Rapid and highly efficient mammalian cell engineering via Cas9 protein transfection
Abstract
CRISPR-Cas9 systems provide a platform for high efficiency genome editing that are enabling innovative applications of mammalian cell engineering. However, the delivery of Cas9 and synthesis of guide RNA (gRNA) remain as steps that can limit overall efficiency and ease of use. Here we describe methods for rapid synthesis of gRNA and for delivery of Cas9 protein/gRNA ribonucleoprotein complexes (Cas9 RNPs) into a variety of mammalian cells through liposome-mediated transfection or electroporation. Using these methods, we report nuclease-mediated indel rates of up to 94% in Jurkat T cells and 87% in induced pluripotent stem cells (iPSC) for a single target. When we used this approach for multigene targeting in Jurkat cells we found that two-locus and three-locus indels were achieved in approximately 93% and 65% of the resulting isolated cell lines, respectively. Further, we found that the off-target cleavage rate is reduced using Cas9 protein when compared to plasmid DNA transfection. Taken together, we present a streamlined cell engineering workflow that enables gRNA design to analysis of edited cells in as little as four days and results in highly efficient genome modulation in hard-to-transfect cells. The reagent preparation and delivery to cells is amenable to high throughput, multiplexed genome-wide cell engineering.
Keywords
CRISPR;
Cas9;
gRNA;
Gene editing;
Transfection;
Multiplex
Abbreviations
CRISPR, clustered regularly interspaced short palindromic repeats;
CAS9, CRISPR associated protein;
gRNA, guide RNA;
crRNA, CRISPR RNA;
tracrRNA, trans-activating crRNA
1. Introduction
CRISPR-Cas9 mediated genome engineering enables researchers to modify genomic DNA in vivo directly and efficiently ( Cho et al., 2013a, Mali et al., 2013, Jiang et al., 2013 and Wang et al., 2013). Three components (Cas9, mature crRNA and tracrRNA) are essential for functional activity. Although the mature crRNA and tracrRNA can be synthesized chemically, the quality of the synthetic RNA is not sufficient for in vivo cell engineering due to the presence of truncated by-products (data not shown). Therefore, templates for the mature crRNA and tracrRNA or a combined single gRNA are often cloned into a Cas9 expression plasmid or built into separate plasmids driven by either U6 or H1 promoters for transcription after transfection of mammalian cells ( Cong et al., 2013 and Mali et al., 2013). However the plasmids have been shown to have toxicity in some cell lines (Kim et al., 2014). Recently, the use of Cas9 delivered as mRNA has led to increases in the rate of genomic cleavage in some cells. For example, a mixture of Cas9 mRNA and a single species of gRNA were co-injected into mouse embryonic stem (ES) cells resulting in biallelic mutations in 95% of newborn mice (Wang et al., 2013). To make guide RNA, often a linearized plasmid containing the T7 promoter and the gRNA sequence is used directly or a linear template is created via PCR amplification of the targeting sequence from a plasmid. If a 5′ T7 promoter does not appear in the plasmid, it is often added at this step and the resulting PCR product can be used in an in vitrotranscription reaction ( Jinek et al., 2012 and Wang et al., 2013). Alternatively, a synthetic DNA fragment containing a T7 promoter, crRNA and tracrRNA can be used as a template to prepare a gRNA by in vitro transcription. Overall, these represent a labor-intensive and time-consuming workflow, which led us to seek a simpler method to synthesize high quality gRNA. To that, 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. The product is then used as template in an in vitrotranscription (IVT) reaction which is followed by a rapid purification step, yielding transfection-ready gRNA in as little as four hours.
To streamline the cell engineering workflow further, we sought to eliminate any remaining cellular transcription or translation by directly introducing Cas9 protein/gRNA ribonucleoprotein (RNP) complexes directly to the cells. Microinjection of Cas9 protein and gRNA complexes into C. elegans was first described in 2013 ( Cho et al., 2013b) and was subsequently used to generate gene-knockout mice and zebrafish with mutation rates of up to 93% in newborn mice (Sung et al., 2014). Following that report, Cas9 protein/gRNA RNP complexes were delivered into cultured human fibroblasts and induced pluripotent stem cells (iPSC) via electroporation with high efficiency and relatively low off-target effects (Kim et al., 2014). In that study, a large amount of Cas9 protein (4.5–45 μg) and gRNA (6–60 μg) were necessary for efficient genome modification (up to 79% indel efficiency). Another recent study delivered Cas9/gRNA RNPs along with donor DNA for homology directed repair into HEK293 T, human primary neonatal fibroblasts and human ESCs cells via electroporation (Lin et al., 2014). Here also, large amounts of Cas9 protein (4.8–16 μg) were necessary for efficient modification. Most recently, delivery of Cas9 protein-associated gRNA complexes via liposomes was reported, in which RNAiMAX was used to deliver Cas9:sgRNA nuclease complexes into cultured human cells and into the mouse inner ear in vivo with up to 80% and 20% genome modification efficiency, respectively ( Zuris et al., 2015).
The CRISPR/Cas system has been demonstrated as an efficient gene-targeting tool for multiplexed genome editing (Wang et al., 2013, Kabadi et al., 2014, Sakuma et al., 2014 and Cong et al., 2013). For example, co-transfections of mouse ES cells with constructs expressing Cas9 and three sgRNAs targeting Tet1, 2, and 3 resulted in 20% of cells having mutations in all six alleles of the three genes based on restriction fragment length polymorphism (RFLP) assay (Wang et al., 2013). Lentiviral delivery of a single vector expressing Cas9 and four sgRNAs into primary human dermal fibroblasts resulted in about 30% simultaneous editing of four genomic loci among ten clonal populations based upon genomic cleavage detection assays (Kabadi et al., 2014). In one recent study, ‘all-in-one’ expression vectors containing seven guide RNA expression cassettes and a Cas9 nuclease/nickase expression cassette were delivered into 293T cells with genome cleavage efficiency ranging from 4 to 36% for each individual target (Sakuma et al., 2014). In general, the efficiency of editing multiple genes in the human genome using plasmid-based delivery methods remains relatively low which subsequently increases the workload for downstream clonal isolation.
In this study, we developed an in vitro gRNA production system and used a systematic approach to optimize the conditions for delivery of Cas9:gRNA complexes via lipid-mediated transfection or electroporation. A variety of mammalian cell lines were tested, including primary cells and other hard-to-transfect cells. Plasmid DNA, mRNA and Cas9 protein transfections were evaluated side by side. Using Cas9 protein transfection via electroporation, we achieved superior genome editing efficiencies even in hard-to-transfect cells. In addition, we explored the genome editing of multiple targets simultaneously using the Cas9 RNPs delivery system described here. We found that delivery of Cas9 RNPs not only led to high indel production at single locus, but supports highly efficient biallelic modulation of at least two genes in a single transfection.
2. Materials and methods
2.1. Materials
293FT cells, Gibco® Human Episomal iPSC line, mouse E14Tg2a.4 embryonic stem cells, primary human keratinocytes cells neonatal, inactivated embryonic fibroblasts, DMEM medium, RPMI 1640 medium, IMDM, McCoy 5A modified medium, DMEM/F-12, KnockOut™ DMEM, Fetal Bovine Serum (FBS), Knockout™ Serum Replacement, Non-Essential Amino Acid solution, basic fibroblast growth factor, Collagenase IV, TrypLE™ Express Enzyme, Geltrex, Opti-MEM Medium, Essential 8™ medium, StemPro®-34 SFM Complete Medium, FluoroBrite™ DMEM, recombinant human leukemia inhibitory factor, GeneArt® Genomic Cleavage Detection Kit (GCD), Lipofectamine® 2000, Lipofectamine® 3000, Lipofectamine® RNAiMAX, Lipofectamine® MessengerMAX, GeneArt® CRISPR Nuclease Vector with OFP Reporter, 2% E-Gel® EX Agarose Gels, PureLink® PCR Micro Kit, TranscriptAid T7 High Yield Transcription Kit, MEGAclear™ Transcription Clean-Up Kit, Zero Blunt® TOPO® PCR Cloning Kit, PureLink® Pro Quick96 Plasmid Purification Kit, Qubit® RNA BR Assay Kit, TRA-1-60 Alexa Fluor® 488 conjugated antibodies, SSEA4 Alexa Fluor®647, and Phusion Flash High-Fidelity PCR Master Mix were from Thermo Fisher Scientific. Jurkat T cells and K562 cells were obtained from the American Type Culture Collection (ATCC). CD34+ cord blood cells were purchased from AllCells. A549 cells, U-2 OS cells, Neuro-2a (N2A) cells were purchased from ATCC. MEF feeder cells and ROCK inhibitor Y-27632 were purchased from EMD Millipore. Monoclonal Cas9 antibody was ordered from Diagenode. Recombinant Cas9 protein with a NLS was initially purified as described (Kim et al., 2014) and later obtained from Thermo Fisher Scientific. All oligonucleotides used for gRNA synthesis and genomic cleavage detection were from Thermo Fisher Scientific (Table S1).
2.2. One-step synthesis of gRNA template
The 80 bp cr/tracrRNA constant region was PCR amplified from the GeneArt® CRISPR Nuclease Vector (1 ng) using the Constant Forward and Universal Reverse oligos (10 μM) and purified via agarose gel extraction. The concentration of PCR product was measured by Nanodrop (Thermo Fisher Scientific) and the molarity was calculated based on the molecular weight of 49.6 kDa. To prepare a mixture of oligonucleotides, the 80 bp cr/tracrRNA PCR product (0.15 μM) was mixed with universal forward and reverse oligos (10 μM) as well as target-specific forward and reverse oligos (0.3 μM).
For each target locus, 2 oligonucleotides that recreate the target sequence and share complementary with the bordering T7 promoter and 80 bp cr/tracr constant region were designed (Fig. 2A). The forward oligo (Target F1) contains the 18 base T7 promoter sequence as well as the first 16 bases of the target and the reverse oligo (Target R1) contains the reverse complement of the first 15 bases of the cr/tracr constant region and the last 19 bases of the target (Table 1s). To set up the synthesis of gRNA template, aliquots of the pooled oligonucleotides were added to a Phusion Flash High-Fidelity PCR Master Mix and amplified using manufacturer's recommended reaction conditions. The PCR product was analyzed by a 2% E-Gel® EX Agarose Gel, followed by purification using Purelink® PCR micro column. The gRNA template was eluted with 13 μl water and the concentration was determined by Nanodrop instrument. To determine the error rate, the PCR product was cloned into Zero Blunt® TOPO® vector, followed by plasmid DNA isolation and sequencing with a 3500xl DNA analyzer (Thermo Fisher Scientific).
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