Blin, Kai

Lee, Sang Yup

Weber, Tilmann

Tilmann Weber’s group at the Novo Nordisk Foundation Center for Biosustainability developed a user-friendly, web server implementation of the sgRNA prediction software, CRISPy, for non-computer scientists

Online Research Database In Technology

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Downloaded from orbit.dtu.dk on: Dec 18, 2017Designing sgRNAs with CRISPy webTips and tricks of the tradeBlin, Kai; Lee, Sang Yup; Weber, TilmannPublished in:Lab TimesPublication date:2017Document VersionPublisher's PDF, also known as Version of recordLink back to DTU OrbitCitation (APA):Blin, K., Lee, S. Y., & Weber, T. (2017). Designing sgRNAs with CRISPy web: Tips and tricks of the trade. LabTimes, (1), 53. www.labtimes.org1/2017© 2015 Cell Signaling Technology, Inc.  Cell Signaling Technology, CST, PTMScan, and SimpeChIP are trademarks of Cell Signaling Technology, Inc.15PADEPIGNONE0079ENG_00© 2016 Cell Signaling Technology, Inc.  Cell Signaling Technology, CST, PTMScan, and SimpeChIP are trademarks of Cell Signaling Technology, Inc.15PADEPIGNONE0079ENG_01CELL SIGNALING TECHNOLOGYEPIGENETICS:DISCOVERY THROUGH VALIDATIONValidated Tools for Discovery:»  SimpleChIP® Kits to facilitate Chromatin IP from cells and tissue.»  PTMScan® Kits and Services to enable MS-based discovery of methylated and acetylated proteins.»  Most ChIP-validated antibodies approved for additional applications like IHC, Flow, IF and WB. Learn more at: www.cellsignal.com/epigeneticdiscoveryCST antibodies for epigenetic-related targets, including histone modifi cations, epigenetic regulators, and general transcription factors.CST antibodies validated for ChIP according to ENCODE* Consortium guidelines.69 22*Landt S.G. et al. (2012) Genome Res. 22, 1813–1831.Molecular model of chromatin.Epigenetics Ad Lab Time.indd   1 11/10/16   2:12 PMLT_117_OC_OC.indd   2 02.02.17   18:39page 44 ProductsLab Times 1-2017Though some experts, such as pi-pette calibration specialist and head of pipette calibration compa-ny  Calibrate-It, Michel Bryce, have already proclaimed the end of manual pipetting, manual micropipettes are still the most prevalent pipetting tools in life science lab-oratories – in spite of liquid handlers and electronic pipettes, which are not only fast-er but also reduce the risk of muscle and strain injuries.Bullet-proof construction A major argument, besides the lower price, still speaking in favour of manual pipettes, is their simple and easy use. Lab technicians and researchers are trained on manual pipettes from day one in the lab – operating a manual pipette has almost be-come second nature to them. The rugged and almost bullet-proofed construction is another advantage of the manual pipette. The mechanics that drive plunger and pis-ton inside the shaft is very robust and may be easily disassembled for cleaning. Even autoclaving at 121°C, to prevent contami-nations, doesn’t bother manual pipettes – in contrast to most electronic pipettes that quit their work after autoclaving.   When calibrated properly, manual pi-pettes are very precise instruments, match-ing the accuracy and precision of electron-ic pipettes and liquid handlers. Let’s face it: the errors in manual pipetting are largely produced by the operator – not by the pi-pette. A group led by Giuseppe Lippi from the University Hospital of Verona, Italy, re-cently analysed the intra- and inter-individ-ual imprecision of manual pipetting (Clin Chem Lab Med: Doi 10.1515). Individual pipetting techniques They randomly chose twenty laboratory operators and let them dispense 1 ml, 100 µl or 10 µl water for ten consecutive times with three recently calibrated, certified Product survey: Manual MicropipettesNot Dead YetManual pipettes are under pressure by faster and more ergonomic electric pipettes and liquid handlers. But they may spend a future life in robots, uti-lising manual pipettes instead of pipetting heads for liquid dispensing.Founded 2006. Issue 1, 2017 Lab Times is published bimonthlyISSN: 1864-2381 Publisher:  LJ-Verlag GmbH & Co. KGOffice:  Merzhauser Str. 177, 79100 Freiburg,  Germany, Phone +49(0)761-286869,  Fax +49(0)761-35738Management:  Kai Herfort, Tel: +49 (0)761-286869Editors: Ralf Neumann (Editor-in-chief), Kathleen Gransalke, Kai Herfort, Winfried Koeppelle, Harald Zähringer, Phone +49(0)761-2925884, editors@labtimes.orgReporters: Alejandrolvido, Steven Buckingham, Bettina Dupont, Jeremy Garwood, Karin Hollricher, Karin Lauschke, Alejandra Manjarrez, Rosemarie Marchan, Alex Reis, Ralf SchreckGraphics, Design and Production: Ulrich Sillmann (Art Director), Kathleen Gransalke, Kai Herfort, Winfried Koeppelle, Ralf Neumann, Harald ZähringerCover Photo: benjaminlion@fotolia, Editing: Kai HerfortSales:Advertising Manager: Bernd BeutelTop-Ad Bernd Beutel, Schlossergäßchen 10,  69469 Weinheim, GermanyPhone:  +49(0)6201-29092-0Fax: +49(0)6201-29092-20info@top-ad-online.deRecruitment adverts: Ulrich SillmannPhone +49 (0)761-2925885jobs@labtimes.orgPrinted at: Hofmann Infocom GmbH  Emmericher Str. 10 90411 Nürnberg, GermanyWeb:  www.labtimes.orgWebmaster: Carsten Rees, Tel.: +49 (0)761-1563461, webmaster@labtimes.orgPrices & Subscription rates:- price per issue: €4.90- research institutes/units: free of charge-  annual subscriptions for companies and  personal subscribers: €27.-Subscribe at http://www.labtimes.org/kontakt/sub.html, or mail to: subscriptions@labtimes.orgBank Account:Fidor-Bank BIC: FDDODEMMXXXIBAN: DE42 7002 2200 0020 1347 47Lab Times Different pipetting styles may lead to intra- and inter-individual imprecision of manual pipetting.Photo: iGEM DenmarkLT_117_Product survey Kopie.indd   44 03.02.17   11:41Lab Times1-2017 page 53 MethodsThe development of CRISPR/Cas9, which originates from a bacterial plasmid/phage defense system, into a powerful, genome-editing tool has been one of the major breakthrough technologies in biotechnology within the last few years. With the RNA-guided endonuclease Cas9 from the Streptococcus pyogenes  CRISPR system, currently the most widely used en-zyme, it is nowadays feasible to highly ef-ficiently edit DNA in a broad variety of or-ganisms. The method works in most organ-isms that allow the expression of the differ-ent components. Simplified, Cas9 can be regarded as a programmable, blunt-cutting restriction endonuclease that recognises its target DNA sequence by Watson-Crick base-pair-ing with the ~20bp protospacer (crRNA) that is bound to the Cas9/tracerRNA com-plex and cleaves the target DNA within this protospacer region, i.e., is complementa-ry to the target region. Cas9 and the RNAs may be used in vitro but may also be ex-pressed within the cell.  Soon, it became evident that the genes encoding the tracer RNA and crRNA, which in the native system bind Cas9 as individual RNAs, can artificially be linked to a single-guide RNA (sgRNA), which still efficiently directs the Cas9 endonuclease to its target, while at the same time being easily cloned and expressed (Science 337:816–821). An-other prerequisite for cleavage by Cas9 is the presence of a Protospacer Adjacent Motif (PAM) that has to directly follow the DNA sequence, to be targeted by the proto-spacer. In the case of the S. pyogenes Cas9, this PAM is NGG. For a biotechnological application of this CRISPR-system it is, therefore, essen-tial to design the target sequence for the sgRNAs in a way that ensures its placement directly in front of a PAM and, on the oth-er hand, is unique within the genome to avoid that Cas9 cleaves at other positions in the genome than desired. In addition, Cas9 unfortunately also displays “off-tar-get” effects, i.e., cleavage activity at posi-tions not 100% identical to the protospacer sequence. These happen mostly at sequenc-es, which still have some similarity to the protospacer but no 100% match. Avoiding these sequences is quite chal-lenging, when designing the sgRNAs by hand – computational tools are highly rec-ommended to be used for finding suitable sequences.CRISPR tool for non-model organisms If you are working with model organ-isms, there are many different programmes and websites available that offer such func-tionality, for example, CHOPCHOP (Nucleic Acids Res 42: 401-07), or CCTop (PLoS ONE, 10:e0124633). However, when we start-ed our work with developing the CRISPR technology for the organisms we work in our lab, no easy usable tool existed, to en-able designing sgRNAs for such non-mod-el organisms, i.e., tools that allow the users to provide an arbitrary genome sequence against which may be searched. Therefore, we have developed CRISpy-web, a web-based tool to design sgRNAs for non-model microorganisms (Synth Syst Bi-otechnol 1:118-21). CRISpy-web is based on the software CRISpy, a web-tool to de-sign sgRNAs for use with Chinese Hamster Ovary (CHO) cells that was previously de-veloped at our institute (Biotechnol Bioeng 111:1604-16). CRISPy-web is freely acces-sible at: http://crispy.secondarymetabolites.org. The first step to use CRISpy-web is to upload the genome sequence of the micro-organism of interest in Genbank format. Al-ternatively, sequences can be directly trans-ferred from the antiSMASH secondary me-tabolite genome mining platform (Nucleic Acids Res 43: 237-43), by entering the ant-iSMASH job ID instead. After selecting and uploading the genome to be analysed, in the next screen, the target region to pre-dict protospacer sequences can be speci-fied. It can be defined either by entering the positions as a range (e.g., 1234-5678), by providing the locus tag, gene name or protein ID (if annotated in the Genbank se-quence), or – if the data was pre-analysed with  antiSMASH – the gene cluster number. In this case, the antiSMASH detected gene cluster can also be directly selected by click-ing on the respective line in the displayed table. When pressing the “Find Targets” but-ton, suitable protospacer sequences are identified in the selected region of the ge-nome of interest. Depending on the size of the genome sequence to analyse, this step can take a few minutes to complete. On the top line of the screen, genes encoded within the selected region of the genome are dis-played as arrows; in the case that the region contains several genes, the user can zoom in on individual genes by clicking on the gene of interest and selecting “show results for this gene only”.  Potential protospacer motifs are indi-cated as little red boxes, depending on their DNA strand orientation (forward strand on top, reverse strand on bottom) and listed in the table – sorted by quality (uniqueness). To select a potential protospacer sequence for export, either click on the red box or click the shopping basket in the list view. On clicking the “checkout” button at top right of the screen, a table containing all se-lected protospacer sequences is displayed and ready for export as a CSV file, and ready to be used in the individual  sgRNA cloning workflows.     KAI BLIN, SANG YUP LEE AND TILMANN WEBER(Novo Nordisk Foundation Center for Bio-sustainability, Technical University of Den-mark) Lab Hint Tips and tricks of the tradeDesigning sgRNAs with CRISPy-webTilmann Weber’s group at the Novo Nordisk Foundation Center for Biosustainability developed a user-friendly, web server implementation of the sgRNA prediction software, CRISPy, for non-computer scientists.Do you have any useful tips? Contact us at:editors@labtimes.orgPhoto: Tilmann WeberLT_117_Tips and Tricks.indd   53 03.02.17   11:43

Designing sgRNAs with CRISPy web:Tips and tricks of the trade

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Designing sgRNAs with CRISPy web:Tips and tricks of the trade

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