Genome Editing TOOLS TO SUPPORT CRISPR/CAS9 APPLICATIONS

Genome Editing TOOLS TO SUPPORT CRISPR/CAS9 APPLICATIONS

Genome Editing: Tools to Support CRISPR/Cas9 Applications Genome editing is enabled by the development of tools to make precise, targeted changes to the genome of living cells. Recent approaches to targeted genome modification -- zinc-finger nucleases (ZFNs) and transcription-activator like effector nucleases (TALENs) enable researchers to generate 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. These genome editing techniques were concurrent with other approaches over the years to manipulate gene function, including homologous recombination and RNA interference. RNAi, in particular, became a laboratory staple enabling inexpensive and high-throughput interrogation of gene function, but is hampered by providing only temporary inhibition of gene function and unpredictable off-target effects. However, methods based on a bacterial CRISPR-associated protein-9 nuclease (Cas9) from Streptococcus pyogenes have generated considerable excitement. CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) and CRISPR-associated (Cas) genes are essential in adaptive immunity in select bacteria and archaea, enabling the organisms to respond to and eliminate invading genetic material. CRISPR/Cas9 in vivo: Bacterial Adaptive Immunity Foreign DNA Acquisition Target NGG PAM Bacterial genome tracrrna cas9 CRISPR loci cas crrna crrna crrna genes tracrrna, trrna Primary transcript crrna biogenesis crrna Cas9 Interference Foreign DNA NGG Cleavage crrna In the acquisition phase, foreign DNA is incorporated into the bacterial genome at the CRISPR locus. The CRISPR locus is then transcribed and processed into crrna during crrna biogenesis. During interference, Cas9 endonuclease complexed with a crrna and separate tracrrna cleaves foreign DNA containing a 20-nucleotide crrna complementary sequence adjacent to the PAM sequence. (Figure not drawn to scale.) 2

CRISPR/CAS9 GENOME EDITING CRISPR/Cas9 Genome Editing The simplicity of the CRISPR nuclease system, with only three components (Cas9, crrna and tracrrna) makes this system attractive for laboratory use. By combining the crrna and tracrrna into a single synthetic guide RNA (), a further simplified two-component system can be used to introduce targeted double-stranded breaks in genomic DNA. Breaks activate repair through error prone Non-Homologous End Joining (NHEJ) or Homology Directed Repair (HDR). In the presence of a donor template with homology to the targeted locus, the HDR pathway may operate, allowing for precise mutations to be made. In the absence of a template, NHEJ is activated, resulting in insertions and/or deletions (indels), which disrupt the target locus. TOOLS & RESOURCES Visit www.neb.com/genomeediting to find our up-to-date listing of products and protocols to support this application. Schematic representation of Cas9 Nuclease, S. pyogenes sequence recognition and DNA cleavage Cas9 Nuclease, S. pyogenes DNA PAM (NGG) CGCTTGTTTCGGCGTGG GTATGG Cleavage Cleavage GCGAACAAAGCCGCACC CATACC CGCUUGUUUCGGCGUGG GUA 20 15 10 5 1 Nucleotide position (upstream of PAM) GLOSSARY Like any new technique, genome editing with CRISPR/Cas9 comes with a list of acronyms and abbreviations. This list should help you to familiarize yourself with the language associated with CRISPR/Cas9 studies. Cas = CRISPR-associated genes Cas9, Csn1 = a CRISPR-associated protein containing two nuclease domains, that is programmed by small RNAs to cleave DNA crrna = CRISPR RNA dcas9 = nuclease-deficient Cas9 DSB = Double-Stranded Break grna = guide RNA HDR = Homology-Directed Repair HNH = an endonuclease domain named for characteristic histidine and asparagine residues Indel = Insertion and/or deletion NHEJ = Non-Homologous End Joining PAM = Protospacer-Adjacent Motif RuvC = an endonuclease domain named for an E. coli protein involved in DNA repair = single guide RNA tracrrna = trans-activating crrna TALEN = Transcription-Activator Like Effector Nuclease ZFN = Zinc-Finger Nuclease 3

TIPS FOR PLANNING YOUR CAS9 EXPERIMENT Tips for Planning Your Cas9 Experiment The CRISPR/Cas9 genome editing technique is a powerful tool for researchers. However, several practical aspects should be carefully considered in order to achieve the best results from this system. These considerations include: whether to opt for wild-type or double nickase, how to achieve multiplexing, which to use and delivery of DNA, RNA or active ribonucleoproteins. One of the most important decision is which to use. A variety of s have been validated for different cells and model organisms, and final application, from cutting or nicking to activating genes and screening libraries. Several groups have provided repositories of these plasmids, which are available through Addgene (www.addgene.org). Examples of NEB products that can be used to support CRISPR workflows are shown below: Featured NEB Products Supporting CRISPR Workflows PRODUCT NAME CRISPR/CAS9 APPLICATION NEB # SIZE NEW Cas9 Nuclease NLS, S. pyogenes Cas9 Nuclease, S. pyogenes Q5 Site-directed Mutagenesis Kit (with or without competent cells) Q5 High-fidelity DNA Polymerases NEBuilder HiFi DNA Assembly Master Mix in vitro cleavage of dsdna. Genome engineering by direct introduction of active nuclease complexes M0641S/L/M 50/250/500 pmol in vitro cleavage of dsdna. Genome engineering by direct introduction of active nuclease complexes M0386S/L/M 50/250/500 pmol Insertion of target sequence into the Cas9- construct and modification of HDR templates E0554S/E0552S 10 rxns High-fidelity construct generation for use with CRISPR workflows Multiple* Multiple* Single-tube, isothermal generation of the Cas9- construct and HDR templates E2621S/L/X 10/50/250 rxns ONLINE RESOURCES Plasmid Repositories www.addgene.org CRISPR-gRNA Design Tools crispr.mit.edu zifit.partners.org/zifit/ www.e-crisp.org/e-crisp/designcrispr.html chopchop.rc.fas.harvard.edu/ Online Forums groups.google.com/forum/#!forum/crispr Organism-specific Resources wormcas9hr.weebly.com www.flyrnai.org TOOLS & RESOURCES Visit neb.com to find: Protocols for applications such as in vitro RNA synthesis and site-directed mutagenesis Online tools, including NEBuilder (NEBuilder.neb.com) for DNA Assembly & NEBaseChanger (NEBaseChanger.neb.com) for the Q5 Site-Directed Mutagenesis Kit NEBuilder HiFi DNA Assembly Cloning Kit HiScribe T7 ARCA mrna Kit (with or without tailing) Single-tube, isothermal generation of the Cas9- construct and HDR templates E5520S 10 rxns Generation of Cas9 mrna with ARCA cap E2060S/E2065S 20 rxns HiScribe T7 High Yield RNA Synthesis Kit Generation of and Cas9 mrna E2040S 50 rxns HiScribe T7 Quick High Yield RNA Synthesis Kit Generation of and Cas9 mrna E2050S 50 rxns T7 Endonuclease I Determination of the targeting efficiency of genome editing protocols M0302S/L 250/1,250 units * Visit Q5PCR.com for ordering information. 4 NEW ENGLAND BIOLABS, NEB, NEBUILDER and Q5 and registered trademarks of New England Biolabs, Inc. HISCRIBE and NEBASECHANGER are trademarks of New England Biolabs, Inc.

TEMPLATE CONSTRUCTION Template Construction for CRISPR/Cas9 Genome Editing Along with Cas9 nuclease, CRISPR experiments require the introduction of an containing an approximately 20 base sequence specific to the target DNA of a non-variable scaffold sequence. can be delivered as RNA or by transforming with a plasmid with the coding sequence under a promoter. A number of strategies have been developed to quickly swap out the 20 base sequences allowing convenient cloning using NEB products. 1 BbsI ENZYME-BASED STRATEGY Annealing Digestion with BbsI expression ~20 bases Phosphorylation & ligation (T4 PNK & T4 DNA Ligase) Target DNA sequence tracrrna sequence Promoter Assembled BbsI R0539S/L T4 DNA Ligase M0202S/M/L/T T4 Polynucleotide Kinase M0201S/L 2 Q5 SITE-DIRECTED MUTAGENESIS STRATEGY Amplification ~20 base insertion Primer expression High-fidelity PCR Treatment with KLD Mix Assembled Q5 Site-Directed Mutagenesis Kit Q5 Site-Directed Mutagenesis Kit (Without Competent Cells) E0554S E0552S 3 NEBUILDER HIFI DNA ASSEMBLY STRATEGY Annealing Linearization with restriction enzyme ~25 bases ~25 bases ~20 bases + expression Synthetic oligonucleotides Assembled Q5 Hot Start High-Fidelity 2X Master Mix Master Mix Cloning Kit M0494S/L E2621S/L/X E5520S Transformation & Purification In vitro transcription NEB 5-alpha Competent E. coli (High Efficiency) NEB 10-beta Competent E. coli (High Efficiency) C2987P/I/H C3019I/H plasmid + + + + HiScribe T7 ARCA mrna Kit HiScribe T7 ARCA mrna Kit (with tailing) E2065S E2060S Cas9 plasmid Cas9 mrna Cas9 protein Homologous repair template (optional) HiScribe T7 High Yield RNA Synthesis Kit E2040S HiScribe T7 Quick High Yield RNA Synthesis Kit E2050S Cells Animals In vitro Anti-Reverse Cap Analog -O-Me-m 7 G() ppp()g Vaccinia Capping System S1411S/L M2080S 5

DESIGNING HOMOLOGOUS REPAIR TEMPLATES Designing homologous repair templates Modifications of Cas9-target sites can be achieved by supplying a homologous repair template, in addition to and Cas9. Homologous repair templates are plasmids containing regions of homology surrounding the target sequence, that are altered to have the desired mutations or knock-ins, such as selectable markers or fluorescent tags. The homologous region can be amplified from genomic DNA using a highfidelity polymerase, and then cloned into your plasmid backbone. NEB suggests Q5 High Fidelity DNA Polymerase products for this amplification, followed by the PCR Cloning Kit (NEB #E1202). Further modifications can then be introduced using Master Mix (NEB #E2621) or by site-directed mutagenesis, using the Q5 Site-Directed Mutagenesis Kit (NEB #E0554). Designing Homologous Repair Templates Q5 Hot Start High-Fidelity 2X Master Mix NEB PCR Cloning Kit Master Mix Cloning Kit Q5 Site-Directed Mutagenesis Kit Q5 Site-Directed Mutagenesis Kit (without Competent Cells) M0494S/L E1202S E2621S/L/X E5520S E0554S E0552S Homology Directed Repair templates can be prepared by PCR amplification of a region of genomic DNA centered around the location of the desired modification. PCR products are then inserted and joined into a backbone. Knock-ins, selectable markers, tags, and other changes can then be introduced through techniques such as NEBuilder or site-directed mutagenesis. In vitro Transcription of Cas9 specificity is dictated by an containing a ~20 base sequence complementary to the target sequence. can be introduced directly, for example through microinjection or transfection, or indirectly through a expression plasmid. Generation of can be achieved through in vitro transcription from plasmid templates or PCR products. NEB provides convenient kits and reagents for in vitro transcription of. HiScribe T7 High Yield RNA Synthesis Kit E2040S HiScribe T7 Quick High Yield RNA Synthesis Kit E2050S T7 Transcription T7 Promoter TAATACGACTCACTATAG ATTATGCTGAGTGATATC Run-off transcript has the top strand sequence. G T7 Transcription DNA template RNA transcript A transcription template contains a T7 promoter sequence followed by the sequence of interest. The T7 promoter is required for transcription to occur. The sequence of the transcript is the same as the top strand of the DNA template and initiates from G residues, encoded in the optimal T7 RNA polymerase promoter. 6

IN VITRO TRANSCRIPTION & TARGETING EFFICIENCY Direct Introduction of Cas9/ Complexes An alternate strategy for genome engineering with CRISPR/Cas9 is direct introduction of Cas9/ complexes (2-5). This method further simplifies CRISPR/Cas9 workflows and, in some cases, has been reported to increase mutagenic activity (2,3), and reduce off-target editing events (2). NEB provides purified Cas9 Nuclease, S. pyogenes (NEB #M0386), with and without a nuclear localization signal, as a standalone enzyme to support direct introduction of Cas9/ complexes in conjunction with our reagents for generation. In vitro DNA cleavage using Cas9 Nuclease, S. pyogenes Cas9 Nuclease, S. pyogenes Cas9 Nuclease NLS, S. pyogenes M0386S/L/M M0641S/L/M 2-Log DNA Ladder DNA Only DNA+ + Cas9 Nuclease DNA+ Cas9 Nuclease DNA+ Input DNA, 4361 bp Fragment 1, 2815 bp Fragment 2, 1546 bp Reactions were set up according to recommended conditions (NEB #M0386), and cleavage products were resolved on a 1% agarose TBE gel. Input DNA is PvuII-linearized pbr322 plasmid DNA. Evaluating Targeting Efficiency with the T7 Endonuclease I Assay A widely used method to identify mutations is the T7 Endonuclease I mutation detection assay (6,7). This assay detects heteroduplex DNA that results from the annealing of a DNA strand, including desired mutations, with a wild-type DNA strand (7). T7 Endonuclease I Targeting Efficiency Assay Isolation of Genomic DNA Targeted cells Modified locus Digestion of Mismatched Duplexes T7 Endonuclease I T7 Endonuclease I M0302L Need to determine targeting efficiencies over 50%? Visit www.neb.com/cas9locusmod to find out how. PCR Amplification Fragment Analysis RFU Denaturation & Annealing Possible re-annealing products References 1. Sander, J.D., and Joung, J.K. (2014) Nat Biotechnol. doi:10.1038/nbt.2842. 2. Kim, S., et al. (2014) Genome Research, 24, 1012 1019. 3. Gagnon, J.A., et al. (2014) PLoS ONE, 9, e98186. 4. Lee, J.S., et al. (2014) G3: Genes, Genomes, Genetics, 4(7), 1291-1295. 5. Cho, S.W., et al. (2013) Genetics, 195, 1177 1180. 6. Fu, Y., et al. (2013) Nat. Biotechnol. 31, 822 826. 7. Fu, Y., et al. (2014) Nat Biotechnol. doi: 10.1038/nbt.2808. Calculate frequency using peak heights Genomic DNA is amplified with primers bracketing the modified locus. PCR products are then denatured and re-annealed yielding three possible structures. Duplexes containing a mismatch are digested by T7 Endonuclease I. The DNA is then electrophoretically separated and fragment analysis is used to calculate targeting efficiency. 7

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