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.
CRISPR/Cas9 formats for highly efficient and high throughput cell engineering applications
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
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.
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.
Arrayed Lentiviral CRISPR library
- Human codon-optimized S. puogenes Cas9 protein
- Blasticidin resistance linked to Cas9 through a self cleaving 2A peptide
- 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
Lentiviral CRISPR particles for CellSensor™ NF-kB-bla ME180 cell line
Workflow solutions for genome editing
- 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.
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