Progress 07/01/23 to 06/30/24
Outputs
Target Audience:Graduate student trained on the culture of bovine embryonic stem cells, recombinant DNA work and gene editing analysis via laboratory instruction. -Postdoctoral scholar gained experience developing a workflow to detect low level gene editing events in a mixed population of bovine embryonic stem cells, after transfection and training students via laboratory instruction. -Undergraduate student trained by graduate student on bovine embryonic stem cell culture and electroporation via practicum experience. Changes/Problems:Covid-19 restrictions and then the departure of a postdoc helping with the work delayed progress on the project as it took time for the graduate student conducting the work to be fully trained to be able to carry out the work. Considerable effort was made to isolate monoclonal lines which to date has proved unsuccessful and also delayed time devoted to completing all objectives. What opportunities for training and professional development has the project provided?One graduate student was trained how to analyze gene editing events to expand professional skills and expertise that include bESC culture and transfection and recombinant DNA techniques. A post-doctoral researcher gained expertise in mentoring graduate students to enhance their professional development in this important area. A graduate student gained experience in training an undergraduate student to culture bESCs, a professional skill the undergraduate is now proficient at. How have the results been disseminated to communities of interest?Work was presented (oral presentation) at the 2022 UC Davis Animal Biology Graduate Group Colloquium on October 25, 2022 Work was presented (oral and poster presentation) and the 2023 international Transgenic Animal Research Conference (TARC XIV) on August 14, 2023 What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
The overall goal of this project is to develop a platform for efficient multiplex gene editing in livestock species. To accomplish this, we are working in embryonic stem cells as once they have been edited, they can be used to derive an animal bearing the desired edits via somatic cell nuclear transfer. The first step is optimizing delivery of gene editing reagents into livestock embryonic stem cells (ESCs). Using multiple lines bovine ESCs (bESCs) previously derived from the inner cell mass of early blastocysts that were cultured and manipulated under feeder-free conditions, we found that some lines were more amenable to transfection and gene editing and that isolation of monoclonal lines of edited cells proved difficult. We established a stable enhanced green fluorescent protein (EGFP) expressing line of bESCs and used them to assess various delivery methods of CRISPR/Cas9 reagents and resulting knockout and knockin efficiencies. As the derivation of ESCs from livestock species has only recently been reported, any knowledge on how to introduce DNA efficiently needs to be generated. A more detailed description of the work is outlined below. Work was completed on the following experiments of Objective 1. Experiment 1.1: Generation of EGFP-bESC reporter line for efficient assessment of CRISPR/Cas9 activity. Three different methods of transfection were tested for efficiency of introduction of a Cas9-GFP plasmid with a single guide RNA (gRNA) for the target H11 locus into bESCs in feeder-free conditions. Use of nulceofection (Amaxa Nucelofector II) resulted in poor bESC survival and/or slow growth rates once nucleofected. NEPA21 electroporation with a 130 V poring pulse yielded 22% EGFP positive cells, which was the highest transfection efficiency observed with electroporation. Transfection efficiency when using Lipofectamine Stem varied between the two lines of bESCs used, averaging 15% in one line and double that (30%) in the other, indicating differences are innate between individually derived bESC lines. Genome editing efficiency at the H11 locus was 73.8% as quantified from both forward and reverse sequences by tracking of indels by decomposition (TIDE). Based on this work, polyclonal lines of EGFP-bEScs were produced by CRISPR-Cas9-mediated knock-in of the EGFP reporter gene at the Bos taurus H11 locus using Lipofectamine Stem to co-deliver Cas9 plasmid carrying the H11 guide sequence and donor plasmid containing EGFP under the CAG promoter in feeder-free conditions. Transfected cells were expanded for more than 7 days and EGFP+ cells were enriched from the heterogeneous population using a cloning ring. Further purification of EGFP+ cells from the enriched population was done using FACS. Single-cell cloning of sorted EGFP+ cells was attempted in 96-well plates in feeder and feeder-free conditions but the bESCs had poor clonogenicity in both cultures. Therefore, polyclonal EGFP-bESCs were subsequently used. In the polyclonal lines, stable integration of the CAG-EGFP construct at the H11 locus was confirmed by junction PCR of the 5' and 3' knock-in regions, followed by Sanger sequencing. The pluripotent integrity of EGFP-bESC reporter cells was verified by immunostaining for pluripotency markers, OCT4 and SOX2, which the EGFP-bESCs positively expressed. This indicates no loss in pluripotency after gene editing. Experiment 1.2: Optimization of CRISPR/Cas9 gene disruption in EGFP-bESCs. The cutting activity of two candidate EGFP guides was tested in cells and the guide with higher editing efficiency was selected. To optimize gene disruption, the EGFP locus was targeted for knockout in reporter EGFP-bESCs by delivering Cas9 and gRNA via a single plasmid DNA or ribonucleoprotein (RNP, synthetic gRNA + Cas9 protein). The EGFP guide sequence was cloned into a Cas9-T2A-mCherry plasmid, and synthetic EGFP guide RNA was complexed with Cas9 protein to determine the optimal amount of DNA plasmid and ratio of Cas9 protein to gRNA for RNP and Lipofectamine Stem or CRISPRMAX was used, respectively, to deliver reagents. Five days after transfection, loss of green fluorescence in EGFP-bESCs was measured by flow cytometry in triplicate. On average, EGFP expression decreased by 48.3% with Cas9-EGFP gRNA RNP and by 28.5% with Cas9-EGFP gRNA plasmid, indicating a higher knockout efficiency with Cas9 as RNP than plasmid. Sanger sequencing of the H11-EGFP locus confirmed knockout efficiency. Knockout efficiency when delivering Cas9 plasmid or RNP via NEPA21 electroporation is in the final stages of analysis. Experiment 1.4: Optimization of HDR mediated single-base substitutions for allele introgression using GFP to BFP conversion model. Homology-directed repair (HDR) templates were designed for the EGFP to blue fluorescent protein (BFP) conversion model for determining which type is the most efficient at introducing small edits. Four single-stranded donor oligonucleotides (ssODNs) were constructed to contain the two base-substitution for BFP and different homology arm lengths (symmetrical 40, 60, 80 bases and asymmetrical 36 bases PAM-distal and 91 bases PAM-proximal). These ssODNS are currently being tested with Cas9-EGFP gRNA RNP in reporter EGFP-bESCs. Work on the remaining experiments of Objective 1 (Experiment 1.3: Determination of off-target effects on edited bESCs, Experiment 1.5: Long HDR with traffic light model, Experiment 1.6: Benchmark in ESCs of other livestock species) and Objective 2 (Optimize multiplex gene editing for knock-in and knock-out) is ongoing. With our work to date, we anticipate that transfection and editing efficiency will vary by cell line both within and between species and target gene, respectively. However, the guidelines we have established (use of lipofection and RNP to delivery CRIPSR/Cas9 reagents) are useful starting points for the ongoing work.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2023
Citation:
Cayabyab, D., Ross, P. J. and Maga, E. A. 2023. A bovine embryonic stem cell reporter line for assessing CRISPR/Cas9 gene editing efficiency. Oral poster pitch and poster presentation at the Transgenic Animal Research Conference XIV, August 13-17, 2023, Granlibakken Conference Center, Tahoe City, CA
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Progress 07/01/20 to 06/30/24
Outputs
Target Audience: University, government and industry scientists working in the fields of genetic engineering and gene editing Federal regulators Livestock producers Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Graduate students had an opportunity for professional development with respect to presentation skills. A total of 10 graduate students and 2 post-doctoral scholars attended the meeting and presented their work in both poster form and in a 3 minute oral poster pitch presentation during a plenary session. Each graduate student and post-doc received full support ($1,500) in grant funds towards their registration fees to enable their participation by offsetting the cost of the conference. How have the results been disseminated to communities of interest?Abstracts from all oral and poster presentations are freely available online at the conference website. https://na-prod-aventri-files.s3.amazonaws.com/html_file_uploads/d0a203453575495859ee7f142a50705a_MASTERTARCXIV2023_BookofAbstracts_forwebsite.pdf?response-content-disposition=inline%3Bfilename%3D"MASTER%20TARC%20XIV%202023_Book%20of%20Abstracts_for%20website.pdf"&response-content-type=application%2Fpdf&AWSAccessKeyId=AKIA3OQUANZMGCIZWZ6F&Expires=1728431358&Signature=A6ypHSfgQFh13hNNmS83dLyfii0%3D What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
A four day international conference focused on genetic engineering and gene editing in livestock, poultry and fish was held in person that brought together scientists from academia, industry and government labs/agencies and regulators from around the world to present and discuss the latest research and developments in the science of using gene biotechnologies in agriculturally important species. A total of 103 people registered and attended the conference with the majority of participants from the US (72) with others from the UK (7), Germany (5), South Korea (5), Australia (4), Netherlands (3) and one person each from the African Union, Canada, China, Italy, Japan, Kenya and New Zealand. Most attendees were from academic institutions (51) with 11 associated with regulatory agencies, 8 from government labs, 31 from biotechnology-related companies and 2 participants from various scientific organizations/commodity groups. The conference consisted of 25 plenary talks, 12 oral student poster pitch presentations and 20 poster presentations. The talks were categorized into four main areas- technology development (2), agricultural applications (6) and applications in birds (2), biomedical applications (3) and Day 2 dedicated solely to regulation (3 talks on getting products to market, 2 talks on policy by USDA personnel and 5 talks on regulatory journeys of those who are working on approvals) along with an opening talk to set the stage and a closing talk looking to the future. With respect to risk assessment, the presentations included topics such as assessment of off-target effects of CRISPR/Cas9 and regulatory issues associated with genetic engineering and gene editing in animals. The final program for the conference can be found here: https://na.eventscloud.com/website/55524/agenda/.
Publications
- Type:
Websites
Status:
Published
Year Published:
2024
Citation:
All abstracts from the conference (invited speakers and poster presentations) are available on the conference website (https://na.eventscloud.com/website/55524/) at: https://na-prod-aventri-files.s3.amazonaws.com/html_file_uploads/d0a203453575495859ee7f142a50705a_MASTERTARCXIV2023_BookofAbstracts_forwebsite.pdf?response-content-disposition=inline%3Bfilename%3D"MASTER%20TARC%20XIV%202023_Book%20of%20Abstracts_for%20website.pdf"&response-content-type=application%2Fpdf&AWSAccessKeyId=AKIA3OQUANZMGCIZWZ6F&Expires=1728431358&Signature=A6ypHSfgQFh13hNNmS83dLyfii0%3D
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Progress 07/01/22 to 06/30/23
Outputs
Target Audience:Graduate student being trained on the culture of bovine embryonic stem cells, recombinant DNA work and gene editing analysis via laboratory instruction. Postdoctoral scholar gaining experience training students via laboratory instruction and transfection of embryonic stem cells. Undergraduate student being trained by graduate student on stem cell culture via practicum experience. Changes/Problems:The departure of a postdoc helping with the work and transferring all work to a graduate student delayed progress on the project as the graduate student needed to be fully trained to carry out the work. What opportunities for training and professional development has the project provided?One graduate student was trained how to analyze gene editing events to expand professional skills and expertise that include bESC culture and transfection. A post-doctoral researcher gained expertise in mentoring graduate students to enhance their professional development in this important area. A graduate student gained experience in training an undergraduate student to culture bESCs, a professional skill the undergraduate is now proficient at. How have the results been disseminated to communities of interest?Work was presented (oral presentation) at the 2022 UC Davis Animal Biology Graduate Group Colloquium on October 25, 2022 What do you plan to do during the next reporting period to accomplish the goals?The GFP-expressing bESC line generated (Experiment 1.1) will be used to optimize delivery of CRISPR-Cas9 reagents to ablate GFP expression (Experiment 1.2), carry out single base substitutions (Experiment 1.4) and add a large DNA insertion at the GFP locus (Experiment 1.5). Data will also be collected on off-target events in ESCs (Experiment 1.3). We will also begin benchmarking the optimal conditions determined for bESCs in ESCs of other species (Experiment 1.6) and begin with multiplexing experiments (Objective 2).
Impacts
What was accomplished under these goals?
The overall goal of this project is to develop a platform for efficient multiplex gene editing in livestock species. To accomplish this, we are working in embryonic stem cells as once they have been edited, they can be used to derive an animal bearing the desired edits via somatic cell nuclear transfer. The first step is optimizing delivery of gene editing reagents into livestock embryonic stem cells (ESCs). Two lines (A and E) of bovine ESCs (bESCs) previously derived from the inner cell mass of early blastocysts were cultured and manipulated under feeder-free conditions and we have found differences in transfection and gene editing efficiency between the lines. We have established a stable green fluorescent protein (GFP) expressing line of bESCs using feeder-free conditions that will be used to further optimize the delivery of CRISPR-Cas9 reagents to achieve knockouts and knockins and to test multiplex editing. As the derivation of ESCs from livestock species has only recently been reported, any knowledge on how to introduce DNA efficiently needs to be generated. A more detailed description of the progress this year is outlined below. Progress has been made toward Objective 1. Objective 1: To optimize CRISPR-Cas9 delivery of genome editing reagents for efficient gene disruption, allele introgression and targeted gene insertion in bovine, ovine and caprine embryonic stem cells. For preliminary assessment of CRISPR-Cas9 activity in bESCs, Line E cells were transfected with Cas9-GFP plasmid containing single guide RNA (sgRNA) for the H11 locus (pX458+H11) using Lipofectamine Stem in feeder-free conditions. Cells positive for green fluorescence were isolated by fluorescence-activated cell sorting (FACS) and collected for sequence analysis. Genomic DNA was extracted from GFP positive cells, the H11 region was PCR amplified and the product was Sanger sequenced. Genome editing efficiency was quantified from both forward and reverse sequences by tracking of indels by decomposition (TIDE), which resulted in an average indel rate of 73.8%, thus confirming efficient activity of Cas9 at the target site. Developing methods of introducing CRISPR/Cas9 gene editing reagents into ESCs under feeder-free conditions would greatly simplify the process and hence we concentrated on lipofection and nucleofection to assess transfection efficiency. While nulceofection (Amaxa Nucelofector II) resulted in poor bESCs survival and/or had slow growth rates once nucleofected, the use of Lipofectamine Stem resulted in a 15% transfection efficiency in Line E and a 30% transfection efficiency in Line A when co-transfected in triplicate with pX458+H11 and donor plasmid with EGFP driven by the CAG promoter and H11 homology arms (pCAG-EGFP-H11). Controls included cells transfected with transfection control plasmid, pmaxGFP and pX458+H11 as a control for transient expression of GFP. Transient expression of GFP from the Cas9-GFP plasmid subsided after 7 days in culture while those transfected with donor plasmid had proliferation of green cells by day 7 indicating stable transfection. These experiments have been replicated in triplicate at least three times with each bESC line. The heterogenous population of EGFP and non-EGFP cells were passaged 7 days after transfection and expanded for 7 additional days to allow bESCs with stable EGFP expression (EGFP-bESCs) to proliferate further. Polyclonal EGFP-bESCs were harvested and their genomic DNA was extracted for validation of targeted EGFP knock-in at the H11 locus. To generate a monoclonal line of EGFP-bESCs, two approaches were explored. 1) Single cell cloning by limiting dilution in a 96-well plate was performed for each replicate. Screening for single EGFP-bESC clones was done 5 days after feeder-free culture in 96-well plates using a 1:1 ratio of fresh NBFR medium to conditioned medium. For each replicate, 1 out of 96 wells had EGFP positive cells. These EGFP-bESCs were allowed to proliferate for an additional 2 days to increase the monoclonal population but cells died, potentially due to edge effect during screening. Single cell cloning of EGFP-bESCs was also attempted on a mouse embryonic fibroblast (MEF) feeder layer, however no EGFP positive colonies were observed. 2) Enrichment of EGFP positive cells by passaging EGFP-bESCs onto 60 mm petri dishes at low seeding density seven days post-transfection. EGFP-bESCs proliferated for 6 days and areas abundant with EGFP positive cells were isolated using a 5 mm cloning ring. Isolated cells were plated onto a smaller culture vessel and maintained for 7 days but no EGFP-bESCs were recovered. At day 5 post-transfection, 24 areas (8 per replicate) that were abundant with EGFP-bESCs were selected for passaging into individual wells on a 24-well plate using a cloning ring. These cells grew for 2 days and each well containing EGFP-bESCs will be used for future single cell cloning in 96-well plates. Once confirmed as monoclonal, these GFP lines will be used for the optimization studies as outlined in Experiments 1.2-1.5.
Publications
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Progress 07/01/21 to 06/30/22
Outputs
Target Audience:Graduate student being trained on the culture of bovine embryonic stem cells, recombinant DNA work and gene editing analysis via laboratory instruction. Postdoctoral scholar gaining experience training students via laboratory instruction. Undergraduate student being trained by graduate student on stem cell culture via practicum experience. Changes/Problems:Due to campus restrictions regarding Covid-19, work was still not able to be performed on a consistent basis this year, hence our request for a no-cost extension. The long doubling time of the stem cells has concentrated efforts on delivering CRISPR/Cas9 reagents via lipofection-based methods which require fewer cells. What opportunities for training and professional development has the project provided?One graduate student was trained how to analyze gene editing events to expand professional skills and expertise that include bESC culture and transfection. A post-doctoral researcher gained expertise in mentoring graduate students to enhance their professional development in this important area. A graduate student gained experience in training an undergraduate student to culture bESCs, a professional skill the undergraduate is now proficient at. How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?The GFP-expressing bESC line generated (Experiment 1.1) will be used to optimize delivery of CRISPR-Cas9 reagents to ablate GFP expression (Experiment 1.2), carry out single base substitutions (Experiment 1.4) and add a large DNA insertion at the GFP locus (Experiment 1.5). Data will also be collected on off-target events in ESCs (Experiment 1.3). We will also begin benchmarking the optimal conditions determined for bESCs in ESCs of other species (Experiment 1.6) and begin with multiplexing experiments (Objective 2).
Impacts
What was accomplished under these goals?
The overall goal of this project is to develop a platform for efficient multiplex gene editing in livestock species. To accomplish this, we are working in embryonic stem cells as once they have been edited, they can be used to derive an animal bearing the desired edits via somatic cell nuclear transfer. The first step is optimizing delivery of gene editing reagents into livestock embryonic stem cells (ESCs). We have tested three different methods to deliver green fluorescent protein (GFP) reporter plasmids to bovine ESCs (bESCs). These include nucleofection, electroporation and two means of lipofection. In all instances, GFP expression was seen with the lipofection-based approach resulting in the highest efficiency with fewer cells (more details are given below). We were able to generate a GFP-bESC reporter line, however, it became contaminated and derivation of other lines are underway in order to optimize the delivery of CRISPR-Cas9 reagents to achieve knockouts and knockins and to test multiplex editing. As the derivation of ESCs from livestock species has only recently been reported, any knowledge on how to introduce DNA efficiently needs to be generated. A more detailed description of the progress this year is outlined below and it should be noted that Covid-19 restrictions still slowed progress this year. Progress has been made toward Objective 1. Objective 1: To optimize CRISPR-Cas9 delivery and genome editing reagents for efficient gene disruption, allele introgression and targeted gene insertion in bovine, ovine and caprine embryonic stem cells. Experiment 1.1 calls for the generation of a line of bESCs that express green fluorescent protein (GFP) by knocking-in a GFP construct driven by an endogenously expressed promoter at a safe harbor locus (H11) using a donor plasmid. To this end, we prepared a donor plasmid for GFP expression driven by the chicken beta-actin promoter (CAG) and containing H11 homology arms and a H11 gRNA sequence. In one of our first attempts, the donor plasmid was co-transfected along with a plasmid expressing Cas9 (pX458) using a novel lipofection reagent (BrPERfect, see in detail below for Experiment 1.2). Treated cells maintained fluorescence 96 hours post-transfection with clusters containing increasing number of fluorescent cells over time (24-96 hours post-transfection). Unfortunately these lines were lost due to contamination of our stock bESC line. We have since derived new lines of bESCs, expanded them and froze some back with which to complete experiment 1.1 and to optimize DNA delivery and compare if any differences in efficiency exist between lines (Experiment 1.2). To date, we have generated at least 4 lines of bESCs stably expressing GFP. Final molecular analysis to confirm insertion at the H11 locus is underway. These GFP cell lines can now be used for the optimization studies as outlined in Experiments 1.2-1.5. Experiment 1.2 calls for the optimization of CRISPR/Cas9 gene disruption in ESCs. Delivery of Cas9 and gRNA as plasmid DNA was optimized using nucleofection (Amaxa Nucleofector II), electroporation (NEPA 21) and lipofection (Lipofectamine Stem and BrPERfect). For nucleofection, two programs were compared using the plasmid pmaxGFP to determine cell survivability and optimal settings for the bESC line. The program with the highest efficiency (30% cell survival 24-hours post-nucleofection) was then used to test different three different amounts of plasmid DNA containing Cas9, GFP and H11 guide sequences. The amount of DNA had no impact on transfection efficiency which was <5% in all cases. Use of the NEPA21 electroporator resulted in a 20% transfection efficiency when using pmaxGFP but <5% transfection efficiency was observed when using our Cas9/GFP/H11 guide plasmid. As each electroporation or nulceofection requires 1x10^6 cells, more parameters can be tested once scalability of bESC culture improves as it takes approximately one month to reach this cell density. This is opposed to lipofection-based methods that require 100,000 cells, a density that can be reached in 1-2 weeks. Transfection of bovine ESCs with pX458 with H11 guide using Lipofectamine Stem resulted in up to 15% transfection efficiency. When pX458 with H11 guide was co-transfected with donor template, transfection efficiency was less than 5%. A 1:1 molar ratio of pX458 with H11 guide and template was tested, as well as different combinations of gene editing reagents: pX458 with no guide with/without template and pX458 with H11 guide with/without template. In addition to Lipofectamine Stem, another cationic polymer-based delivery reagent, BrPerfect, was tested in bovine ESCs which had similar or higher transfection efficiency than Lipofectamine Stem. Both delivery reagents were tested at different ratios of DNA to polymer. Cells treated with these different combinations of reagents were collected for DNA extraction. PCR was then done on genomic DNA to amplify the H11 region for sequencing to check for edits at the target site. We developed a digital droplet PCR-based assay to detect gene editing at the H11 locus in a mixed population of cells (ie transfected and non-transfected cells (cells with and without edits)) and found gene editing to occur at a rate of less than 10% with the delivery of CRISPR-Cas9 reagents via plasmids. Our test plasmid uses the CMV promoter to express Cas9 and GFP and this could account for the low efficiencies we are seeing as this promoter is known to be silenced in stem cells. We are currently working on the optimization of the delivery of RNP (Cas9 protein and gRNA complex).
Publications
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Progress 07/01/20 to 06/30/21
Outputs
Target Audience:Graduate student being trained on the culture of bovine embryonic stem cells and recombinant DNA work via laboratory instruction. Changes/Problems:Due to campus restrictions regarding Covid-19, work was not able to be performed on a consistent basis until June of this year. What opportunities for training and professional development has the project provided?One graduate student was mentored on the culturing of bESCs and the processes of lipofection and nucleofection as well as recombinant DNA techniques to generate the constructs that will be required. How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?The GFP-expressing bESC line will be generated (Experiment 1.1) and used to optimize delivery of CRISPR-Cas9 reagents to ablate GFP expression (Experiment 1.2), carry out single base substitutions (Experiment 1.4) and add a large DNA insertion at the GFP locus (Experiment 1.5). Data will also be collected on off-target events in ESCs (Experiment 1.3). We will also begin benchmarking the optimal conditions determined for bESCs in ESCs of other species (Experiment 1.6) and begin with multiplexing experiments (Objective 2).
Impacts
What was accomplished under these goals?
The overall goal of this project is to develop a platform for efficient multiplex gene editing in livestock species. To accomplish this, we are working in embryonic stem cells as once they have been edited, they can be used to derive an animal bearing the desired edits via somatic cell nuclear transfer. The first step is optimizing delivery of gene editing reagents into livestock embryonic stem cells (ESCs). We have tested three different methods to deliver green fluorescent protein (GFP) reporter plasmids to bovine ESCs (bESCs). These include a piggyBac transposon system, lipofection and nucelofection. In all instances, GFP expression was seen with the lipofection-based approach resulting in the highest efficiency (more details are given below). With base knowledge now of how best to introduce DNA into bESCs, we will now be able to efficiently generate a GFP-bESC reporter line to use for the optimization of the delivery of CRISPR-Cas9 reagents to achieve knockouts and knockins and to test multiplex editing. As the derivation of ESCs from livestock species has only recently been reported, any knowledge on how to introduce DNA efficiently needs to be generated. A more detailed description of the progress this year is outlined below and it should be noted that Covid-19 restrictions delayed much of this work. Progress has been made toward Objective 1. Objective 1: To optimize CRISPR-Cas9 delivery and genome editing reagents for efficient gene disruption, allele introgression and targeted gene insertion in bovine, ovine and caprine embryonic stem cells. Experiment 1.1 calls for the generation of a line of bESCs that express GFP. To aid in the efficient production of this reporter line, we first optimized the delivery of plasmid DNA to the cells. Passage 15-20 bESCs were cultured in feeder-free conditions and GFP reporter plasmids were introduced using lipofection or nucleofection. All experiments were carried out in triplicate. The expression vector pmaxGFP was introduced using both Lipofectamine 2000 and Lipofectamine Stem reagents using a ratio of 3:4 DNA (μg) to Lipofectamine (μL). Cells were 75% confluent on the day of lipofection and 24hr after transfection, cells treated with Lipofectamine 2000 had 50% confluence and 30% fluorescence. Non-transfected, Lipofectamine 2000-treated cells had 70% confluence and no fluorescence as expected. Cells transfected using Lipofectamine Stem had higher survival and transfection efficiency with 70% confluence and 50% fluorescence. Non-transfected, Lipofectamine Stem-treated cells had 100% confluence and no fluorescence as expected. At 48hr post-transfection, fluorescence decreased in cells that were transfected using either Lipofectamine 2000 or Lipofectamine Stem as was expected due to the transient nature of the transfection. Overall, given that the use of Lipofectamine Stem resulted in more surviving and fluorescent cells, it is the better option for lipofecting bovine ESCs. The Amaxa Nucleofector was also used to introduce pmaxGFP in bESCs and preliminary results have demonstrated that this method is feasible and results in efficiency equivalent to that seen with Lipofectamine 2000. Work is currently being carried out to replicate and optimize these findings. Finally, to assess a method that would result in the integration of the vector in the genome, the Super PiggyBac Transposase expression vector and PB-CMV-MCS-EF1α-GreenPuro PiggyBac cDNA cloning and expression vector were co-transfected in bESCs using Lipofectamine Stem Transfection Reagent and the recommended ratio of 1:2 for DNA (μg) to lipofectamine (μL). This resulted in two colonies expressing GFP 24hr post-transfection. The detection of fluorescence in cells suggests that the PiggyBac transposon system can enter bESCs and the expression cassette can integrate into the genome. More work is currently being carried out to optimize this system as well as the use of electroporation. The optimization of conditions with plasmids will be the starting point for the optimization of the delivery of gene editing reagents in Experiment 1.2.
Publications
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