SUREGUIDE CRISPR LIBRARIES

Transcription

1 SUREGUIDE CRISPR LIBRARIES Fidelity The Agilent Advantage Guide Representation Customization

2 The Agilent Advantage Fidelity What is fidelity? Fidelity in DNA synthesis is simple to understand but can be difficult to achieve. Loosely speaking, fidelity is the number of errors that are introduced during the synthesis process. Typically this is measured by referencing the error rate, which is the ratio of the number of incorrect bases to the number of correct bases. Multiple types of errors can occur during DNA synthesis including deletions, insertions and base identities. Why is it important for grna libraries? CRISPR libraries are designed based on having perfect sequence input. While feasible for applications requiring the delivery of a single guide, creating a sequence perfect library of guides is near impossible. Fortunately, some of this can be accounted for in the screening process as guides are typically selected by phenotype and sequenced, allowing errors or examples with excessive off-target effects to be eliminated. It is important not to mistake sequence verified libraries for sequence perfect libraries. Sequence verified libraries do contain an abundance of the correct sequence, but they nearly always contain low levels of incorrect sequence. Genomic DNA Genome specific tracrrna-crrna chimera Targeted genome editing PAM Matching genomic sequence RNA-guided Cas9 protein Genome specific crrna sequence Site-specific dsdna break Figure 1. CRISPR/Cas9 is a two component nuclease using a protein as the functional component and a guide RNA as the targeting component. The sequence of the guide RNA defines the cut site of the nuclease, thus the fidelity of the guide is critical to obtaining high efficiency cleavage and low off-target effects for in vivo CRISPR genome editing. Superior fidelity of SureGuide CRISPR libraries Advantages Industry leading fidelity - Consistently low error rates improves your functional results including reduced screening time and offtarget effects. Consistent, high-quality synthesis for both catalog and custom applications High complexity, parallel synthesis without sacrificing fidelity Library synthesis process Generating pooled CRISPR libraries starts with synthesizing high-complexity pools of oligonucleotides. Agilent uses an arraybased synthesis, based on ink-jet technology to deposit individual bases into defined positions on a slide. Oligonucleotides are built up one base at a time until the desired length is reached. The constructs are then cleaved from the slide. Oligo pools can then be resuspended, amplified and cloned in batch using the SureVector library cloning kit or in vitro transcribed to generate RNA. A B 7.% 75.% 8.% 85.% 9.% 95.% Figure 2. A) A comparison of the % of sequence perfect constructs in a SureGuide CRISPR library (blue) constructed with the SureVector library cloning kit (92%) and the same library from a competitor (orange, 78%). B) A comparison of the number of errors/kb in the same library for Agilent (blue, 4) and a competitor (orange, 12). SureGuide CRISPR libraries contain less than half the number of errors as competitors leading to clearer results and reduced screening times. 1

3 Guide Representation The Agilent Advantage What is guide representation? CRISPR libraries are complex mixtures of many DNA sequences. This complexity introduces a level of quality that is often unfamiliar to users of individual or small oligo pools. Guide representation refers to the uniformity of quantities of guides across all sequences in the pool. A common metric used to express the quality of the representation is the 1/9 ratio, a ratio of the number of guides in the 9th percentile to the number of guides in the 1th percentile. First the oligo pool is sequenced and the number of reads for each member are mapped back. These read counts form a distribution where the tails of the distribution are the over and under represented guides. A ratio near one indicates that the under-represented and over-represented guides have similar read counts. Why is guide representation important for SureGuide libraries? When working with pooled libraries, the typical approach is to set up a selection based screen that will allow you to segregate CRISPR modified cells by phenotype. This can require screening of up to 1x the number of cells as you have guides in your pool. If guides are over-represented, they will show up disproportionally often in the screen. An even bigger problem is when guides are under-represented. Having even 1-2% of guides under-represented in a genome-wide screen can increase the screening requirements by an order of magnitude. Factors affecting guide representation The first critical component of creating even guide representation in a CRISPR library is the method of DNA synthesis. Maintaining these precise ratios of concentration while pipetting multiple aliquots into a single library is challenging. Array-based synthesis is typically a better choice for these types of applications because oligos can be uniformly printed and the copy number of each oligo can be adjusted to account for any bias. As one moves past the oligo synthesis into plasmid generation and viral transfection, there is a significant danger of decreasing quality of representation. The cloning method and levels of viral titer have a large effect on the overall outcome of the experiment and the final levels of representation you can expect to achieve. Guide Representation Comparison with Competitor Libraries Libraries Missed Guides 9 th /1 th percentile 95 th /5 th percentile 99.5 th /.5 th percentile Agilent option Agilent option Agilent option Agilent option GeCKO (Broad)? NA GeCKO (Competitor) GeCKO (Competitor) expanded Table 1. With <.1% missed guides, you can be sure the guides in your library will be present in your delivered library. The uniformity of our libraries has an average 9/1 ratio of 2.29, which is less than half that of our competitor s libraries (5.29). Normalized Abundance (Fractions of Total).25.2 GeCKOv2 plasmid libraries GeCKOv2-A plasmid libraries.15 Agilent option Normalized Bins (1=Average) Commercial Commercial Amplified Agilent option 2 Agilent option 1 Agilent option 4 Advantages Array-based synthesis refined to provide the best possible representation in complex libraries Fewer missed guides and improved representation in SureGuide libraries leads to reduced screening time and effort 95% of all guides are represented above the underrepresentation threshold Figure 3. Distribution of sequenced guides in a pool. Blue and green curves represent the normalized abundance of reads in 4 Agilent libraries. A value of.5 indicates guides for which the number of reads are ½ as abundant as the average. The red curves are the same data for a commercially available competing library. 2

4 The Agilent Advantage Customization What is customization? Customization refers to the ability to fully define all aspects of the CRISPR library. CRISPR libraries are specific to the application and you do not want to waste time and resources screening variants which are not of interest. With genome-wide validated libraries you do not have control over the guide design, the cloning strategy or any of the sequence features contained in your libraries. Customization allows you to tune each of these parameters with the precision and control required for your carefully designed experiments. Why is it important for CRISPR libraries? A pooled approach to phenotypic selection experiments using CRISPR has the potential to vastly reduce the difficulty of generating modified cells. While the applications are extensive, this can be restricted by the lack of availability of CRISPR libraries for custom applications. Many companies, limited by their production process, offer only standard content. At Agilent we provide full customization and every oligo pool, even our validated GeCKO libraries, uses the same production process. Components of a custom CRISPR library Guide content CRISPR guide design is a rapidly evolving field. To make sure you are always using the best possible set of guides for your application, customization can be necessary. Guide structure Alternative guide structures have been shown to reduce off-target effects and improve editing efficiency in certain cell types. Library complexity Phenotypic screening can be laborious and expensive. Customization allows you to screen only the variants you are interested in. Comparison of Agilent s oligo printing to conventional printing technologies. Get to your oligo pools in one easy step. Oligo Pool Oligo Pool A G C T Oligo Pool Figure 4. Agilent s process of generating pooled libraries differs significantly from conventional methods. While additional steps are required to customize a conventional library, this is done in a single step using Agilent s oligo printing technology. Advantages Fully customize your CRISPR libraries for any application, delivery system or target Create your own genomewide library or design small subsets targeting individual pathways Our application scientists can help you design your CRISPR libraries 3

5 SUREPRINT PLATFORM Both catalog and custom CRISPR libraries are manufactured using a proprietary, non-contact industrial inkjet printing process in which oligo monomers are deposited uniformly onto specially-prepared glass slides. This in situ synthesis process prints oligonucleotide probes, base-by-base, from digital sequence files. The precise inkjet process enables the delivery of extremely small, accurate volumes of the chemicals to be spotted. In contrast to traditional synthesis, Agilent libraries are directly synthesized as highly complex pools enabling a high level of customization. Oligo Synthesis by Agilent Agilent has been a technological leader in the synthesis of longer oligos for the past decade. Conventional synthesis begins to accumulate a large number of deletions once the length exceeds around 5 nucleotides. At this point, the yields become too low to efficiently synthesize and purify the oligos. Our process allows synthesis of long oligos exceeding 2 nucleotides in length. SurePrint oligo printing on array QC Process Agilent s oligo libraries have been utilized for a vast range of precision applications. Each pool of Agilent oligos is synthesized alongside a control batch, allowing a full assessment of the printing process prior to shipping the CRISPR library. Solutions offered by Agilent Agilent s oligo library synthesis platform offers the world s most advanced and reliable manufacturing scale array-based DNA synthesis. We have harnessed the SurePrint platform to refine the DNA printing process for CRISPR libraries, giving us the ability to generate libraries far superior to those that have previously been available. Every library we build is custom, so we can be sure the quality and consistency of your custom libraries are up to the same standard as our predefined content. You can design your own library from the ground up or, for example, add additional guides to the GeCKO library. The content of Agilent s CRISPR libraries is free to design and define, making the start of your functional genomics experiments more accessible than ever. Figure 5. (A) Agilent s ink-jet based printing uses the four DNA bases as inks. (B) Bases are deposited in small volumes on the chip. (C) Oligonucleotides are built up one base at a time across the entire chip. Depurination side reaction 3) Deblock HO N 1 1) Coupling Repeat n times =P- RO N 2 =P- RO =P- RO N 1 GuideRNA synthesis 2) Oxidation Full Length (%) Conventional Processes 98% cycle yield, no depurination 99.5% cycle yield, no depurination 98% cylce yield, with depurination 99.5% cycle yield, with depurination Agilent Oligo Length (mer) Figure 6. Agilent s unique chemistry for array-based DNA synthesis significantly improves per cycle yields. The curve at bottom right shows the fraction of full length construct as a function of oligo length for conventional (red) and Agilent s (blue) process. 4

6 Instructions to Order GeCKOv2 Libraries GeCKOv2 libraries are available for both mouse (G7554A) and human (G7555A) applications. The table below provides a summary of the libraries including the number of guides per gene, non-coding targets, and control sequences. Kits also include a control appropriate for the organism the library is designed for. If you require additional information, including the sequences of each component of the library please contact your local Agilent representative or technical support. Instructions to Order Custom Libraries Custom ready-to-clone libraries (G7555A) are available in three sizes, up to 1, guides, up to 3, guides and up to 6, guides. Promoter elements are included in the construct and are a U6 promoter optimized for mammalian applications (alternative promoters can be ordered in an unamplified format). You will provide to your Agilent representative either: (1) a text file containing the variable sequence of the guide corresponding to your targets, (2) a list of genomic regions preferably in a.bed format or (3) a list of target gene names using the NCBI gene nomenclature. Options 2 and 3 are only available for S. Pyogenes Cas9. Custom ready-to-amplify libraries (G7555B) are available in four sizes, up to 5, guides, up to 25, guides, up to 5, guides and up to 1, guides. To order a custom library you will provide the full sequence of the construct you will be cloning. Ordering Information SureGuide GeCKOv2 CRISPR Libraries SureGuide Amplified Custom CRISPR Libraries SureGuide Unamplified Custom CRISPR Libraries Part Numbers G7553A - Human G7554A - Mouse G7555A - Human (hu6 promoter) G7555B - Any species Library Format Ready-to-package plasmid library Ready-to-clone amplified DNA library Ready-to-amplify DNA library Delivery Lentivirus Designed for lentivirus, user choice Users choice Number of Guides Human - 123,411; Mouse - 13,29 Up to 6, Up to 1, User Supplies Lentivirus packaging, delivery, screening Cloning, viral packaging, delivery screening Amplification, cloning, viral packaging, delivery, screening Sequence Verified Yes No No Quantity 2 ug of plasmid DNA split into A and B libraries 125 ng of linear, amplified, pooled library 1 pmol of linear, unamplified, pooled library Also Included N/A N/A Amplification kit Custom Design Input None Up to 5 nt sequence for each guide Complete sequence up to 2 nt Control Mal1-C User designed User designed LEARN MORE OR BUY ONLINE: Trusted Answers. Together. For Research Use Only. Not for use in diagnostic procedures. PR7-247 Agilent Technologies, Inc. 216 Printed in USA, October 2, EN Find an Agilent customer center in your country: U.S. and Canada agilent_inquiries@agilent.com Europe info_agilent@agilent.com Asia Pacific info_agilent@agilent.com