NEB UK Expressions 2014 JULY Cell Signaling Technology: New Antibodies Genome Editing for PD-L1 & Feature Article New Cas9 Nuclease LKB1 Tips for CRISPR/Cas9 Experiments Page 14 sgRNA Template Construction Pages 3-7 The Importance of a Well Validated Antibody Page 12 CRISPR/Cas9 and Targeted Genome Editing: A New Era in Molecular Biology Page 3 NEW PRODUCTS INTRODUCTORY PRICING ! CONTENTS NEW New Protein Standards Scan for product We are pleased to introduce two new protein molecular weight info. Blue Protein Standard, standards which offer increased stability and sharper bands. These new Broad Range 03 CRISPR/Cas9 and Targeted Lot: 0011403 standards replace our prestained protein markers and ladders (P7708, P7706S Exp: 3/16 100 mini gel lanes S Store at -20°C Genome Editing: A New Era in P7709, P7710 and P7711). The load volume is 5 µl for a mini gel, 0.5 ml and 10 µl for a full sized gel. These products will remain stable for Scan for product Molecular Biology. up to 2 weeks at room temperature (25°C) and for up to 3 months info. at 4°C. However, we recommend storing your Protein Standard at Color Protein Standard, 06 Tips For Planning Your Broad Range -20°C, for a guaranteed shelf life of 2 years. Lot: 0011403 P7712S Exp: 3/16 100 mini gel lanes For Research Use Only. S Store at -20°C CRISPR/Cas9 Experiments. New England Biolabs is an ISO 9001, 0.5 ml ISO 14001 and ISO 13485 Color Protein Standard, Blue Protein Standard, certified facility. 07 sgRNA Template Construction Broad Range, P7712 Broad Range, P7706 For CRISPR/Cas9 Genome • 12 bands • 11 bands Editing. • broad range: 11-245 kDa • broad range: 11-190 kDa For Research Use Only. New England Biolabs is an ISO 9001, • 25 kDa and 75 kDa ISO 14001 and ISO 13485 • 25 kDa and 80 kDa certified facility. 08 SplintR Ligase: a DNA ligase reference bands reference bands that ligates adjacent ssDNA oligos • replacing P7709 and P7711 • replacing P7708 and P7710 splinted by a complementary RNA. kDa kDa 245 09 NGS Technical Tips: Getting 190 ADVANTAGES 190 135 Started with RNA-Seq. 135 100 • Convenience – No need to boil 100 before first use; simply thaw, mix 10 Application Note: Automated 75 80 and load Size Selection of NEBNext Small 58 RNA libraries with the SAGE 58 • Stability – Longer term storage; 2 year shelf life at -20oC Pippin Prep™. 46 46 • Greater resolution – Sharper low mol. wt. bands 11 NEBNext Reagents for Small 32 RNA Library Prep. 32 12 IHC Validation: The Importance 25 P7708. P7709, P7710 and P7711 will be of a Well Validated Antibody. 25 22 discontinued and replaced by the new standards as indicated but will continue to be available 13 IHC Controls: Be in Control of 22 for a short time while supplies last. Because 17 the proteins used in the prestained standards are different than those used in the prestained your IHC Analysis. 17 protein markers and ladders, it is important 11 14 Tumour Immunology: New to note that the apparent molecular weights will be different. Please refer to the apparent ™ 11 PD-L1 Antibody from CST . 10–20% Tris-glycine molecular weight chart to find the appropriate 10–20% Tris-glycine SDS-PAGE Gel molecular weights for your gel. 15 Cancer Research: New LKB1 SDS-PAGE Gel IHC Formulated Antibody from Ordering Information CST. PRODUCT NEB # SIZE INTRODUCTORY PRICE Color Protein Standard, Broad Range P7712S 100 lanes £83 Antibodies for Flow Cytometry Blue Protein Standard, Broad Range P7706S 100 lanes £74 FROM CST Page 16 Cas9 Nuclease, S. pyogenes NEW Cas9 Nuclease, S. pyogenes, is an RNA-guided endonuclease that catalyzes site-specific cleavage of double stranded DNA. Cas9 Nuclease is a central component of CRISPR-based immunity, a mechanism used to protect a bacterial or archaeal cell from invading viral and foreign DNA and has been adapted for use in genome engineering, because it can be easily programmed for target specificity by supplying sgRNAs of any sequence. In cells and animals, genome targeting is performed by expressing Cas9 nuclease and sgRNA from DNA constructs (plasmid or virus), supplying RNA encoding Cas9 nuclease and sgRNA, or by introducing RNA-programmed Cas9 Nuclease directly. See pages 2-7 of this issue for more information on CRISPR/Cas9 and genome editing Ordering Information PRODUCT NEB # SIZE PRICE Read Me, Recycle Me New England Biolabs (UK) Ltd Cas9 Nuclease, S. pyogenes M0386S 150 units £138 100% 100% recycled paper, 75-77 Knowl Piece, Hitchin, Herts SG4 0TY vegetable based inks. Tel: 0800 318486 | Email: [email protected] www.neb.uk.com FEATURE ARTICLE CRISPR/Cas9 and Targeted Genome Editing: A New Era in Molecular Biology The development of efficient and reliable ways to make precise, targeted changes to the genome of living cells is a long-standing goal for biomedical researchers. Recently, a new tool based on a bacterial CRISPR-associated protein-9 nuclease (Cas9) from Streptococcus pyogenes has generated considerable excitement (1). This follows several attempts over the years to manipulate gene function, including homologous recombination (2) and RNA interference (RNAi) (3). RNAi, in particular, became a laboratory staple enabling inexpensive and high-throughput interrogation of gene function (4, 5), but it is hampered by providing only temporary inhibition of gene function and unpredictable off-target effects (6). Other recent approaches to targeted genome modification – zinc-finger nucleases [ZFNs, (7)] and transcription-activator like effector nucleases [TALENs (8)]– enable researchers to generate permanent ARTICLE FEATURE mutations by introducing double-stranded breaks to activate repair pathways. These approaches are costly and time-consuming to engineer, limiting their widespread use, particularly for large scale, high-throughput studies. Alex Reis, Bitesize Bio Figure 1. Cas9 in vivo: Bacterial Adaptive Immunity Breton Hornblower, Ph.D., Brett Robb, Ph.D. and George Tzertzinis, Ph.D., New England Foreign Biolabs, Inc. DNA NGG The Biology of Cas9 Acquisition Target PAM The functions of CRISPR (Clustered Regularly CRISPR loci Interspaced Short Palindromic Repeats) and Bacterial genome CRISPR-associated (Cas) genes are essential in tracrRNA cas9 cas crRNA crRNA crRNA genes adaptive immunity in select bacteria and archaea, enabling the organisms to respond to and elimi- nate invading genetic material. These repeats tracrRNA, trRNA Primary transcript were initially discovered in the 1980s in E. coli (9), but their function wasn’t confirmed crRNA until 2007 by Barrangou and colleagues, who biogenesis demonstrated that S. thermophilus can acquire resistance against a bacteriophage by integrating Cas9 crRNA a genome fragment of an infectious virus into its CRISPR locus (10). crRNA Interference Three types of CRISPR mechanisms have been NGG identified, of which type II is the most studied. In Foreign Cleavage this case, invading DNA from viruses or plasmids DNA is cut into small fragments and incorporated into a CRISPR locus amidst a series of short repeats In the acquisition phase, foreign DNA is incorporated into the bacterial genome at the CRISPR loci. CRISPR loci is then transcribed and processed into crRNA during crRNA biogenesis. During interference, Cas9 endonuclease complexed with (around 20 bps). The loci are transcribed, and a crRNA and separate tracrRNA cleaves foreign DNA containing a 20-nucleotide crRNA complementary sequence adjacent transcripts are then processed to generate small to the PAM sequence. (Figure not drawn to scale.) RNAs (crRNA – CRISPR RNA), which are Genome Editing Glossary used to guide effector endonucleases that target invading DNA based on sequence complemen- Cas = CRISPR-associated genes Indel = insertion and/or deletion tarity (Figure 1) (11). Cas9, Csn1 = a CRISPR-associated protein NHEJ = Non-Homologous End Joining One Cas protein, Cas9 (also known as Csn1), containing two nuclease domains, that is PAM = Protospacer-Adjacent Motif has been shown, through knockdown and programmed by small RNAs to cleave DNA RuvC = an endonuclease domain named for rescue experiments to be a key player in cer- crRNA = CRISPR RNA an E. coli protein involved in DNA repair tain CRISPR mechanisms (specifically type II dCAS9 = nuclease-deficient Cas9 sgRNA = single guide RNA CRISPR systems). The type II CRISPR mecha- nism is unique compared to other CRISPR DSB = Double-Stranded Break tracrRNA, trRNA = trans-activating crRNA systems, as only one Cas protein (Cas9) is gRNA = guide RNA TALEN = Transcription-Activator Like required for gene silencing (12). In type II HDR = Homology-Directed Repair Effector Nuclease systems, Cas9 participates in the processing HNH = an endonuclease domain named ZFN = Zinc-Finger Nuclease of crRNAs (12), and is responsible for the for characteristic histidine and asparagine destruction of the target DNA (11). Cas9’s residues function in both of these steps relies on the presence of two nuclease domains, a RuvC-like nuclease domain located at the amino terminus and a HNH-like nuclease domain that resides in the mid-region of the protein (13). 03 FEATURE ARTICLE continued… To achieve site-specific DNA recognition and Cas9 and CRISPR as a New Tool in the homology-directed repair (HDR) pathway cleavage, Cas9 must be complexed with both a Molecular Biology allowing for precise replacement mutations to crRNA and a separate trans-activating crRNA be made (Figure 2A) (17, 18). The simplicity of the type II CRISPR nuclease, (tracrRNA or trRNA), that is partially comple- with only three required components (Cas9 Cong and colleagues (1) took the Cas9 system mentary to the crRNA (11). The tracrRNA is along with the crRNA and trRNA) makes this a step further towards increased precision required for crRNA maturation from a primary system amenable to adaptation for genome by developing a mutant form, known as transcript encoding multiple pre-crRNAs. This editing. This potential was realized in 2012 Cas9D10A, with only nickase activity.