...characterized two Cpf1 nucleases, distant cousins of well-known Cas9. Cpf1 cleaves DNA in a staggered pattern...detectable off-target cleavage. Most gRNAs directed low-frequency Cpf1 cleavage at 1-12 off-target sites...proximity to the cleavage site, preventing future edits. In contrast, since Cpf1 cleaves relatively far ...Cpf1-mediated cleavage, the continued presence of the PAM may give Cpf1 the ability to cleave again and possibly...the two (tracrRNA and crRNA) needed by Cas9 for cleavage. Now, two new studies show that Cpf1 displays ..., Bernd, et al. "Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system." Cell (2015)...blunt ends 3 nt upstream of the PAM site, Cpf1 cleaves in a staggered fashion, creating a 5 nucleotide...

... strands hybridize, the nuclease can detect strands with mismatches and cleave them. The results are then...surveyor mismatch cleavage assays to detect mutations triggered by engineered nucleases." G3: Genes| Genomes...profiling of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity." Nature biotechnology...genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases." Nature biotechnology 33.2 (2015)...genotyping. The most common options include mismatch cleavage assays, such as Surveyor™, T7E1, and Sanger sequencing...adequate standards for validating edits.  Mismatch cleavage assays rely on pairing between the edited strand... in the genome predicted to contain off-target cleavage events. Unbiased techniques search the whole genome...

...sequence! In bacteria, Cas13d-mediated cleavage promotes collateral cleavage of other RNAs. As with other Cas13s...and two nuclear long non-coding RNAs. One interesting twist to Cas13d enzymes is their cleavage pattern...EsCas13d favors cleavage at uracils, but a more detailed exploration of this cleavage pattern is necessary...a CRISPR repeat array near a putative effector nuclease. The Cas13d proteins they identified have little...identified Cas13a-c orthologs, but they do include HEPN nuclease domains characteristic of the Cas13 superfamily...their own CRISPR arrays into guide RNAs. crRNA cleavage is retained in dCas13d and is thus HEPN-independent...Cas13s, this collateral cleavage does not occur when Cas13d is expressed in a mammalian system. Since Cas13d...

...and structural integrity; cleavable. Not great for purification; too cleavable in eukaryotes TAP GRRIPGLINP...FLAG) are cleaved by specific proteases without requiring the addition of an independent cleavage recognition...cell. FLAG tags can be cleaved by enterokinase, which recognizes DDDDK^X, cleaving after the lysine. The...Notes TEV ENLYFQS Cleaves between the Gln and Ser residues Thrombin LVPRGS Cleaves between Arg and Gly...maltose-binding protein (MBP). Overexpression can also lead to insolubility, and aggregated protein is not useful...sequence of amino acids, is basically a molecular leash for your protein. If you’re working with an uncharacterized...-based purification; has inherent enterokinase cleavage site GST Large Protein 26 Purification and Stability...

...have two endonuclease domains: the n-terminal RuvC-like nuclease domain and the HNH-like nuclease domain...conformational change that positions the nuclease domains to cleave opposite strands of the target DNA. Thus...and Cas9, bind to these transcripts and C) cleave them leaving mature crRNAs bound to the Cas9/tracrRNA ...Cas9 complex to the target DNA which is E) cleaved leaving a F) double-strand break. A “gRNA” is a researcher-designed...Cas9 genome editing systems use a non-specific endonuclease (Cas9 or closely related Cpf1) to cut the genome...genome and a small RNA (gRNA) to guide this nuclease to a user-defined cut site. After reading this post...functions of the various CRISPR/Cas9 components. Please note that while this post is intended to provide...

...every team should have a goal, or at the very least, a clear reason for existing. Here are a few types of...own experience: Leadership Few things are more frustrating than being on a leaderless team. Sometimes ...team, I try to make it very clear why that person is there. If it becomes clear after a few meetings that...strong sense of purpose, an excellent leader, and perfectly clear lines of communication, but if teammates...this gap by both highlighting that researchers do learn MANY transferable skills while working in the lab...The famous discoveries and experiments I’d been learning about for years were usually the products of teams...forward nearly 20 years and I am known by some colleagues as the “cross-team guy” at Addgene, an unofficial...

...serve as slaves.  Meganucleases In 1989 the first ever DNA-editing enzymes, meganucleases, were discovered...created by genetics.    Zinc finger nucleases Zinc finger nucleases (ZFNs) are artificial restriction enzymes...DNA-binding domain to a DNA-cleavage domain (FokI). Zinc finger nucleases were first designed in 2005....activator-like effector nucleases (TALENs) were engineered. TALENs follow a DNA-binding and DNA-cleaving strategy similar...The major drawback of all the endonuclease-based gene editors (meganucleases, ZFNs, TALENs, and CRISPR-Cas...2002) Mutations altering the cleavage specificity of a homing endonuclease. Nucleic Acids Research 30:3870...in 1990, just a year after the discovery of meganucleases. The storyline involves the resurrection of ...

...these authors were able to reconstruct a cleared guinea pig cochlea in 3D. The basic configuration of the...passes through the specimen, which often leads to photobleaching.  Furthermore, point-by-point scanning ... of clear specimens and for in vivo imaging of transparent organisms. Advances in tissue clearing Similar...modified by others to produce Murray’s clear solution.  Murray's clear (11) was mostly used to study the development...with diameters less than 4nm. Lateral view of a cleared rat pons, medulla and the first segments of the...fluorescence microscopy was first pioneered by Voie and colleagues and originally named orthogonal-plane fluorescence...reconstruction (5). In order to image large tissue such as cleared rodent brain, the Ultramicroscope was developed...

... follow a lead and when to terminate a project are fundamental skills that all group leaders must master... 8. Learn to mentor, manage, communicate, and teach The dirty secret about becoming a group leader is ...teaching experience and learn about modern pedagogy techniques, especially active learning. Serve as a daily...Assuming you want to become competitive for a Group Leader position, what will you need? Multiple first ...universities and institutes annually advertise for Group Leader positions starting in August, the year before the...completed. Therefore, to be ready to apply for a Group Leader position, key first author manuscripts need to ...perspective for your project. Furthermore, Group Leader position applications require 3 letters of reference...

...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....Alex Chavez Figure 1: Schematic of native (nuclease-competent) Cas9 interacting with either a full-length...native Cas9's two catalytic domains, HNH and RuvC, cleave DNA strands complementary and non-complementary...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...

...Cpf1 CRISPR Nucleases   Cas9 Cpf1 Components crRNAtracrRNACas9 crRNACpf1 Endonuclease size spCas9... multiple targets compared to Cas9.  Read on to learn more about Cpf1 multiplexing.  For an in-depth review...crNA: ~42 nt Requires tracrRNA? Yes No dsDNA cleavage Blunt end 5' overhang PAM sequence NGG TTTV ...PAM site preserved? Usually Destroyed Yes, Cpf1 cleaves 5' of the protospacer Multiplexing CRISPR-Cas9...express the gRNAs and Cas9. Co-transfections can lead to variable expression levels due to differences...allow for expression of multiple gRNAs (i.e. Csy4 cleavage sequence, tRNAs, multiple individual promoters...requires its own promoter   Kabadi et al Csy4-Cleavable Cassettes transfection gRNAs expressed from a ...

...multiplexing gRNAs used the RNA nuclease Csy4 from Pseudomonas aeruginosa to cleave 4 gRNAs separated by 28 base...cell, this system is clearly also robust enough to ensure high levels of gRNA cleavage.   Xie et. al constructed...sequences preceding gRNAs, to test for gRNA cleavage. gRNAs were cleaved precisely as predicted with no addition...schematics. Scissors indicate cleavage sites. After confirming gRNA cleavage, they examined gRNA expression...with Cas9 and the polycistronic transcript. As nucleases may have off-target effects and/or toxicity, it...every living organism. Precursor, or pre-tRNAs are cleaved by RNase P and Z in eukaryotes (E in bacteria),...elements of tRNAs are necessary for recognition and cleavage. This sequence flexibility indicates that gRNAs...

...a fluorophore will bleach. This is, however, not the full story. The photobleaching rate does not depend... 2-fold does not reduce photobleaching by half. How exactly the photobleaching rate depends on power is...to select a photostable fluorescent protein. Photobleaching is the irreversible destruction of a fluorophore...influence of light. Any fluorescent molecule will photobleach at some point. For live-cell imaging, it is desirable...fluorescent proteins that are photostable. On top of photobleaching, fluorescent proteins may display reversible...destroyed. Factors that affect photostability The photobleaching rate of a fluorophore primarily depends on ...excitation wavelength. A fluorophore is less likely to bleach when it is excited off the peak wavelength, since...

...over time! Communicate often and clearly As a manager you need to clearly state expectations and goals. ...this gap by both highlighting that researchers do learn MANY transferable skills while working in the lab... be patient and keep an open mind. First let’s clear up a misconception about being a manager: yes, being...professional career of. Like everyone else I mostly learned through experience and from my mistakes. Here I... people! More specifically, they had different learning styles. One was a “Doer” and the other was a “... the best protocol;  he wanted to get going and learn through doing things. Had I realized that at the...fits all”. Manage the individuals in your team: learn what motivates them (keep in mind that it can be...

... a collection that is greater than 75 plasmids, please let us know by contacting [email protected]. ...plasmid can be added on subsequent pages. Type - please choose from one of the following descriptions for...e-mail us at  [email protected] Special cases Please contact us at [email protected] or +1 (617)-ADDGENE...must be verified by next-generation sequencing. Please see the help center article, "Can I deposit a pooled... any genomic alterations unique to your strain. Please contact us at [email protected] or +1 (617)-ADDGENE...bacterial strain(s) through Addgene. Description – please indicate the gene cloned into your plasmid, any... not lost.   Step 1: Sequences, Maps, and Files Please provide any full or partial plasmid sequences and...

... of restriction endonucleases and methyltransferases. Restriction endonucleases cleave DNA at or near ...system is MazE/MazF. The toxin MazF is a ribonuclease that cleaves specific sequences in RNA resulting in...restriction enzyme from cleaving the DNA. A role for restriction endonucleases in plasmid addition was...chromosome. The more stable endonuclease can then cut the chromosome leading to cell death. Antisense RNA-regulated...antitoxin. However, the antitoxin will be degraded leaving the cell to be killed by the more stable toxin....GyrA subunit of DNA gyrase and trapping it in the cleaved complex. This results in DNA breakage and cell ... which binds and inhibits MazF, is continually cleaved by the ClpAP protease so without continual production...

...connect one concept to another which leads us to… Framework Learning to think like an immunologist, a geneticist...that you think are clearly connected, step back and ask yourself, “are these clearly connected because ...the way information or tasks are presented to a learner; and germane cognitive load refers to the work ...which can drive away even the most enthusiastic of learners.  What contributes to cognitive load?  Everything...use up your audience’s mental bandwidth.  Jargon Learning, using, and retaining new vocabulary takes time...others. But sometimes we’re unaware that we are leaning on an unspoken expectation of our audience knowing...details to communicate. It’s okay (and encouraged) to leave blank spaces or to gloss over some aspects when ...

...its method of DNA cleavage. Instead of cleaving DNA at a defined site to form a clean DNA break, Cas3 locates...non-specific RNA cleavage and 2). cell toxicity. Cas7-11 does not display collateral RNA cleavage and is significantly...We are all oh so familiar with the nuclease Cas9, but how does it stack up to the new Cas enzymes on ...without shortly thereafter hearing ‘Cas9’. The Cas9 nuclease is a genome engineering powerhouse, and for good...staggered breaks, also known as sticky ends. Cas12a cleavage results in 5 bp overhangs and is particularly ...enzymes, Cas14 targets ssDNA instead of dsDNA. It cleaves ssDNA with high fidelity - sensitive to even a ... to Cas12a is that Cas14 also indiscriminately cleaves non-complementary ssDNA when engaged by its target...

...eukaryotic polyadenylation signal directs cleavage at the cleave signal and addition of a poly-A tail to...Resources on the Addgene Blog Learn how the Promoter Controls Gene Expression Learn about New Cloning Techniques...transcription stops, or termination. Read on to learn more! Download our Plasmids 101 eBook to get all... (such as a gene) and initiate the process of releasing the newly synthesized RNA from the transcription...destabilize the transcription complex, initiating cleavage of the transcript.   Figure 1: A predicted...transcription of the template and initiating the desired cleavage event, although some engineered terminators come...and RNA secondary structure to induce transcript cleavage, similar to the Rho-independent termination found...

...with them and remaining close colleagues of the PI? Or do lab alumni leave the field or get pushed out ...association? Will you learn how to run a lab if that is your aspiration? Will you help or at least observe grant...Scientists are lifelong learners. I believe we won’t be happy if we aren’t learning so before you take any...very communal. That didn’t suit my collaborative learning style at all. Finally I rotated in a small lab... members stay around? Is it too long or do they leave prematurely? Do lab alumni stay in the same field...organizational skills? Are they involved with colleagues and the department? Are they successful at getting...teamwork that brings out your best performance and learning? Everyone’s different so give some thought to ...