Lipofection-mediated genome editing using DNA-free delivery of the Cas9/gRNA ribonucleoprotein into plant cells.

KEY MESSAGE: A novel and robust lipofection-mediated transfection approach for the use of DNA-free Cas9/gRNA RNP for gene editing has demonstrated efficacy in plant cells. Precise genome editing has been revolutionized by CRISPR/Cas9 systems. DNA-based delivery of CRISPR/Cas9 is widely used in various plant species. However, protein-based delivery of the in vitro translated Cas9/guide RNA (gRNA) ribonucleoprotein (RNP) complex into plant cells is still in its infancy even though protein delivery has several advantages. These advantages include DNA-free delivery, gene-edited host plants that are not transgenic, ease of use, low cost, relative ease to be adapted to high-throughput systems, and low off-target cleavage rates. Here, we show a novel lipofection-mediated transfection approach for protein delivery of the preassembled Cas9/gRNA RNP into plant cells for genome editing. Two lipofection reagents, Lipofectamine 3000 and RNAiMAX, were adapted for successful delivery into plant cells of Cas9/gRNA RNP. A green fluorescent protein (GFP) reporter was fused in-frame with the C-terminus of the Cas9 protein and the fusion protein was successfully delivered into non-transgenic tobacco cv. 'Bright Yellow-2' (BY2) protoplasts. The optimal efficiencies for Lipofectamine 3000- and RNAiMAX-mediated protein delivery were 66% and 48%, respectively. Furthermore, we developed a biolistic method for protein delivery based on the known proteolistics technique. A transgenic tobacco BY2 line expressing an orange fluorescence protein reporter pporRFP was targeted for knockout. We found that the targeted mutagenesis frequency for our Lipofectamine 3000-mediated protein delivery was 6%. Our results showed that the newly developed lipofection-mediated transfection approach is robust for the use of the DNA-free Cas9/gRNA technology for genome editing in plant cells.

Improved delivery of Cas9 protein/gRNA complexes using lipofectamine CRISPRMAX.

OBJECTIVES: To identify the best lipid nanoparticles for delivery of purified Cas9 protein and gRNA complexes (Cas9 RNPs) into mammalian cells and to establish the optimal conditions for transfection. RESULTS: Using a systematic approach, we screened 60 transfection reagents using six commonly-used mammalian cell lines and identified a novel transfection reagent (named Lipofectamine CRISPRMAX). Based on statistical analysis, the genome modification efficiencies in Lipofectamine CRISPRMAX-transfected cell lines were 40 or 15 % higher than those in Lipofectamine 3000 or RNAiMAX-transfected cell lines, respectively. Upon optimization of transfection conditions, we observed 85, 75 or 55 % genome editing efficiencies in HEK293FT cells, mouse ES cells, or human iPSCs, respectively. Furthermore, we were able to co-deliver donor DNA with Cas9 RNPs into a disrupted EmGFP stable cell line, resulting in the generation of up to 17 % EmGFP-positive cells. CONCLUSION: Lipofectamine CRISPRMAX was characterized as the best lipid nanoparticles for the delivery of Cas9 RNPs into a variety of mammalian cell lines, including mouse ES cells and iPSCs.

Synthetic TAL effectors for targeted enhancement of transgene expression in plants.

Transcription activator-like effectors (TALEs), secreted by the pathogenic bacteria Xanthomonas, specifically activate expression of targeted genes in plants. Here, we designed synthetic TALEs that bind to the flanking regions of the TATA-box motif on the CaMV 35S promoter for the purpose of understanding the engineerable 'hot-spots' for increasing transgene expression. We demonstrated that transient expression of de novo-engineered TALEs using agroinfiltration could significantly increase reporter gene expression in stable transgenic tobacco expressing the orange fluorescent protein reporter gene pporRFP under the control of synthetic inducible, minimal or full-length 35S promoters. Moreover, the additive effects of a combination of two different synthetic TALEs could significantly enhance the activation effects of TALEs on reporter gene expression more than when each TALE was used individually. We also studied the effects of the C-terminal domain and the activation domain of synthetic TALEs, as well as the best 'hot-spots' on the 35S promoter on targeted transgene activation. Furthermore, TALE activation of the Arabidopsis MYB transcription factor AtPAP1 (PRODUCTION OF ANTHOCYANIN PIGMENT 1) in stable transgenic tobacco gave rise to a dark purple colour on infiltrated leaves when driven by four copies of cis-regulatory elements of pathogenesis-related gene (PR1) with enhancer motifs B and A1 from the 35S promoter. These results provide novel insights into the potential applications of synthetic TALEs for targeted gene activation of transgenes in plants.

Generation of iPS cells using a BacMam multigene expression system.

Generation of iPS cells from mouse embryonic fibroblasts (MEF) was achieved using a BacMam transduction system containing a polycistronic plasmid expression vector for coincident and optimized expression of four defined reprogramming transcription factors. The sequences for Oct4, Klf4, Sox2 and c-Myc, were cloned as a fusion gene (OKSM) in a single open reading frame (ORF) via self-cleaving 2A peptides and expressed under the control of the CAG promoter. The transduction efficiency of primary MEF cells with BacMam particles carrying CAG-directed Venus reporter gene is 64-98%. After three successive transductions (at intervals of 3 days) of MEF cells with BacMam particles carrying a OKSM or OSKM cassette, the iPS cell colonies are observed in 15-24 days. A single transduction of MEF cells is also effective in generating sufficiently reprogrammed iPS cell lines. The iPS cell lines from colonies picked were positively stained by Nanog, SSEA-1 immunofluorescence and alkaline phosphatase substrate markers. The advantage of using the EOS-S(4+)-EmGFP reporter to identify sufficiently reprogrammed iPS cell lines is discussed by representing experimental results obtained with electroporated plasmids, such as a mixture of 2 tandem OS and KM plasmids and a polycistronic OKSM expression plasmid.

Development of a Proline-Based Selection System for Reliable Genetic Engineering in Chinese Hamster Ovary Cells.

Chinese hamster ovary (CHO) cells are the superior host cell culture models used for the bioproduction of therapeutic proteins. One of the prerequisites for bioproduction using CHO cell lines is the need to generate stable CHO cell lines with optimal expression output. Antibiotic selection is commonly employed to isolate and select CHO cell lines with stable expression, despite its potential negative impact on cellular metabolism and expression level. Herein, we present a novel proline-based selection system for the isolation of stable CHO cell lines. The system exploits a dysfunctional proline metabolism pathway in CHO cells by using a pyrroline-5-carboxylate synthase gene as a selection marker, enabling selection to be made using proline-free media. The selection system was demonstrated by expressing green fluorescent protein (GFP) and a monoclonal antibody. When GFP was expressed, more than 90% of stable transfectants were enriched within 2 weeks of the selection period. When a monoclonal antibody was expressed, we achieved comparable titers (3.35 ± 0.47 µg/mL) with G418 and Zeocin-based selections (1.65 ± 0.46 and 2.25 ± 0.07 µg/mL, respectively). We further developed a proline-based coselection by using S. cerevisiae PRO1 and PRO2 genes as markers, which enables the generation of 99.5% double-transgenic cells. The proline-based selection expands available selection tools and provides an alternative to antibiotic-based selections in CHO cell line development.

Simultaneous single cell stable expression of 2-4 cDNAs in HeLaS3 using psiC31 integrase system.

An important consideration in the design of multigene delivery technology is the availability of suitable vectors to introduce multiple genes stably and stoichiometrically into living cells and co-express these genes efficiently. As a promising system for this purpose, we developed multi-cDNA expression constructs harboring two to three tandemly situated cDNAs in a single plasmid. The utility of this vector system is amplified by combining it with the psiC31 recombinase system which mediates site-specific integration of the genes into naturally occurring chromosomal sequences. By analyzing 55 psiC31-mediated integration events with five different constructs, each carrying one, two or three tandem cDNA expression cassettes, we identified 39 pseudo attP sites in the HeLaS3 chromosomes. All these sites share a common motif containing an inverted repeat and showing a similarity to the native psiC31 attP. The 36 integration events represented 27 different pseudo attP sites, suggesting the possibility of duplicate integration of the multigene expression plasmids into different genomic loci in a single cell. We demonstrated successive introduction of two different multi-cDNA expression plasmids into definite chromosomal pseudo attP sites, attaining integration of four cDNAs of known genomic constitution at precise genomic loci of a single HeLaS3 cell. The expression levels of these several transgenes were enhanced and made equally stable and robust by inserting the cHS4 insulator between genes.

Creation of engineered human embryonic stem cell lines using phiC31 integrase.

It has previously been shown that the phage-derived phiC31 integrase can efficiently target native pseudo-attachment sites in the genome of various species in cultured cells, as well as in vivo. To demonstrate its utility in human embryonic stem cells (hESC), we have created hESC-derived clones containing expression constructs. Variant human embryonic stem cell lines BG01v and SA002 were used to derive lines expressing a green fluorescent protein (GFP) marker under control of either the human Oct4 promoter or the EF1alpha promoter. Stable clones were selected by antibiotic resistance and further characterized. The frequency of integration suggested candidate hot spots in the genome, which were mapped using a plasmid rescue strategy. The pseudo-attP profile in hESC differed from those reported earlier in differentiated cells. Clones derived using this method retained the ability to differentiate into all three germ layers, and fidelity of expression of GFP was verified in differentiation assays. GFP expression driven by the Oct4 promoter recapitulated endogenous Oct4 expression, whereas persistent stable expression of GFP expression driven by the EF1alpha promoter was seen. Our results demonstrate the utility of phiC31 integrase to target pseudo-attP sites in hESC and show that integrase-mediated site-specific integration can efficiently create stably expressing engineered human embryonic stem cell clones.

Expression profiling of human glial precursors.

BACKGROUND: We have generated gene expression databases for human glial precursors, neuronal precursors, astrocyte precursors and neural stem cells and focused on comparing the profile of glial precursors with that of other populations. RESULTS: A total of 14 samples were analyzed. Each population, previously distinguished from each other by immunocytochemical analysis of cell surface markers, expressed genes related to their key differentiation pathways. For the glial precursor cell population, we identified 458 genes that were uniquely expressed. Expression of a subset of these individual genes was validated by RT-PCR. We also report genes encoding cell surface markers that may be useful for identification and purification of human glial precursor populations. CONCLUSION: We provide gene expression profile for human glial precursors. Our data suggest several signaling pathways that are important for proliferation and differentiation of human glial precursors. Such information may be utilized to further purify glial precursor populations, optimize media formulation, or study the effects of glial differentiation.

Multi-gene gateway clone design for expression of multiple heterologous genes in living cells: modular construction of multiple cDNA expression elements using recombinant cloning.

Much attention has been focused on manipulating multiple genes in living cells for analyzing protein function. In order to perform high-throughput generation of multi-gene expression clones, gateway cloning technology (which represents a high-throughput DNA transfer from vector to vector) can be anticipated. In the conventional strategy for gateway cloning, the construction of two or more expression elements into tandem elements on a single plasmid requires the recombination of multiple entry clones with a destination vector in a single reaction mixture. Use of increasing numbers of entry clones in a single reaction is inefficient due to the difficulty in successfully recognizing multiple pairs of matched att signals simultaneously. To address this problem, a "Modular Destination" vector has been devised and constructed, whereby cDNA inserts are sequentially introduced, resulting in a tandem structure with multiple inserts. Whereas the standard destination vector contains only Cm(R) and ccdB genes flanked by two attR signals, this destination vector contains, in addition, one or two cDNA expression elements. Here, we show the rapid construction of expression vectors containing three or four tandemly arrayed cDNA expression elements and their expression in mammalian cells.

Multi-gene gateway clone design for expression of multiple heterologous genes in living cells: eukaryotic clones containing two and three ORF multi-gene cassettes expressed from a single promoter.

Two types of eukaryotic operon-type Expression clones were constructed using the Multisite Gateway system employing six types of att signals. These clones harbored a DNA cassette containing two heterologous ORFs (cDNAs) or three heterologous ORFs in tandem downstream of a single promoter. The most promoter-proximal ORF was translated via a Kozak signal and the downstream one or two ORF(s) were translated as directed by internal ribosome entry site(s) (IRES). These clones were observed to produce two or three different proteins at levels that depended on the activities of the translational initiation signals used. With the intention of modulating the expression level of the first ORF, the translational initiation signals including a Kozak sequence and 11 different IRESs were investigated for their efficiency using a single ORF. The translational activity of these signals varied within a 10-fold magnitude. Using these results, expression at pre-described relative levels was achieved from the optional IRES of the respective ORFs in the cassette. Controllable expression at desired levels of two different ORFs directed by optional IRESs on a bicistronic construct, transcribed from a single promoter, was demonstrated.

Isolation of neural stem and precursor cells from rodent tissue.

Isolation and characterization of neural stem cells and lineage-specific progenitors provide important information for central nervous system development study and regenerative medicine. We describe methods for dissection of rodent embryonic spinal cords by enzymatic separation, and isolation and enrichment (or purification) of neuronal and glial precursors at different developing stages by fluorescence-activated cell sorting.

TEG-seq: an ion torrent-adapted NGS workflow for in cellulo mapping of CRISPR specificity.

GUIDE-seq was developed to detect CRISPR/Cas9 off-target. However, as originally reported, it was associated with a high level of nonspecific amplification. In an attempt to improve it, we developed target-enriched GUIDE-seq (TEG-seq). The sensitivity level reached 0.1-10 reads-per-million  depending on the NGS platform used, which was equivalent to 0.0002-1% measured by Targeted Amplicon-seq. Application of TEG-seq was demonstrated for the evaluation of various Cas9/gRNA configurations, which suggests delivery of Cas9/gRNA ribonucleoprotein results in significantly fewer off-targets than Cas9/gRNA plasmid. TEG-seq was also applied to 22 gRNAs with relatively high in silico ranking score that targeted the biological relevant SNPs. The result indicated the initial selection of gRNAs with high score is important, although it cannot exclude the possibility of off-target.

MicroRNA expression pattern of undifferentiated and differentiated human embryonic stem cells.

Many of the currently established human embryonic stem (hES) cell lines have been characterized extensively in terms of their gene expression profiles and genetic stability in culture. Recent studies have indicated that microRNAs (miRNAs), a class of noncoding small RNAs that participate in the regulation of gene expression, may play a key role in stem cell self-renewal and differentiation. Using both microarrays and quantitative PCR, we report here the differences in miRNA expression between undifferentiated hES cells and their corresponding differentiated cells that underwent differentiation in vitro over a period of 2 weeks. Our results confirm the identity of a signature miRNA profile in pluripotent cells, comprising a small subset of differentially expressed miRNAs in hES cells. Examining both mRNA and miRNA profiles under multiple conditions using cross-correlation, we find clusters of miRNAs grouped with specific, biologically interpretable mRNAs. We identify patterns of expression in the progression from hES cells to differentiated cells that suggest a role for selected miRNAs in maintenance of the undifferentiated, pluripotent state. Profiling of the hES cell "miRNA-ome" provides an insight into molecules that control cellular differentiation and maintenance of the pluripotent state, findings that have broad implications in development, homeostasis, and human disease states.

Micro RNA profiling: an easy and rapid method to screen and characterize stem cell populations.

MicroRNAs (miRNAs) are small regulatory RNAs varying in length between 20 and 24 nucleotides. They are thought to play a key role during development by negative gene regulation at the post-transcriptional level. Recent studies using quantitative polymerase chain reaction (QPCR) and northern blot analysis have reported the presence of several miRNA unique to specific cell types. The NCode multispecies miRNA array provides a means for simultaneously profiling the expression patterns of hundreds of known miRNAs in a given cell type or biological sample. Using this method, miRNA expression patterns in embryonic and adult stem cell lines can be characterized and compared with each other. The accuracy of NCode miRNA array data can be further confirmed by QPCR analysis of putative array hits. This array-based screening platform is a fast and easy to use analytical tool that allows one to asses the state of stem cell lines following multiple passages in culture as well as a discovery tool that eliminates the need to screen large numbers of candidate regulatory miRNAs by northern blot or PCR. In this chapter, we describe in detail the method to carry out miRNA array analysis in human embryonal carcinoma cells and confirm the array results using QPCR.

Enhanced CRISPR/Cas9-mediated precise genome editing by improved design and delivery of gRNA, Cas9 nuclease, and donor DNA.

While CRISPR-based gene knock out in mammalian cells has proven to be very efficient, precise insertion of genetic elements via the cellular homology directed repair (HDR) pathway remains a rate-limiting step to seamless genome editing. Under the conditions described here, we achieved up to 56% targeted integration efficiency with up to a six-nucleotide insertion in HEK293 cells. In induced pluripotent stem cells (iPSCs), we achieved precise genome editing rates of up to 45% by co-delivering the Cas9 RNP and donor DNA. In addition, the use of a short double stranded DNA oligonucleotide with 3' overhangs allowed integration of a longer FLAG epitope tag along with a restriction site at rates of up to 50%. We propose a model that favors the design of donor DNAs with the change as close to the cleavage site as possible. For small changes such as SNPs or short insertions, asymmetric single stranded donor molecules with 30 base homology arms 3' to the insertion/repair cassette and greater than 40 bases of homology on the 5' end seems to be favored. For larger insertions such as an epitope tag, a dsDNA donor with protruding 3' homology arms of 30 bases is favored. In both cases, protecting the ends of the donor DNA with phosphorothioate modifications improves the editing efficiency.

Multi-gene gateway clone design for expression of multiple heterologous genes in living cells: conditional gene expression at near physiological levels.

Using Multisite Gateway five-DNA-fragment constructs vectors that enable expression of two tandemly situated cDNAs on a single plasmid were developed. Heterologous protein production in cells was achieved by modulating respective cDNA expression to pre-determined and different levels. Optimization of cDNA expression at near physiological protein levels was achieved using promoters from four cell cycle-dependent genes. In comparison with conventionally available promoters, EF-1alpha or CMV, the promoters used in this study were able to modulate cDNA expression levels over a magnitude of approximately 10 or 100-fold, respectively. In transiently transfected cells, two different proteins (CPalpha1 and CPbeta2), which form a heterodimer, each labeled with a different-colored fluorescent protein, were successfully synthesized at pre-determined levels from their respective cDNAs. The above vectors were designed to contain an FRT/Flp recombination site for integration onto chromosomes and for establishment of stable clones in HeLa cells by site-specific recombination. In the stable transformant cells produced only about 4% of the protein production levels measured in the transiently transformed cells. The biological significance of these observations is discussed.

Rapid and highly efficient mammalian cell engineering via Cas9 protein transfection.

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.

Evidence for high specificity and efficiency of multiple recombination signals in mixed DNA cloning by the Multisite Gateway system.

Six types of recombination signal DNA sequences of the Multisite Gateway cloning system were investigated as to their specificity and efficiency in the LR and BP recombination reactions. In the LR reaction to generate an Expression clone by recombination between attL and attR signals which are contained in the Entry clone and the Destination vector, respectively, the cross-reactivity of various attL and attR pairs on six types of respective signal sequences was examined. In the BP reaction to create an Entry clone by transferring the target DNA segment in the Expression clone or the attB-flanked PCR product into a Donor vector, various combinations of attB and attP pairs were tested for their reactivities in recombination. The results obtained indicate a markedly higher specificity and efficiency of cross-reactivity with only the matched att signal pairs, such as attL3-attR3, attB5-attP5, and so on, compared to unmatched signal pairs, such as attL3-attR5, attB5-attP3, and so on, thus verifying a high-throughput production of the positive clones in the Gateway system in which multiple recombination signals exist together in one reaction system. Examples of rapid construction of a three or four DNA-fusion structure in the plasmid are shown.

Systematic transfer of prokaryotic sensors and circuits to mammalian cells.

Prokaryotic regulatory proteins respond to diverse signals and represent a rich resource for building synthetic sensors and circuits. The TetR family contains >10(5) members that use a simple mechanism to respond to stimuli and bind distinct DNA operators. We present a platform that enables the transfer of these regulators to mammalian cells, which is demonstrated using human embryonic kidney (HEK293) and Chinese hamster ovary (CHO) cells. The repressors are modified to include nuclear localization signals (NLS) and responsive promoters are built by incorporating multiple operators. Activators are also constructed by modifying the protein to include a VP16 domain. Together, this approach yields 15 new regulators that demonstrate 19- to 551-fold induction and retain both the low levels of crosstalk in DNA binding specificity observed between the parent regulators in Escherichia coli, as well as their dynamic range of activity. By taking advantage of the DAPG small molecule sensing mediated by the PhlF repressor, we introduce a new inducible system with 50-fold induction and a threshold of 0.9 µM DAPG, which is comparable to the classic Dox-induced TetR system. A set of NOT gates is constructed from the new repressors and their response function quantified. Finally, the Dox- and DAPG- inducible systems and two new activators are used to build a synthetic enhancer (fuzzy AND gate), requiring the coordination of 5 transcription factors organized into two layers. This work introduces a generic approach for the development of mammalian genetic sensors and circuits to populate a toolbox that can be applied to diverse applications from biomanufacturing to living therapeutics.

Chromatin insulator elements block transgene silencing in engineered human embryonic stem cell lines at a defined chromosome 13 locus.

Lineage reporters of human embryonic stem cell (hESC) lines are useful for differentiation studies and drug screening. Previously, we created reporter lines driven by an elongation factor 1 alpha (EF1α) promoter at a chromosome 13q32.3 locus in the hESC line WA09 and an abnormal hESC line BG01V in a site-specific manner. Expression of reporters in these lines was maintained in long-term culture at undifferentiated state. However, when these cells were differentiated into specific lineages, reduction in reporter expression was observed, indicating transgene silencing. To develop an efficient and reliable genetic engineering strategy in hESCs, we used chromatin insulator elements to flank single-copy transgenes and integrated the combined expression constructs via PhiC31/R4 integrase-mediated recombination technology to the chromosome 13 locus precisely. Two copies of cHS4 double-insulator sequences were placed adjacent to both 5' and 3' of the promoter reporter constructs. The green fluorescent protein (GFP) gene was driven by EF1α or CMV early enhancer/chicken ß actin (CAG) promoter. In the engineered hESC lines, for both insulated CAG-GFP and EF1α-GFP, constitutive expression at the chromosome 13 locus was maintained during prolonged culture and in directed differentiation assays toward diverse types of neurons, pancreatic endoderm, and mesodermal progeny. In particular, described here is the first normal hESC fluorescent reporter line that robustly expresses GFP in both the undifferentiated state and throughout dopaminergic lineage differentiation. The dual strategy of utilizing insulator sequences and integration at the constitutive chromosome 13 locus ensures appropriate transgene expression. This is a valuable tool for lineage development study, gain- and loss-of-function experiments, and human disease modeling using hESCs.