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Tech Note

Efficient gene knockins in iPS cells using long ssDNA templates for homology-directed repair

  • ssDNA HDR templates were designed for knockin of the AcGFP1 coding sequence at the SEC61B or TUBA1A locus in human iPS cells
  • Relative to dsDNA, electroporation of iPS cells using ssDNA templates resulted in dramatically lower toxicity
  • Analysis of edited cell populations by FACS indicated AcGFP1 knockin efficiencies ranging from 3% to 13%
  • Analysis of clonal populations confirmed the occurrence of both monoallelic and biallelic knockins, and correct localization of AcGFP1-tagged fusion proteins
Introduction Results Conclusions References

Introduction  

A common application of CRISPR/Cas technology involves engineering gene knockins in which DNA sequences are substituted or inserted at specific genomic loci. In contrast with CRISPR-mediated indels, which result from the error-prone non-homologous end joining (NHEJ) pathway, gene knockins are often engineered via homology-directed repair (HDR), typically through the use of CRISPR reagents (Cas enzyme and guide RNA) in tandem with a DNA template that shares homology with the target site and encodes for the desired modification (Hsu et al., 2014; Figure 1, below). The template used for HDR may be double-stranded DNA (dsDNA, linear or plasmid) or single-stranded DNA (ssDNA), and recent findings have suggested that the repair mechanism varies depending on the type of template used; dsDNA triggers a RAD51-dependent mechanism that mirrors meiotic homologous recombination (HR), whereas HDR involving ssDNA (referred to as single-stranded template repair or SSTR) is RAD51-independent and requires multiple components of the Fanconi Anemia (FA) repair pathway (Richardson et al., 2018).

Method for engineering gene knockins via CRISPR-mediated cleavage followed by homology-directed repair. 
Figure 1. Engineering CRISPR-mediated gene knockins via homology-directed repair. Following formation of a double-stranded break at a genomic target site via Cas9-gRNA-mediated cleavage, a DNA template sharing homology with the targeted region can trigger homology-directed repair (HDR), resulting in knockin of exogenous sequences encoded in the DNA template (purple).

This approach has been used to engineer small edits as short as single-nucleotide substitutions, and also for the insertion of longer sequences, including fluorescent reporters or gene promoters. Knockin efficiencies can vary widely, depending in part on the species or cell type, the genomic region being targeted, and the size of the insertion. A common method for delivery of the reagents involves coelectroporation of Cas9-gRNA ribonucleoprotein complexes (RNPs) in tandem with DNA HDR templates, because it can be applied to a wide array of cell types and the transience of Cas9-gRNA expression reduces the likelihood of off-target effects. HDR templates consisting of either dsDNA or ssDNA can be used, although ssDNA has been associated with lower toxicity and a reduced likelihood of off-target integration relative to dsDNA (Roth et al., 2018). Smaller ssDNA templates (i.e., ≤200 nt) are typically obtained via oligo synthesis. For longer ssDNA, however, synthesis can be cost prohibitive, and an increasingly popular method is the Guide-it Long ssDNA Production System, which allows for on-demand production of high-quality ssDNA up to 5 kb in length from a double-stranded PCR product via selective digestion of either the sense or antisense strand. 

A powerful application of this CRISPR-mediated knockin technology involves site-specific insertion of fluorescent protein sequences to obtain fusion proteins that are expressed under the same promoter and subject to the same regulatory and cell-signaling mechanisms as their endogenous counterparts, as opposed to expressing the fusion protein from a plasmid under a different promoter (e.g., CMV) or from a non-native genomic context via random integration. To demonstrate the suitability of the Guide-it Long ssDNA Production System for this application, we used it to generate two independent hiPS cell lines expressing AcGFP1 fused to the N-termini of SEC61B and Tubulin, respectively.

Results  

Design and production of ssDNA templates

Sense and antisense single-stranded DNA (ssDNA) templates for AcGFP1 knockin at the SEC61B and TUBA1A loci were designed to include homology arms of ~350 nt flanking the AcGFP1 sequence (Figure 2), and were produced using the Guide-it Long ssDNA Production System. For comparison purposes, unprocessed dsDNA PCR products corresponding to each HDR template were retained for electroporation in parallel. It has been demonstrated that there is an exponential relationship between homology arm length and knockin efficiency, with ssDNA templates including homology arms 350–700 nt in length providing optimal performance (Li et al., 2018). In our own studies involving hiPSCs, optimal performance was observed with 350-nt homology arms, and the use of longer arms did not substantially increase knockin efficiencies.

Design of ssDNA template for knockin of AcGFP1 at SEC61B.

Figure 2. Schematics depicting Cas9-sgRNA cleavage sites and ssDNA constructs designed for knockin of AcGFP1 at the SEC61B and TUBA1A loci. The ssDNA constructs included the AcGFP1 coding sequence (~700 nt in length) flanked on either side by ~350-nt homology arms. Binding sites for PCR primers used to detect the inserted AcGFP1 sequence at the SEC61B locus are depicted by blue arrows. As indicated, neither of the ssDNA constructs included a promoter sequence, such that expression of the resulting AcGFP1-tagged fusion proteins would be driven by the respective endogenous promoters.

Electroporation of hiPSCs with ssDNA results in markedly lower toxicity vs. dsDNA

Coelectroporation of Cas9 complexed with single guide RNA (sgRNA) in tandem with HDR templates was performed according to the protocol specified for the Neon system in the Guide-it Recombinant Cas9 (Electroporation-Ready) User Manual. Cas9-sgRNA RNPs were prepared by co-incubating recombinant Cas9 protein with in vitro-transcribed sgRNAs designed to target the SEC61B or TUBA1A locus, and the RNPs were then combined with corresponding HDR templates consisting of either dsDNA or ssDNA (sense or antisense). The various RNP-HDR template combinations and negative-control RNPs without associated HDR templates were then electroporated into ChiPSC18 cells using standard conditions. After 48 hours of culturing, imaging was performed on each cell population to assess the relative toxicity associated with each RNP-HDR template combination (Figure 3). While coelectroporation of ssDNA or dsDNA HDR templates and RNPs yielded noticeable toxicity relative to the application of RNPs alone, the level of toxicity associated with dsDNA was much greater than what was observed for ssDNA. This finding is consistent with the results of previous studies performed by Takara Bio and by others (Roth et al., 2018).

Electroporation of ssDNA results in lower toxicity relative to electroporation of dsDNA.

Figure 3. Toxicity comparison for electroporation of dsDNA or ssDNA into iPS cells. ChiPSC18 cells were electroporated with Cas9-sgRNA RNPs targeting the TUBA1A locus in the absence or presence of homology-directed repair (HDR) templates consisting of dsDNA or ssDNA, and then cultured for 48 hours. While electroporation of either DNA template resulted in increased toxicity compared to electroporation of Cas9-sgRNA RNPs alone (as evidenced by lower cell densities), application of ssDNA resulted in greatly reduced toxicity relative to dsDNA.

Analysis of AcGFP1 knockin efficiencies by flow cytometry

Following culturing, the edited populations were analyzed using flow cytometry (Figure 4). In these analyses, the percentage of fluorescent-positive cells is equivalent to the percentage of successful knockins (i.e., knockin efficiency). For each locus, it was observed that the use of sense or antisense ssDNA templates yielded different knockin efficiencies; in the case of SEC61B, the sense template resulted in a higher knockin efficiency than the antisense template (12% vs. 7%, respectively; Figure 4 and data not shown), whereas for TUBA1A, the antisense template provided a higher efficiency (2.7% vs. 1.3%; Figure 4 and data not shown). Currently, there is no clear rule regarding the preferential use of sense or antisense ssDNA for HDR templates longer than 200 nt, but in the case of shorter single-stranded oligodeoxynucleotide (ssODN) templates, template polarity has been found to have an effect on efficiency (Bollen et al., 2018).

FACS analysis of AcGFP1 knockin efficiences at SEC61B and TUBA1A.

Figure 4. Analysis by flow cytometry of AcGFP1 knockin efficiencies at SEC61B and TUBA1A in iPS cells using ssDNA donors. Following electroporation and culturing, edited cell populations and unelectroporated negative control cells were analyzed for AcGFP1 expression by flow cytometry. Measuring the proportion of fluorescent cells in each population allowed for determination of knockin efficiencies: 13% knockin efficiency at SEC61B using a sense ssDNA, and 2.75% efficiency at TUBA1A using an antisense ssDNA.   

From the AcGFP1-positive populations sorted by flow cytometry, single cells were isolated using limiting dilution and expanded into clonal cell lines using the Cellartis iPSC rCas9 Electroporation and Single-Cell Cloning System.

Detection of monoallelic or biallelic knockin by PCR

One of the most important steps in characterizing edited clonal cell lines involves determining their zygosity. When the inserted sequence is of sufficient length (e.g., AcGFP1), zygosity can be easily assessed via PCR amplification of the target locus using primers that anneal outside of the genomic region that shares homology with the HDR template (Figure 5). In such analyses, a wild-type allele will generate a shorter amplicon than an allele carrying a successful knockin. To determine the zygosity of the different clonal cell lines encoding for an AcGFP1 fusion to SEC61B, genomic DNA was extracted from several of the cell lines and amplified by PCR. Agarose gel analysis of the resulting PCR products indicated that the majority of the clonal cell lines carried biallelic knockins and that comparable editing outcomes were obtained using sense (ss) or antisense (as) ssDNA templates (Figure 5).  

PCR analysis of AcGFP1 knockin at the SEC61B locus in clonal populations isolated by FACS.

Figure 5. Detection of monoallelic or biallelic knockin by PCR amplification of the target site. Genomic DNA was extracted and analyzed by PCR using forward and reverse primers designed to anneal outside the genomic region sharing homology with the HDR template. The AcGFP1 and homology arm sequences encoded in the ssDNA templates are shown in green and light blue, respectively. The PCR primers were designed such that presence or absence of an inserted AcGFP1 sequence would yield PCR products of different sizes (1.9 kb or 1.2 kb, respectively). Gel electrophoresis of PCR products confirmed the occurrence of monoallelic AcGFP1 knockins, represented by double bands (e.g., ssDNA-as, Lane C5), and biallelic AcGFP1 knockins, represented by single bands corresponding to the larger product size (e.g., ssDNA-as, Lane C1). Two wild-type clones lacking any copies of the AcGFP1 insert were also identified (ssDNA-as, Lane C10 and ssDNA-ss, Lane C11).

Analysis of AcGFP1-tagged protein localization by fluorescence microscopy

Following isolation and expansion, AcGFP1-positive clones were imaged using fluorescence microscopy (Figure 6). In each clonal population, AcGFP1 fluorescence matched the canonical localization pattern of the protein encoded by the intended genomic target: targeting of the SEC61B locus yielded AcGFP1 expression in endosomal compartments, while targeting of the TUBA1A locus resulted in fluorescent labelling of microtubules. These results suggest that electroporation of RNPs and ssDNA templates targeting SEC61B or TUBA1A resulted in successful insertion of AcGFP1 at the corresponding locus, such that the resulting fusion proteins localize in the same manner as their untagged, endogenous counterparts.

In vivo expression of AcGFP1 fusion proteins in cells edited using Cas9-sgRNA RNPs and ssDNA HDR templates.

Figure 6. Analysis of AcGFP1 localization in clonal cell lines following editing. AcGFP1-positive clonal cell lines derived from each edited population were fixed and counterstained with DAPI and analyzed by fluorescence microscopy. In each case, AcGFP1 localized in a manner consistent with the function of the endogenous protein: endosomal compartments (SEC61B locus) and microtubules (TUBA1A locus), respectively.

Conclusions  

The combination of CRISPR/Cas technology with the application of DNA templates for homology-directed repair has given rise to a powerful method for precise genome editing that can be employed in a wide array of experimental contexts. However, the efficiency of this approach varies greatly, depending on the cell type(s) and the genomic loci being targeted, and a persistent challenge involves delivering HDR templates in a manner that minimizes toxicity and the likelihood of off-target integration. Here we have presented data demonstrating that electroporation of Cas9-sgRNA RNPs in tandem with ssDNA generated using the Guide-it Long ssDNA Production System enables efficient and accurate knockin of long sequences in hiPSCs with minimal toxicity. Given the relative simplicity and cost-effectiveness of the approach presented here, we anticipate that it will continue to drive greater adoption and application of CRISPR/Cas9 technology for precise genome editing. 

References  

Bollen et al., How to create state-of-the-art genetic model systems: strategies for optimal CRISPR-mediated genome editing. Nuc. Acids Res. 46, 6435–6454 (2018).

Hsu et al., Development and applications of CRISPR-Cas9 for genome engineering. Cell 157, 1262–1278 (2014).

Li et al., Design and specificity of long ssDNA donors for CRISPR-based knockin. bioRxiv, https://doi.org/10.1101/178905 (2017).

Richardson et al., CRISPR-Cas9 genome editing in human cells occurs via the Fanconi anemia pathway. Nat. Gen. 50, 1132–1139 (2018).

Roth et al., Reprogramming human T cell function and specificity with non-viral genome targeting. Nat. Lett. 559, 405–409 (2018).

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Production of sgRNA by in vitro transcription and testing for cleavage using recombinant Cas9 protein

Production of sgRNA by in vitro transcription and testing for cleavage using recombinant Cas9 protein
Schematic highlighting the key steps for using Guide-it kits for the production of high amounts of single guide RNA (sgRNA) and screening for target site cleavage efficacy using recombinant Cas9 protein.

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sgRNA yield and quality for different gene targets using the Guide-it Complete sgRNA Screening System

sgRNA yield and quality for different gene targets using the Guide-it Complete sgRNA Screening System
sgRNA yield and quality for different gene targets using the Guide-it Complete sgRNA Screening System. Panel A. 20 µl of each sgRNA in vitro transcription (IVT) reaction was incubated for 4 hr at 37°C. The yield for each transcribed sgRNA was quantified using a NanoDrop spectrophotometer. Panel B. Each sgRNA IVT reaction was a run on an Agilent Bioanalyzer to assay for sample quality.

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sgRNA quality produced via in vitro transcription

sgRNA quality produced via in vitro transcription
sgRNA quality produced via in vitro transcription. sgRNAs produced using the Guide-it sgRNA In Vitro Transcription Kit were compared with sgRNAs produced using a competitor’s kit. The Guide-it kit produced a high-quality, single band for each reaction, while the competitor’s kit showed unwanted byproducts.

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Flow chart describing how the Guide-it sgRNA In Vitro Transcription Kit (including the Guide-it IVT RNA Clean-Up Kit) and Guide-it sgRNA Screening Kit work together in the Guide-it Complete sgRNA Screening System, which can be used to synthesize and test the efficacy of sgRNAs

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Flow chart describing how the Guide-it sgRNA In Vitro Transcription Kit (including the Guide-it IVT RNA Clean-Up Kit) and Guide-it sgRNA Screening Kit work together in the Guide-it Complete sgRNA Screening System, which can be used to synthesize and test the efficacy of sgRNAs.
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391 LIMITED USE LABEL LICENSE: RESEARCH USE ONLY Notice to Purchaser: This product is the subject to a license granted to Takara Bio USA, Inc. and its Affiliates from Caribou Biosciences, Inc., and this product is transferred to the end-user purchaser (“Purchaser”) subject to a “Limited Use Label License” conveying to the Purchaser a limited, non-transferable right to use the product, solely as provided to Purchaser, together with (i) progeny or derivatives of the product generated by the Purchaser (including but not limited to cells), and (ii) biological material extracted or derived from the product or its corresponding progeny or derivatives (including but not limited to cells) (collectively, the product, and (i) and (ii) are referred to as (“Material”) only to perform internal research for the sole benefit of the Purchaser. The Purchaser cannot sell or otherwise transfer Material to a third party or otherwise use the Material for any Excluded Use. “Excluded Use” means any and all: (a) commercial activity including, but not limited to, any use in manufacturing (including but not limited to cell line development for purposes of bioproduction), product testing, or quality control; (b) preclinical or clinical testing or other activity directed toward the submission of data to the U.S. Food and Drug Administration, or any other regulatory agency in any country or jurisdiction where the active agent in such studies comprises the Material; (c) use to provide a service, information, or data to a third party; (d) use for human or animal therapeutic, diagnostic, or prophylactic purposes or as a product for therapeutics, diagnostics, or prophylaxis; (e) activity in an agricultural field trial or any activity directed toward the submission of data to the U.S. Department of Agriculture or any other agriculture regulatory agency; (f) high throughput screening drug discovery purposes (i.e., the screening of more than 10,000 experiments per day) as well as scale-up production activities for commercialization; (g) modification of human germline, including editing of human embryo genomes (with the sole exception of editing human embryonic stem (ES) cell lines for research purposes) or reproductive cells; (h) self-editing; and/or (i) stimulation of biased inheritance of a particular gene or trait or set of genes or traits (“gene drive”). It is the Purchaser’s responsibility to use the Material in accordance with all applicable laws and regulations. For information on obtaining additional rights, including commercial rights, please contact licensing@cariboubio.com or Caribou Biosciences, Inc., 2929 7th Street, Suite 105, Berkeley, CA 94710 USA, Attn: Licensing.
396 Sigma-Aldrich CRISPR Use License Agreement This Product and its use are the subject of one or more of the following issued patents and patent applications: Australia Patent Nos. 2013355214; 2017204031; and 2018229489; Canada Patent Nos. 2,891,347 and 2,977,152; China Patent No. CN105142669; European Patent Nos. EP 2 928 496 B1; EP 3 138 910 B1, 3 138 911 B1, EP 3 138 912 B1, EP 3 360 964 B1, EP 3 363 902 B1; Israel Patent No. IL238856; Singapore Patent No. 11201503824S; South Korea Patent Nos. 10-1844123 and 10-2006880; and U.S. Patent Application Serial Nos. 15/188,911; 15/188,924; 15/188,927; 15/188,931; and 15/456,204 (the “Patent Rights”). The purchase of this Product conveys to you (the “Buyer”) the NON-TRANSFERABLE right to use the Product for Licensed Research Use (see definition below) subject to the conditions set out in this License Agreement. 1. “Licensed Research Use” means any use for research purposes, except: (i) Buyer may not sell or otherwise transfer the Product (including without limitation any material that contains the Product in whole or part) or any Related Material to any other third party (except that you may transfer the Product, or any Related Material to a bona fide collaborator or contract research organization), or use the Products or any Related Material to perform services for the benefit of any other third party; (ii) Buyer may use only the purchased amount of the Product and components of the Product, and shall use any Related Material, only for your internal research within the Field, and not for any Commercial Purposes; (iii) Buyer shall use the Product and any Related Material in compliance with all applicable laws and regulations, including without limitation applicable human health and animal welfare laws and regulations; and (iv) the Buyer shall indemnify, defend, and hold harmless SIGMA and their current and former directors, officers, employees and agents, and their respective successors, heirs and assigns (the “Indemnities”) against any liability, damage, loss, or expense (including without limitation reasonable attorneys’ fees and expenses) incurred by or imposed upon any of the Indemnitees in connection with any claims, suits, investigations, actions, demands or judgments arising out of or related to the exercise of any rights granted to the Buyer hereunder or any breach of this License Agreement by such Buyer. 2. For purposes of Section 1 above, the following definitions shall apply: “Commercial Purposes” means (a) the practice, performance or provision of any method, process or service, or (b) the manufacture, sale, use, distribution, disposition or importing of any product, in each case (a) or (b) for consideration, or on any other commercial basis. “Field” means use as a research tool for research purposes; provided, however, that notwithstanding the foregoing, the Field shall expressly exclude (a) any in vivo and ex vivo human or clinical use, including, without limitation, any administration into humans or any diagnostic or prognostic use, (b) the creation of transgenic rodent models and/or derivatives thereof (including, but not limited to, rodents’ cells and rodents’ organs) by for-profit entities, (c) any in vivo veterinary or livestock use, or non-research agricultural use, or (d) use as a testing service, therapeutic or diagnostic for humans or animals. “Related Materials” means any progeny, modification or derivative of a Product. 3. Your right to use the Product will terminate immediately if you fail to comply with these terms and conditions. You shall, upon such termination of your rights, destroy all Product, Related Materials, and components thereof in your control, and notify SIGMA of such in writing. For information on purchasing a license to this Product for purposes other than Licensed Research Use, contact your local SIGMA sales representative, or call +1 800-325-3010.

The Guide-it In Vitro Transcription Kit contains the necessary reagents for the transcription and cleanup of your sgRNA sequences. It is combined with the Guide-it sgRNA Screening Kit as part of the Guide-it Complete sgRNA screening system which can be used for the simple production, cleanup, and evaluation of single guide RNAs (sgRNAs) for CRISPR/Cas9 studies.

Notice to purchaser

Our products are to be used for Research Use Only. They may not be used for any other purpose, including, but not limited to, use in humans, therapeutic or diagnostic use, or commercial use of any kind. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without our prior written approval.

Documents Components Image Data

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Production of sgRNA by in vitro transcription and testing for cleavage using recombinant Cas9 protein

Production of sgRNA by in vitro transcription and testing for cleavage using recombinant Cas9 protein
Schematic highlighting the key steps for using Guide-it kits for the production of high amounts of single guide RNA (sgRNA) and screening for target site cleavage efficacy using recombinant Cas9 protein.

Back

sgRNA yield and quality for different gene targets using the Guide-it Complete sgRNA Screening System

sgRNA yield and quality for different gene targets using the Guide-it Complete sgRNA Screening System
sgRNA yield and quality for different gene targets using the Guide-it Complete sgRNA Screening System. Panel A. 20 µl of each sgRNA in vitro transcription (IVT) reaction was incubated for 4 hr at 37°C. The yield for each transcribed sgRNA was quantified using a NanoDrop spectrophotometer. Panel B. Each sgRNA IVT reaction was a run on an Agilent Bioanalyzer to assay for sample quality.

Back

sgRNA quality produced via in vitro transcription

sgRNA quality produced via in vitro transcription
sgRNA quality produced via in vitro transcription. sgRNAs produced using the Guide-it sgRNA In Vitro Transcription Kit were compared with sgRNAs produced using a competitor’s kit. The Guide-it kit produced a high-quality, single band for each reaction, while the competitor’s kit showed unwanted byproducts.

Back

Flow chart describing how the Guide-it sgRNA In Vitro Transcription Kit (including the Guide-it IVT RNA Clean-Up Kit) and Guide-it sgRNA Screening Kit work together in the Guide-it Complete sgRNA Screening System, which can be used to synthesize and test the efficacy of sgRNAs

Flow chart describing how the Guide-it sgRNA In Vitro Transcription Kit (including the Guide-it IVT RNA Clean-Up Kit) and Guide-it sgRNA Screening Kit work together in the Guide-it Complete sgRNA Screening System, which can be used to synthesize and test the efficacy of sgRNAs
Flow chart describing how the Guide-it sgRNA In Vitro Transcription Kit (including the Guide-it IVT RNA Clean-Up Kit) and Guide-it sgRNA Screening Kit work together in the Guide-it Complete sgRNA Screening System, which can be used to synthesize and test the efficacy of sgRNAs.
632638 Guide-it™ IVT RNA Clean-Up Kit 50 Rxns USD $389.00

The Guide-it IVT RNA Clean-Up Kit is included as part of the Guide-it In Vitro Transcription Kit and is used to purify sgRNA transcribed using the Guide-it In Vitro Transcription Kit.

Notice to purchaser

Our products are to be used for Research Use Only. They may not be used for any other purpose, including, but not limited to, use in humans, therapeutic or diagnostic use, or commercial use of any kind. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without our prior written approval.

Documents Components Image Data

Back

Production of sgRNA by in vitro transcription and testing for cleavage using recombinant Cas9 protein

Production of sgRNA by in vitro transcription and testing for cleavage using recombinant Cas9 protein
Schematic highlighting the key steps for using Guide-it kits for the production of high amounts of single guide RNA (sgRNA) and screening for target site cleavage efficacy using recombinant Cas9 protein.

Back

sgRNA yield and quality for different gene targets using the Guide-it Complete sgRNA Screening System

sgRNA yield and quality for different gene targets using the Guide-it Complete sgRNA Screening System
sgRNA yield and quality for different gene targets using the Guide-it Complete sgRNA Screening System. Panel A. 20 µl of each sgRNA in vitro transcription (IVT) reaction was incubated for 4 hr at 37°C. The yield for each transcribed sgRNA was quantified using a NanoDrop spectrophotometer. Panel B. Each sgRNA IVT reaction was a run on an Agilent Bioanalyzer to assay for sample quality.

Back

sgRNA quality produced via in vitro transcription

sgRNA quality produced via in vitro transcription
sgRNA quality produced via in vitro transcription. sgRNAs produced using the Guide-it sgRNA In Vitro Transcription Kit were compared with sgRNAs produced using a competitor’s kit. The Guide-it kit produced a high-quality, single band for each reaction, while the competitor’s kit showed unwanted byproducts.

Back

Flow chart describing how the Guide-it sgRNA In Vitro Transcription Kit (including the Guide-it IVT RNA Clean-Up Kit) and Guide-it sgRNA Screening Kit work together in the Guide-it Complete sgRNA Screening System, which can be used to synthesize and test the efficacy of sgRNAs

Flow chart describing how the Guide-it sgRNA In Vitro Transcription Kit (including the Guide-it IVT RNA Clean-Up Kit) and Guide-it sgRNA Screening Kit work together in the Guide-it Complete sgRNA Screening System, which can be used to synthesize and test the efficacy of sgRNAs
Flow chart describing how the Guide-it sgRNA In Vitro Transcription Kit (including the Guide-it IVT RNA Clean-Up Kit) and Guide-it sgRNA Screening Kit work together in the Guide-it Complete sgRNA Screening System, which can be used to synthesize and test the efficacy of sgRNAs.

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Takara Bio USA, Inc. provides kits, reagents, instruments, and services that help researchers explore questions about gene discovery, regulation, and function. As a member of the Takara Bio Group, Takara Bio USA is part of a company that holds a leadership position in the global market and is committed to improving the human condition through biotechnology. Our mission is to develop high-quality innovative tools and services to accelerate discovery.

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  • PCR master mixes
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  • Baculovirus titration kits early access program
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