...disliked, some that everyone loved, and some that ran the spectrum. I recently presented Kurt Vonnegut’...without compromising on the relevant science."     - Randall Carter, Contracts Administrator Inferior: How Science... Non-science favorites  The Mistborn series by Brandon Sanderson "The Mistborn series is the first 3 books...Associate Director of Quality Control Jonathan Strange & Mr. Norrell by Susanna Clarke "A quasi-historical...Vonnegut’s Slaughterhouse-Five, and the responses ranged from “one of my favorite books” to “I barely got through...

...wanting to know our latest results as soon as the gels ran.” Dr. Ellis tries to lead his team as a scientists.... “Being a PI is more than just experiments and grant writing. You have to enjoy the mentoring role.” ...emphasis on genetic engineering, sequencing, and translation of results to companies.” Take home message Starting...

...Davidson AR.  Nature. 2013 Jan; 493, 429–432. Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu...and are not translatable to the other CRISPR subtypes. Still, the large and dynamic range of CRISPR systems...well-suited for various innovative biological and translative technologies. Addgene has empowered researchers...http://en.wikipedia.org/wiki/File:Crispr.png Barrangou R, Horvath P. Science. 315, 2007 Jan; 1709–1712...CRISPRdb. Date: 2013-09-18. Makarova K, Haft DH, Barrangou R, Brouns SJJ, Charpentier E, Horvath P, Moineu...

... to focus, which was extremely effective. Others ran experiments at odd hours, used more secluded workspaces...

...842 . https://doi.org/10.1016/j.molcel.2014.08.002 Ran FA, Hsu PD, Lin C-Y, Gootenberg JS, Konermann S, ... single strand break; when two such breaks are introduced near each other, in opposite strands. This “...Cas9 can be easily programmed to make a DNA double strand break (DSB) in the genome wherever the user chooses....org/10.1371/journal.pgen.1000683 Bétermier M, Bertrand P, Lopez BS (2014) Is Non-Homologous End-Joining...Giovannangeli C, Jasin M, Brunet E (2014) Chromosomal Translocations in Human Cells Are Generated by Canonical Nonhomologous.../doi.org/10.1016/j.cell.2013.08.021 Waters CA, Strande NT, Pryor JM, Strom CN, Mieczkowski P, Burkhalter...

...complex with guide RNA and target DNA. Nishimasu H, Ran FA, Hsu PD, Konermann S, Shehata SI, Dohmae N, Ishitani... sgRNAs for each transcription start site in 15,977 human genes. The transcriptional repressor (CRISPRi...SAM is an engineered protein complex for the transcriptional activation of endogenous genes and it consists...The next insight was to assemble a synthetic transcription activation complex consisting of multiple distinct...together synergistically, modeled after natural transcription activation processes. Konermann and her colleagues...consequences of repressing or inducing individual transcripts. The activator (CRISPRa) sgRNA library uses the...CRISPRi) library contains 10 sgRNAs for each transcription start site in those 15,977 human genes. Weissman...

...fragments of these transcripts. I remember when small RNA was just something we ran off the bottom of ...’t give tRNAs much thought. High throughput transcriptome sequencing methods including work done by the...hypothesized that life started with RNA and a simple cell membrane. His recent work shows that they are making fairly...knew…I had barely heard of these). These single stranded, covalently closed RNAs seem to be conserved in...

...that produce GMO food plants. When a press piece ran on the detector plants, we expected to see that same...below. We’re happy to answer questions about this transition (so far) and what we’ve learned.  Thank you ...

... same output regardless of who or which computer ran the process. Another design choice we appreciated...automating the bioinformatics steps needed to transform the raw sequencing data into a complete plasmid...appropriate place in our workflow without having to translate or convert programs to a uniform language.  The...concepts and vocabulary of Nextflow so that we could translate our pipeline steps into processes and then connect...

...just make their own buffers when the kits buffers ran out. Homemade buffers performed the same as those...rather a glass powder slurry that has a milk-like appearance. To make DNA stick to the glass particles in ...the Patterson Protocol was shown to purify DNA ranging in size from 100 bp to 48 kb with high yields and...could interfere with interpreting the results of transfection assays. Go forth and perform your DNA extractions...

... 556:57–63 . https://doi.org/10.1038/nature26155 Ran FA, Cong L, Yan WX, Scott DA, Gootenberg JS, Kriz...SpCas9 variants effectively double the targeting range of CRISPR/Cas9 within the human genome. Another ...known to produce off-target editing with SpCas9 (Amrani et al., 2018). When choosing a Cas9, remember to...formerly Cpf1), a nuclease that generates double strand breaks in target genes resulting in the formation...nature24049 Find plasmids from this paper at Addgene. Amrani N, Gao XD, Liu P, Edraki A, Mir A, Ibraheim R, ...018-1591-1 Cox DBT, Gootenberg JS, Abudayyeh OO, Franklin B, Kellner MJ, Joung J, Zhang F (2017) RNA editing...

...Genome engineering using the CRISPR-Cas9 system. Ran FA, et al. Nature Protocols Nov;8(11):2281-308; (2013)...dHJs are four-stranded branched structures that form when elongation of the invasive strand “captures” and...lesion results from breakage of both DNA strands - a double-strand break (DSB). DSBs can be caused by intracellular...proteins required for strand invasion and a primer for DNA repair synthesis. The ssDNA strand then displaces ...displaces one strand of the homologous DNA donor and can template repair off the other strand; this results in...pathways: classical double-strand break repair (DSBR), synthesis-dependent strand-annealing (SDSA), and break-induced...on the crossing strand (horizontally at the purple arrows) or on the non-crossing strand (vertically at...

...the most out of their meetings. Amanda Gunn ran randomized coffee trials for us at ASCB|EMBO 2019, matching...within a bin as Horizontal Transfer. This comes from horizontal gene transfer, which occurs when bacteria...in Atlanta, GA, in 2018. A lot of my experiences rang true with theirs.  It was at this dinner and the...Singletons logo. Image: James et al., 2020. Help me stranger - Finding mentors for the meeting Just like analysis...

...that better human health.     References Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu...3.4% of founders establishing germline transmission of the transgene, this technique is still in need of ...and chimeric adults mated to produce completely transgenic offspring. Although powerful, this technology... a mouse model, and its success is not always guaranteed. To learn more about this process check out our...the Cas9 endonuclease that creates the DNA double-strand break (DSB) at the target site, and a donor oligonucleotide...

... . https://doi.org/10.1016/s0764-4469(00)01250-6 Ran FA, Hsu PD, Wright J, Agarwala V, Scott DA, Zhang...of the DNA. Finally, in 1953, Franklin along with James Watson and Francis Crick resolved the DNA double...writers have been curious about the capability of transforming the genetic code of living organisms and its...that Streptococcus pneumoniae bacteria could be transformed from one organism to the other. This is now famed...Hershey-Chase experiment. In the same year, Rosalind Franklin and her student Raymond Gosling took the first...double helix. In the 1960s, Har Gobind Khorana, Marshall W. Nirenberg, and Robert W. Holley cracked the genetic...explained how the four letters of the DNA get translated into the twenty amino acids. Once scientists ...

...10.1146/annurev-biophys-062215-010822 Nishimasu, H., Ran, F. A., Hsu, P. D., Konermann, S., Shehata, S. I.... NUC HNH Cut DNA target strand RuvC Cut DNA non-target strand CTD (or PI) Recognize PAM...information on these regions.   Cas9’s transition from weak, random binding to precision gene editing requires...left) and transparent (right). The RNA guide sequence is extended as a single unpaired strand in the center...10 of the guide RNA, prearranged for base pairing with a complementary DNA strand. These 10 nucleotides...the non-target strand for the PAM sequence and doesn’t mind if the PAM complementary strand is mismatched...of the PAM in the non-target DNA strand (dark gray). Target DNA strand, light gray.   Once it has found...

...titrating the transposase to transposon ratios. High ratios of transposase to transposon result in a greater...]. Like other transposons, piggyBac™ has two components, a transposon and a transposase. The piggyBac™...piggyBac™ transposase facilitates the integration of the transposon specifically at ‘TTAA’ sites randomly dispersed... piggyBac™ transposon. These plasmids, when combined with a source of piggyBac™ transposase (available...distributor) allow you to quickly transfer a DNA sequence from the transposon vector to one of many TTAA sequences...folks at Transposagen. This post was contributed by Amar Singh and Mathew Goodwin from Transposagen What ...What is the piggyBac™ transposon? The piggyBac™ transposon was originally identified in moths in 1983, but...

...Cre-dependent transgenes can also be paired with Cre-expressing transgenic mouse lines to restrict transgene expression...with broad CNS or PNS transduction, cell type specificity, or retrograde transport in neurons. (b) Genetic...capsids or transgenes. Sensory neurons and liver appear especially susceptible to off-target transgene expression-induced...It has a similar transduction rate to AAV-PHP.eB in the brain, and a lower transduction rate in the spinal...2022). AAV-PHP.N specifically transduces NeuN+ neurons, with transduction efficiency varying from 10-70%... effectively transduces BALB/cJ, CBA/J, and FVB/NJ mice, with minimal liver transduction observed in both...effectively transduces neurons in the peripheral nervous system (PNS). It has been shown to transduce 80% of...

..., and enzymes for transmitter synthesis. Retrograde transport is used for transport of endocytosed material... facilitate transport: retrograde transport relies on dynein, while anterograde transport relies on kinesin...take advantage of existing transport pathways in neurons. Anterograde transport is typically used for the...mechanisms of trans-synaptic trafficking are not fully understood and may be a form of transcytosis where the...after intracellular transport without actually causing infection or leading to transgene expression within...endothelial transcytosis (Foust et al., 2009; Merkel et al., 2017), and these two modes of transport may share...optogenetically controlled transgenes, in combination with conditional transgenic mice, to reveal not only...

...contains the transposase gene (red) for expressing the transposase enzyme.2.) The transposase enzyme is ...which cuts the DNA.3.) The released transposon can now bind a strand of DNA with a TA dinucleotide (there...removal of the transposon; the plasmid is then degraded by the cell.4.) The transposase creates a double...double strand break in the DNA and allows the tranposon to integrate. Additionally, the TA sequence is duplicated....   The Sleeping Beauty transposon system The Sleeping Beauty (SB) transposon system is one such system...perform gene transfer in vertebrates. The system is comprised of 2 components: 1) an SB transposase, the enzyme...enzyme required for catalysis of transposition; and 2) a transposon containing the gene-expression cassette...