Genome Engineering: The CRISPR/Cas Revolution (2015) Cold Spring Harbor, NY, US

by X. Liang, J. Potter, J. Yang, J. Carte , Y. Zou, W. Chen, J. Braun, N. Roark, V. Blackston, S. Kumar, S. Szumowski, L. Wong, C. Revankar, S. Ranganathan, N. Ravinder and J. Chesnut; Thermo Fisher Scientific, Carlsbad, CA, USA- 09/24/2015

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Abstract

CRISPR-Cas9 systems provide a platform for high efficiency genome editing in broad host types thereby enabling innovative applications in cell engineering. However, the delivery of Cas9 and synthesis of guide RNA (gRNA) are two steps that limit overall efficiency and general ease of use. Here we describe novel methods for rapid synthesis of gRNA and delivery of Cas9 protein/gRNA RNP complexes into a variety of cells through optimized and robust liposome-mediated transfection or Neon™ electroporation. Presented here is a streamlined cell engineering workflow that goes from gRNA design to analysis of edited cells in as little as 3-4 days. Delivery of Cas9 RNPs not only led to high indel production at single locus, but supports highly efficient bi-allelic modification of multiple genes simultaneously in hard-to-transfect cells. The reagent preparation and delivery to cells requires no plasmid manipulation so is amenable for high throughput library and multiplexed genome engineering. In a second application, we show data using lentivirusbased CRISPR delivery for high-throughput screening of mammalian cell populations. Using CRISPR technology, we can perform complete gene knock-out studies which hold promise for clearer phenotypes and fewer false readouts compared with the variable knock-down of expression using RNAi. We are creating gene family-specific arrayed libraries of CRISPR-lenti particles which will enable high throughput, arrayed gene knockout screens using various cell types, in comparison with our proprietary Cell Sensor lines that give a fluorescent read out.

Introduction

CRISPR-Cas9 mediated genome engineering enables researchers to modify genomic DNA directly and efficiently. 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 synthetic RNA is often not sufficient for cell engineering due to the presence of truncated by-products. We describe here a streamlined modular approach for gRNA production in vitro. Starting with two short single stranded oligos, the gRNA template is assembled in a ‘one pot’ PCR reaction followed by a rapid in vitro transcription and purification step, yielding transfection-ready gRNA in as little as 4 hours (Figure 1). To streamline the cell engineering workflow further, we sought to eliminate the need for cellular transcription and translation by directly introducing Cas9/gRNA ribonucleoprotein (RNP) complexes to the cells. We used a systematic approach to optimize the conditions for delivery of Cas9/gRNA RNP complexes via lipid-mediated transfection or electroporation. Using Cas9 protein transfection via electroporation, we achieved superior genome editing efficiencies even in hard-to-transfect cells.. 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 (7). Lentiviral vectors circumvent this issue 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-lenti particles for application in loss of function screenings. The subsequent Lenti-CRISPR library is amenable to screening workflows in a variety of cell types, including our proprietary CellSensor lines. Included here is also some data from our purified ready to transfect gRNA libraries that are generated using our streamlined HTP gRNA synthesis process with a rapid turn around time for custom libraries or gene sets. Both library formats discussed here are offered in 96 well format with four gRNAs per gene per well and the target specific gRNAs to a gene subset of choice are designed using our 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 CRISPR workflow

Figure 1. Streamlined CRISPR workflow. Cas9 protein workflow: design to cleavage in 4 days.

Results

Figure 2. Rapid and highly efficient gRNA synthesis

Figure 2. Rapid and highly efficient gRNA Synthesis. (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. 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. Robust editing efficiencies with purified gRNA libraries generated in 96 well format

Figure 3. Robust editing efficiencies with purified gRNA libraries generated in 96 well format. Target specific cleavage efficiency for13 different  genes with 2 target specific gRNA tested in each case alongside HPRT gene target control (synthesized from the control oligo in the gRNA synthesis kit) and one irrelevant target (with no PAM). In vitro synthesized gRNA was generated using crRNA specific DNA oligo and our new gRNA synthesis reagents in 96 well format. Following synthesis gRNA was purified using MEGAclear-96 transcription cleanup kit. gRNA was transfected into 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 show over 40% cleavage efficiency. We designed two different targets using our design algorithm and in each case we had successful target loci cleavage.

Figure 4. Transfection reagent -mediated delivery and editing results
Figure 4. Transfection reagentmediated delivery and editing results. (A) Three separate genomic loci were edited via Cas9 plasmid DNA, mRNA or protein transfection of HEK293FT cells using Lipofectamine™ RNAiMAX. For the HPRT target, transfection was performed in the presence or absence of serum. The efficiency of genome modification was determined by a genomic cleavage assay. (B) HEK293FT cells were transfected with either plasmid DNA, Cas9 mRNA/gRNA or Cas9 RNPs directed to the HPRT loci. Cell samples were taken at different time points and analyzed by genomic cleavage assays. (C) Western blot analysis of samples taken at different time points. (D) Off-target mutation of VEGFA T3 target caused by Cas9 plasmid DNA, mRNA or protein transfection. Percentages of on-target mutation as well as OT3-2 and OT3-18 off-target mutations were determined by DNA sequencing.
Figure 5. Multiplex knockout with Cas9 in Jurkat T cells (male)

Figure 5. Multiplex Knockout with Cas9 in Jurkat T Cells (Male). Three genes (5 alleles since HPRT is on the X chromosome and Jurkat T is male) targeted in a multiplex knockout. (A) Genomic Cleavage Detection gel images of percent indel cleavage at each loci. (B) Clonal populations of edited cells were sequenced. White bars indicate wild type, red bars indicate knockout, green bars indicate heterozygotes.

Table 1. Transfection efficiency in variety of cell lines

Table 1. Transfection efficiency in variety of cell lines. Cas9 based genomic editing efficiencies using Lipofectamine™ RNAiMAX and Neon™ electroporation mediated transfection formats among multiple cell lines. Targeted HPRT for human cell lines and Rosa 26 for mouse cell lines. Forty eight hours post-transfection, the percent locus-specific indel formation was measured by GeneArt ™ Genomic Cleavage Detection Kit. iPSC pluripotency markers were confirmed after transfection (data not shown). Jurkat and iPSC cleavage efficiencies were sequence confirmed.

Results—LentiCRISPR Library

Figure 6. Library screening

Figure 6. Library Screening. The arrayed library screening approach provides controlled delivery of each gRNA, eliminating a time-consuming deconvolution step and requires some level of automation. Screening pooled libraries is less expensive than arrayed formats and does not require special infrastructure.

Figure 7. Lentiviral particles design

Figure 7. Lentiviral particles design. Components of the pLenti-Cas9-P2A-Bsd Vector: human codon-optimized, S. pyogenes Cas9 gene with a Blasticidin resistance linked to a Cas9 cassette through a self cleavage 2A peptide. The pLentiCRISPR-EFS-Puro vector encodes specific sgRNA targets expressed from a U6 promoter under Puromycin resistance from the EF-1a promoter. Not shown are pLentiCRISPR-EFS-Puro control vectors containing the target sequence to a validated safe harbor site and a GFP-Puro fusion cassette.

Figure 8. LentiCRISPR for CellSensor NFkB-bla ME-180 cell line

Figure 8. LentiCRISPR for CellSensor™ NFkB-bla ME-180 cell line. A subset of kinase genes screened in NFkB ME-180 cells with a FRET based reporter assay. In the absence of TNFa, green fluorescence indicates an unstimulated pathway. Upon treatment with TNFa, cells with gRNA that effectively knockout the NFkB pathway remain green.

Conclusions

  • Design to analysis in 3-4 days using in vitro transcribed gRNA and Cas9 protein or mRNA
  • Electroporation of Cas9 RNPs using the Neon™ Transfection system works in all cells lines tested so far with 20-94% indel efficiency including Jurkat, iPSC, CD34+
  • IVT gRNA synthesis protocol is amenable to 96-well format
  • LentiCRISPR library offers an improved loss-of-function screening platform for human genome functional characterization
  • CellSensor cell lines offer simple high-throughput screening assay for LentiCRISPR–mediated gene knockout

References

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Acknowledgements

Rene Quintanilla, Mahalakshmi Sridharan, Huimin Xie, and Xin Yu