. Here, we will mostly use the gamma-retroviruses...

...overlap between them (Table 1). In each construct, 3 copies of the fluorescent protein were fused in-frame...from the tyrosine-protein kinase Lyn) Membranes 3 mCherry 587nm/ 610 nm human β-Actin Actin 4 Citrine...sites of the MCS are only regenerated at the 5’ and 3’ termini of the ligation product, and the restriction... them, but rather, can only be added to the 5’ or 3’ terminus using the newly generated MCS. MXS-chaining...the third module is added to the 5’ terminus or the 3’ terminus of the ligation product. Comparison to other...chaining-based cloning methods are listed in Table 3. The goal of high throughput cloning is to facilitate...Chaining by emailing us at [email protected]. Table 3: Chaining Based Cloning Methods Method Restriction...

... (Nishimasu et al., 2014). Figure 3: pegRNAs whose 3′ extension begins with a C base can cause...Created with BioRender.com. The first base of the 3′ extension of the pegRNA should not be C. A C base...Add silent mutations near point mutations to create 3-base (or longer) tracts of edited bases. DNA mismatch...targets single-base mismatches (left), but “bubbles” of 3 or more mismatched bases can often evade MMR (right...epegRNAs: epegRNAs include structured motifs at their 3′ end to protect them from degradation. When designing...protein La to protect the end of the pegRNA. Adding 3′ polyU tracts to the end of pegRNAs (but not epegRNAs...prime editing efficiency. Nature Biotechnology, 40(3), 402–410. https://doi.org/10.1038/s41587-021-01039...

...approach of CeNGEN, check out their publication.   3’UTRome The 3’UTRs of mRNA are important for post-... the role of 3’UTRs, the Arizona State University (ASU) Biodesign institute developed a 3’UTR database...mutations with unc- 119 + selection." MicroPublication 1 (2018): 2–3. Wang, Han, et al. "cGAL, a temperature-robust...database provides detailed information about C. elegans 3’UTRs structures, alternative polyadenylation for all...Mangone, Marco, et al. "UTRome. org: a platform for 3′ UTR biology in C. elegans." Nucleic acids research...the OpenWorm website. microPublication microPublication is a unique publication for the worm community...into larger publications and would otherwise be lost in lab notebooks forever. microPublications are peer-reviewed...

...2010 3 Nature  3.3% 10.5%   3.2  9.6 2 2010 167  Nature 3.3%  10.5%  3.2  534.1 3 2010 ...plasmids in our data were published in Nature, but only 3 percent of papers without Addgene plasmids were. Thus...journal-adjusted citation measure we re-weight the 3 percent of Nature papers without Addgene plasmids ...2010 3 Applied and Environmental Microbiology  1.6%  1.2% 0.75   2.3 4 2010 167  Applied and Environmental...deposited at Addgene still accrue on average 2 to 3 times as many citations as other papers in the same...databases available, Web of Science. For each publication, Web of Science provides basic bibliographic ...Science, PLOS One, and PNAS. We then broke the publications from these journals into two groups: those containing...

... within the lentiviral 3’ long terminal repeats (LTR). The 3’ LTR is duplicated during lentiviral integration...both for use in western blots (Figure 3).      Figure 3: HeLa cells transduced with SARS-CoV-2...were encoded on the lentiviral minus strand in the 3’ LTR. These optimizations produced 90% accurate integration...least in part due to La binding and stabilizing the 3’ end of the pegRNA, which is not well protected by...epegRNAs), which add a structural motif to stabilize the 3’ end of the pegRNA. As research has just begun with...endogenous small RNA-binding protein. Nature. 2024 Apr 3. doi: https://doi.org/10.1038/s41586-024-07259-6. ...mice. B) Quantifications showing high microglia specificity of GFP expression for the indicated ssAAV5s...

...PMID: 27347757. Find plasmids from this publication at Addgene. 3. Zetsche B, Gootenberg JS, Abudayyeh ...01200-3. doi: 10.1016/j.cell.2015.09.038. PubMed PMID: 26422227 Find plasmids from this publication at ...preferred PAM is 5’-TTN, differing from that of Cas9 (3’-NGG) in both distribution and GC-content, which might.... Additionally, whereas Cas9 generates blunt ends 3 nt upstream of the PAM site, Cpf1 cleaves in a staggered...Cpf1 activity, except when they were present in the 3’ end of the target sequence (bases 19-23). Cpf1 is...gRNA positions 1, 8, 9, and 19-23. Accordingly, the 3’ end of the gRNA target sequence does not have an ...decrease in Cpf1 activity with 4-6 base deletions at the 3’ end of the target sequence. Cpf1’s strength may lie...

...-TTN, differing from that of Cas9 (3’-NGG) in both genomic location and GC-content. After sequencing and...PMID: 27918548. Find plasmids from this publication at Addgene. 3. Kim, Daesik, et al. Genome-wide analysis...for a surprise! Whereas Cas9 generates blunt ends 3 nt upstream of the PAM site, Cpf1 cleaves in a staggered... AT-rich regions or AT-rich genomes that lack the 3’-NGG PAM sites favored by SpCas9. Since Cpf1 doesn...Cpf1 activity, except when they were present in the 3’ end of the target sequence (bases 19-23). Cpf1 is...gRNA positions 1, 8, 9, and 19-23. Accordingly, the 3’ end of the gRNA target sequence does not have an ...decrease in Cpf1 activity with 4-6 base deletions at the 3’ end of the target sequence. Figure 2: Effects...

...thing ask incredulously, “You seriously didn’t add 3 microliters of 5 mM star anise?” This is something...expression was across the different cells (Figure 3).   Figure 2: Cas9 expression in monoclonal...stable cell pool were then expanded for an additional 3 weeks under blasticidin selection. Anti-Cas9 Western... control (WT). Vinculin is a loading control. 3. In terms of making the monoclonal line: I had never...with many additional tips can be found here. Figure 3: A surprise for the author. Checking Cas9 expression...would still be able to identify the cleavage product. 3. The Surveyor kit had a lot of good tips for first...from control (GFP gRNA-targeted) BRAF sites (lanes 3, 4) were used as controls. Duplexes were generated...

...spending time with her horse, and wondering what her cat is thinking. Find her on Twitter @JessicaSacher. ... step involves digesting phage genomic DNA into 1-3 kb (approximately gene-sized) fragments so that these...bulk of resultant fragments falling between 1 and 3 kb results in optimal likelihood of capturing a whole...filter paper and saturate each with lysis solution (~3 ml each), pouring off any excess. When your protein...Expression Vectors in the FX Cloning System Kit  Step 3: Probe random phage DNA fragments with live host cells...PMID: 27536293. PubMed Central PMCID: PMC4971087. 3. Simpson, David J., Jessica C. Sacher, and Christine..." Chemical Reviews (2016). PubMed PMID: 27319741. 3. Simpson, David J., Jessica C. Sacher, and Christine...