Creating a CRISPR Community: The Sanger Institute-AstraZeneca Conference (2016) Cambridge, UK

by N. Ravinder, J. Yang, Y. Zou, S. Szumowski, J. Braun, L. Wong, W. Chen, X. Liang, N. Roark, V. Blackston, H. Xie, C. Revankar, S. Ranganathan, J. Potter, and J. Chesnut; Thermo Fisher Scientific, Carlsbad, CA, USA - 01/23/2016

Download PDF version

Abstract

The CRISPR-Cas9 system is a powerful tool for manipulating multiple genes simultaneously thereby enabling rapid generation of cell models and bio-therapeutic research. They also serve as high throughput loss-offunction screening platforms for investigating genes that are involved in various biological processes in mammalian cells. While the utility of these tools are improving, there are several factors, including design, synthesis, purity and optimal delivery methods that should be taken into account to ensure maximum editing efficiency and specificity.

Here we report streamlined workflows and data using CRISPR/Cas9 formats for large-scale gene knock-out studies. This includes development of purified transfection-ready gRNA libraries and Cas9 nuclease for transient expression and robust editing across broad cell types. In a second application, we show data using lentivirus based CRISPR delivery for high throughput loss-of-function screening of mammalian cell populations. This format provides efficient transduction of a wide array of dividing and non-dividing cells and holds great promise for clearer phenotypes and less false readout compared to variable knock-down of expression using RNAi. Together the methods and CRISPR library capabilities described here enhance our ability to perform high throughput genetic screening and cell engineering.

Introduction

Arrayed purified guide RNA (gRNA) libraries: Cas9 protein together with crRNA and tracrRNA (usually combined as a single gRNA) are essential for activity and although the RNA components can be synthesized chemically, the quality of the standard synthetic RNA is often not sufficient for cell engineering due to the presence of truncated by-products. In addition the turn around time and cost or synthesis is long. We describe here a streamlined modular approach for gRNA production in vitro. Starting with two short single stranded oligo and with the method described here you can generate transfectionready gRNA in as little as 4 hours (Figure 1 and 2). The reagents for production of gRNA is now available as GeneArt™ Precision gRNA synthesis kit. In addition with our robust HTP synthesis capability we can provide normalized custom ready to transfect guide RNA as single tubes or libraries in 9 well format at >200ng/ul. Together with the gRNA design and ordering tool, our transfection grade Cas9 nuclease protein or mRNA and respective delivery reagents we can design gRNA and analyze cleavage efficiencies in as little as 4 days.

GeneArt™ Arrayed Lentiviral CRISPR Libraries: Functional characterization studies of the human genome have relied on RNA interference (RNAi) as the leading method for genome-wide loss of function screening but its utility is limited by the inherent lack of protein depletion by RNAi and observed off-target effects. Lentiviral vectors circumvent this challenge as they can be easily titrated to control transgene copy number and are stably maintained as genomic integrants during subsequent cell replication. Here we describe the design and optimization of gene family libraries of CRISPR-lentiviral particles for application in loss of function screenings. The subsequent Lentiviral CRISPR library is amenable to screening workflows in a variety of cell types, including our proprietary CellSensor lines.

Both library formats discussed here are offered in 96 well format with four guide RNAs per gene. Each of these are designed using our cloud based CRISPR design tool. Together the CRISPR/Cas9 offerings described here provide a complete genome editing solution for rapid and highly efficient cell engineering workflows.

Figure 1. Streamlined workflow with Cas9 RNP.
-Guide RNA design and reagent ordering through GeneArt™ CRISPR design tool
-Robust gRNA synthesis and transfection-grade Cas9 nuclease protein
-Design-to-cleavage in 4 days

Results

Figure 2. Rapid and highly efficient gRNA Synthesis kit. (A) The oligonucleotide pool consists of one 80 nt tracrRNA PCR fragment, two end primers, and two overlapping 34 bp oligonucleotides containing the unique target. (B) One-step PCR synthesis of gRNA template. Lane 1, gRNA template prepared from all-in-one plasmid served as control. Lanes 2, 3 PCR assembly. (C) In vitro transcription (IVT). Aliquots of PCR product (Lanes 2 and 3) along with the control (Lane 1) were subjected to IVT and analyzed by denaturing gel.


Figure 3. Purified, ready-to-transfect gRNA libraries: consistently streamlined synthesis with high yield
-Purified guide RNA for single genes or gene sets in 96 well format
-Out of 2000 targets tested in this sample set >97% had 20ug or more yield with >200ng/ul concentration
-Any low yield targets can be scaled up or concentrated to desired concentration
-gRNA amounts are quantified and normalized per well

Figure 4. Stability of gRNA at different time points (from 1 day up to 2 months) and temperature conditions tested by Tapestation QC. RNA at room temperature (RT) and after 20 freeze thaw cycles (20X) were similar to freshly thawed samples from -80°C or -20°C.

Figure 5. The cleavage efficiency for samples from different storage conditions and freeze thawed samples were similar to freshly thawed gRNA from -80°C.

Figure 6. Robust editing efficiencies with purified gRNA libraries generated in 96 well format. Target specific cleavage efficiency for 13 different genes with 2 target-specific gRNA tested in each case and one irrelevant target (no PAM). Purified gRNA synthesized in 96 well format was transfected into U2OS Cas9 stable cell lines using Lipofectamine™ RNAiMAX transfection reagent. GeneArt Cleavage detection assay was performed 48 hours post transfection. >80% of the targets from this list showed over 40% cleavage efficiency.

Figure 7. Purified gRNA: low toxicity. Thirty one different gRNAs were transfected into their respective wells containing U2OS Cas9 stable cells using either 20ng/well normalized gRNA samples or non-normalized amounts with samples between 10 to 50 ng /well range. Untransfected well containing cells treated with RNAiMAx only was used as a control. 48 hours post transfection cells were harvested and analyzed for live vs dead cells using SYTOX™ green based flow cytometry analysis. In both normalized and non-normalized conditions we saw >80% viability.

Arrayed Lentiviral CRISPR library

  • Human codon-optimized S. puogenes Cas9 protein
  • Blasticidin resistance linked to Cas9 through a self cleaving 2A peptide
Figure 8. Lentiviral CRISPR Arrayed Library.
  • Specific guide RNA from U6 promoter
  • Puromycin resistance from EF1-α promoter
  • 4 targets per each gene in 96 well format
  • Guide RNA targeting 5’ exons and filtered off-target sites
  • Controlled delivery of each gRNA per well, eliminating a time-consuming deconvolution step and requires some level of automation

Figure 9. High gene targeting efficiency with lentiviral transduction. Lentiviral transduction to validate the gene targeting efficiency by coinfection with Cas9 lentiviral particles or alternatively in Cas9 stable expression cell line. High genome editing efficiency was observed in both single infection and co-infection as measured by GeneArt™ Genomic Cleavage Detection (GCD) kit.

Lentiviral CRISPR particles for CellSensor™ NF-kB-bla ME180 cell line

Figure 10. Screening results. A subset of kinases screened in NF-kB ME180 cells with a ratiometric reporter assay. After stimulation with TNFa, the ratio of blue/green fluorescence increased. Cells infected with lentiviral particles carrying gRNA that effectively knocked-out the NF-kB pathway remained green with low ratio of blue/green fluorescence.

Workflow solutions for genome editing

Figure 11. Thermo Fisher Scientific workflow solutions for genome editing. We offer a collection of optimized and validated end-to-end solutions for your entire genome editing workflow, aimed at optimizing conditions and validating the functional efficiency of genome editing.

Conclusions

  • Design to cleavage analysis in 3-4 days using IVT gRNA and Cas9 protein (as RNP complex) or mRNA.
  • Ready to transfect purified gRNA for gene sets of choice with consistently high yield, purity and stability.
  • Purified guide RNA and Lentiviral CRISPR arrayed libraries available with 4 sequence-verified distinct gRNA constructs per gene in a 96-well format.
  • GeneArt arrayed lentiviral CRISPR library offers an improved loss-of-function screening platform for human genome functional characterization.
  • The titer of lentiviral CRISPR library particles is > 106 TU/ml.
  • CellSensor cell lines offer simple high throughput screening assay for Lentiviral CRISPR-mediated gene knockout.

References

  1. Cong, L., Ran, F.A., Cox, D., Lin, S., Barretto, R., Habib, N., Hsu, P.D., Wu, X., Jiang, W., Marraffini, L.A. and Zhang F. (2013) Multiplex genome engineering using CRISPR/Cas systems. Science. 339, 819-823.
  2. Berns K., Hijmans E.M., Mullenders J., Brummelkamp T.R., Velds A, Heimerikx M., Kerkhoven R.M., Madiredjo M., Nijkamp W., Weigelt B., Agami R., Ge W., Cavet G., Linsley P.S., Beijersbergen R.L., Bernards R. (2004) A large-scale RNAi screen in human cells identifies new components of the p53 pathway. Nature. 428, 431-437.
  3. Kim, S., Kim, D., Cho, S.W., Kim, J. and Kim, J.S. (2014) Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins. Genome Res. 24, 1012-1019.
  4. Kabadi, A.M., Ousterout, D.G., Hilton, I.B. and Gersbach, C.A. (2014) Multiplex CRISPR/Cas9-based genome engineering from a single lentiviral vector. Nucleic Acids Res. Oct 29;42(19):e147. doi:10.1093/nar/gku749.
  5. Liang, X., Potter, J, Kumanr, S., Zou, Y., Quintanilla, R., Sridharan, M., Carte, J., Chen, W., Roark, N., Ranganathan, S., Ravinder, N., Chesnut, JD. (2015) Rapid and highly efficient mammalian cell engineering via Cas9 protein transfection. J Biotech. S0168-1656.
  6. Mali, P., Yang L., Esvelt KM., Aach J., Guell M., DiCarlo JE., Norville JE., Church GM. (2013) RNA-guided human genome engineering via Cas9. Science. 339, 823-826.
  7. Shalem, O., Sanjana, N., Hartenian, E., Shi, X., Scott, D., Mikkelson, T., Heckl, D., Ebert, B., Root, D., Doench, J., Zhang, F. (2014) Genome-Scale CRISPR-Cas9 Knockout Screening in Human Cells. Science. 343(6166): 84-87.
  8. Wang, H., Yang, H., Shivalila, C.S., Dawlaty, M.M., Cheng, A.W., Zhang, F. and Jaenisch, R. (2013) One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering. Cell. 153, 910-918.
  9. Zuris, J.A., Thompson, D.B., Shu, Y., Guilinger, J.P., Bessen, J.L., Hu, J.H., Maeder, M.L., Joung, J.K., Chen, Z.Y. and Liu, D.R. ( 2014) Cationic lipid-mediated delivery of proteins enables efficient proteinbased genome editing in vitro and in vivo. Nat Biotechnol. Oct 30. doi: 10.1038/nbt.3081.