Progress 09/01/18 to 08/31/23
Outputs
Target Audience:Graduate and undergraduate students being trained in the area of gene editing via laboratory instruction. Changes/Problems:As it was not possible to get AR knockout pigs to go to term for reasons described above, we also did work targeting a different gene hypothesized to have a similar impact to AR knockout. For this alternative, we targeted the gene SRD5A2 wich encodes the enzyme 5-alpha reductase type 2, the enzyme required for the final step of converting testosterone into androstenone, an androgen metabolite, in the testis. The reduction of this enzyme should impede development of the reproductive tract. An embryo transfer with SRD5A2 edited embryos carried out in May of this year (2023) resulted int he birth of six pigs, two of which (one male and one female) were gene edited at the SRD5A2 locus. We have obtained campus funding to maintain the pigs until puberty and characterize their reproductive capabilities. What opportunities for training and professional development has the project provided?Four graduate students were mentored on both embryo production and manipulation as well as on the analysis of gene editing events, one was mentored on analysis of gene editing and two on bioinformatic analyses. How have the results been disseminated to communities of interest?Work was presented in poster form at the 2022 Society for the Study of Reproduction (SSR) annual meeting (in person) and at the 2023 Transgenic Animal Research Conference (TARC) XIV (in person). What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
This project addressed two concerns associated with genetic engineering/gene editing in animals- the containment of genetically engineered animals and the specificity of gene editing events. To address these concerns, we proposed a platform to render males sterile and methodologies to assess off-target cutting by the CRISPR/Cas9 gene editing system. The goal was to use CRISPR/Cas9 to disrupt the androgen receptor (AR) gene in the pig that is responsible for the production of male sex hormones and thus render males infertile as a means of containment as they would be unable to pass along any other genetic modifications in the animals. Knockout of the pig AR gene via electroporation of zygotes was incompatible with development past 25 days of gestation. Furthermore, all resulting AR edited fetuses past 21 days of gestation were female, leading to questions of the importance of AR expression in early embryo development in the pig if this approach is a viable option for genetic containment in the pig. Multiple off-target events with the AR guide RNAs were detected using in vitro assays but two or less were detected in vivo (edited fetuses and blastocysts). This suggests that it is more difficult for guides to cut off-target in the context of genomic DNA in vivo and that blastocysts can be used to assess the true prevalence of off-target events. Work was completed on Objectives 1 and 3 as we could not produce live-born gene edited AR knockout pigs to characterize (Objective 2). Objective 1: Generation of pigs lacking the ability to make male reproductive hormones Aim 1.1: Design and validation of sgRNAs targeting the pig androgen receptor gene Three candidate guide RNAs specific to the pig androgen receptor gene were evaluated. Two guides targeting exon 2 (classical signaling) and one guide targeting exon 5 (non-classical signaling) were assessed in parthenotes. Combinations of the guides were also assessed. Guides and Cas9 protein (1:2 ratio) were microinjected or electroporated into porcine zygotes and mutation rates determined by DNA sequencing after development to the blastocyst stage. Mutation rate at the desired site ranged from 57-100% for individual guides. Guide 2A resulted in a 93% rate of biallelic mutations when using electroporation, guide 2B 75% and the single guide for exon 5 had a rate of 66% biallelic mutation and 33% monoallelic mutation. Aim 1.2: Generation of pigs lacking a functional androgen receptor. Recipient gilts carrying AR-edited embryos failed to maintain pregnancy past 25 days of gestation. A total of 14 embryo transfer surgeries were done, with 13 recipients confirmed pregnant. Two recipients aborted between days 26-28 of pregnancy and four others had no fetuses at day 25. Other recipients carrying in vitro produced embryos with unrelated edits maintained pregnancies with normal fetuses recovered at days 45 and 49, indicating our system of embryo production and editing was not the cause of the losses. As such, two pregnancies were ended at day 22, two at day 23 and one each on days 25, 21 and 19 to collect fetuses for further analysis. Surprisingly, all day 21 and older fetuses (n= 23) were female. This sex bias was not seen in AR-edited blastocysts and male AR-edited fetuses were present at day 19 (9/13). Thus, CRISPR/Cas9 editing of the AR gene led to a sex biased loss of male fetuses between day 19 and 21 of gestation and subsequent loss of female fetuses. In wild-type fetuses, we found that AR is expressed earlier in development (day 19) than previously known (day 35) and that expression of the rate-limiting enzyme in testosterone synthesis could be detected in the placenta on day 19 (n=4) and day 23 (n=2) but is absent by day 48 (n=3) and 54, indicating that AR may be needed in early development in the pig. However, the most recent update of the pig genome annotation places an U2.35 spliceosome in the intronic region between exons 3 and 4. Expression of the spliceosome was lower in day 19 AR-edited fetuses (n=2) compared to day 19 wild-type male fetuses (n=3) while levels were similar in edited (n=5 at day 21, n=2 at day 23) and wild-type (n=7 at day 19, n=3 at day 23, n=1 at day 27) females. The current thought that some of the genes on the X-chromosome partially escape X-chromosome inactivation in conjunction with partial rather than complete reduction of transcription with edits of the AR, might explain the observed sex bias. If this were the case, reduced expression of this U2 spliceosome would be expected in the 19-day male fetuses with edited AR compared with control male (and female) fetuses as we observed. Objective 3: Assessment of the fidelity of the gene editing tools used Aim 3.1: Screen most likely off-target sites by PCR Potential off-target cutting sites in the pig genome were analyzed using three different open source software programs, CRISPOR, CCTOP and CASOFFinder, to identify the top 20 probable off-target sites for g2A and g2B and in exon 2. For each guide, 15 sites that were common between each of the three programs and had the highest scores for cutting in intron/exon regions were chosen as were 5 sites unique to CRISPOR and CASSOFFinder and one from CCTOP. The number of putative off-target sites ranged from 91-894 for g2A, 143-1,138 for g2B across the three programs. Primers were designed for 24 potential off-target sites for g2A and 25 for g2B. Three sites for g2A and five sites for g2B either had numerous mismatches or confirmed SNPs and were excluded from in vitro testing. Five of the 20 sites tested for g2A were cut and four of 20 sites for g2B indicating that these guides can cut elsewhere in the genome. Aim 3.2: Whole genome sequencing of founder and F1 animals. As we could not produce live founder animals, sequencing was carried out on knockout fetuses that were collected. See aim 3.3 for details. Aim 3.3: Whole genome sequencing of parthenote-derived blastocysts. CHANGE-Seq (circularization for high-throughput analysis of nuclease genome-wide effects by sequencing) was used to assess genome-wide off-target events. After optimization, CHANGE-Seq was carried out with AR guides g2A and g2B using genomic DNA from three unrelated wild-type pigs, generating paired 150 bp reads using both the Illumina MiniSeq and NextSeq platforms. The total number of off-target sites detected across the three replicates was 132 (12 sites in common in all replicates) for g2A and 335 (68 common across replicates) for g2B. Of the 20 off-target sites tested for each guide with the in Aim 3.1, CHANGE-Seq identified 3 of them for g2A (2 of which cut in vitro) and 6 for g2B (4 cut in vitro). The results of CHANGE-Seq were used to investigate these putative off-target sites in gene edited blastocysts and collected fetuses to determine if this can be a screening option to test for off-target events prior to generating animals. We used the rhAmp-Seq technique which allows for multiplex PCR resulting in hundreds of amplicons in a single reaction that can be used to create a sequencing library for the Illumina platform. We generated rhampSeq CRISPR panels of 100 off-target sites for each guide (g2A and g2B) including off-target sites that were common to all 3 wild-type animals, sites present at high frequency(read count) and off-target sites that were not observed in the CHANGE-seq but cut amplicon DNA in the in vitro experiments. A total of 8 blastocysts with mutation efficiency of greater than 93% were analyzed for each guide as were 8 gene edited fetuses. Two different approaches for alignment of reads were compared and both found only three off-target mutations (1 with g2A, 2 with g2B) in both the blastocysts and fetuses. This data was validated using whole genome sequencing of 5 edited fetuses and 8 edited and 2 wild-type whole genome amplified blastocysts. The average number of reads per sample was 4E08 and the percentage of reads aligned to the sites chosen for the rhAmpSeq varied from 4.2%-7.9%.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2023
Citation:
Zacanti, K., Park I,. McNabb, B. R., Urbano, T. M., Maga, E. A., Nitta-Oda, B. J., Rowe, J. D., Hennig, S. L., Ross, P. J. and Berger, T. Gender disparity in survival of early porcine fetuses due to altered androgen receptor or associated U2 spliceosome component. Scientific Reports 13:15072.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2023
Citation:
J. L. Jankovitz and E. A. MAga. Androgen Receptor developmental timeline and expression patterns in fetal pigs. Abstract and poster presentation at the Transgenic Animal Research Conference XIV held in August 2023.
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Progress 09/01/21 to 08/31/22
Outputs
Target Audience:Graduate and undergraduate students being trained in the area of gene editing via laboratory instruction. Changes/Problems:While there were fewer Covid-19 restrictions on campus this year, we were still impacted by personnel having to quarantine at various times. We also loss use of our male pigs (one to injury, one to illness) for a period of 4 months, limiting our ability to obtain semen on a consistent basis for production of embryos. We also had complications with several embryo transfer surgeries, limiting the number we could perform. Both these issues for semen availability and embryo transfers have now been resolved and we anticipate being able to work at full capacity starting this fall. What opportunities for training and professional development has the project provided?Three graduate students were mentored on both embryo production and manipulation as well as on the analysis of gene editing events. How have the results been disseminated to communities of interest?Work was presented in poster form at the 2022 Society for the Study of Reproduction (SSR) annual meeting (in person). What do you plan to do during the next reporting period to accomplish the goals?New guide RNAs are being designed to introduce a defined mutation in the AR gene in an attempt to mitigate the embryonic lethality we are currently seeing. New guides will be validated, delivery with template DNA optimized and embryo transfers carried out in an attempt to generate live born AR-edited pigs. If successful, characterization of AR-edited pigs will begin (Aims 2.1 and 2.2).We will conclude the analysis of off-target events (Objective 3).
Impacts
What was accomplished under these goals?
This project addresses two concerns associated with genetic engineering/gene editing in animals- the containment of genetically engineered animals and the specificity of gene editing events. To address these concerns, we are working on a platform to render males sterile and methodologies to assess off-target cutting by the gene editing system CRISPR/Cas9. Using CRISPR/Cas9, we are working in the pig to disrupt a gene responsible for the production of male sex hormones, thereby rendering males infertile as a means of containment (as they would be unable to pass along any other genetic modifications in the animals). Introduction of CRISPR-Cas9 protein and guide RNAs specific to the androgen receptor (AR) gene via electroporation and transfer to recipient gilts has failed to maintain pregnancies past 28 days while transfer of non-manipulated or electroporated zygotes maintained development for at least 48 days. Furthermore, all resulting AR edited fetuses past 21 days of gestation were female, leading to questions of the importance of AR expression in early embryo development in the pig and if this approach is a viable option for genetic containment in the pig. This project addresses two concerns associated with genetic engineering/gene editing in animals- the containment of genetically engineered animals and the specificity of gene editing events. To address these concerns, we are working on a platform to render males sterile and methodologies to assess off-target cutting by the gene editing system CRISPR/Cas9. Using CRISPR/Cas9, we are working in the pig to disrupt a gene responsible for the production of male sex hormones, thereby rendering males infertile as a means of containment (as they would be unable to pass along any other genetic modifications in the animals). Introduction of CRISPR-Cas9 protein and guide RNAs specific to the androgen receptor (AR) gene via electroporation and transfer to recipient gilts has failed to maintain pregnancies past 28 days while transfer of non-manipulated or electroporated zygotes maintained development for at least 48 days. Furthermore, all resulting AR edited fetuses past 21 days of gestation were female, leading to questions of the importance of AR expression in early embryo development in the pig and if this approach is a viable option for genetic containment in the pig. Progress has been made towards Objectives 1 and 3 as we have yet to produce live-born gene edited AR knockout pigs to characterize (Objective 2). Objective 1: Generation of pigs lacking the ability to make male reproductive hormones Aim 1.2: Generation of pigs lacking a functional androgen receptor. Work in previous years has shown that recipient gilts carrying AR-edited embryos fail to maintain pregnancy past 28 days of gestation. Briefly, a total of 11 embryo transfer surgeries were done, with 10 recipients confirmed pregnant. Four of these pregnancies were ended at days 22 (n=2) and 23 (n=2) of gestation to collect any fetuses for molecular analysis and it was found 14 of the 15 fetuses recovered were edited at the AR locus. Work this year revolved around investigating systemic issues, collecting fetuses at earlier time points and determining the sex of the fetuses and expression of other genes involved with androgen production to explain the early pregnancy losses with AR knockout in the pig. Embryo transfers with control (non-electroporated) and null electroporated embryos cultured in different media demonstrated that pregnancies could be established and maintained until at last day 48 of gestation, eliminating the prospect that our embryo culture and production system was causing the early losses of the AR knockout edited embryos. Three additional embryo transfers were done to collect fetuses at days 19 (n=13 fetuses), 21 (n=12 fetuses) and 25 (n=8 fetuses) of gestation. Interestingly, all of the 21 day and older AR-edited fetuses (n=16, days 21-25) were female. This sex bias was not seen in AR-edited blastocysts and male AR-edited fetuses were present at day 19 (9/13). Thus, CRISPR/Cas9 editing of the AR gene led to a sex biased loss of male fetuses between day 19 and 21 of gestation and subsequent loss of female fetuses. We are currently evaluating the expression of genes involved with testosterone synthesis and non-coding RNAs in the vicinity of the porcine AR gene to help explain these results and offer an alternative approach to editing the AR gene. Objective 3: Assessment of the fidelity of the gene editing tools used Aim 3.1: Screen most likely off-target sites by PCR. We had previously screened 20 potential off-target sites for each of two AR guide RNAs (g2 and g3) using an in vitro assay and found that 5 of the 20 sites cut for g2 and 4 of the 20 for g3. When these sites were screened in blastocysts (n=5 for g2, n=5 for g5) and collected fetuses (n=8) with on-target mutations, no cutting at these off-target sites was evident. This data tells us that while guides can cut off-target amplicons in vitro, it is more difficult for them to cut off-target in the context of genomic DNA (blastocysts and fetuses). Aim 3.2: Whole genome sequencing of founder and F1 animals. As we have yet to produce live founder animals, sequencing is being carried out on knockout fetuses that have been collected. See aim 3.3 for details. Aim 3.3: Whole genome sequencing of parthenote-derived blastocysts. CHANGE-Seq (circularization for high-throughput analysis of nuclease genome-wide effects by sequencing) was used to assess genome-wide off-target events. First, Cas9/gRNA concentrations were optimized using qPCR on AR amplicons to determine the most efficient Cas9 protein (two different brands tested) and Cas9/guide RNA ratio (three ratios tested for each guide (g2 and g3)) to use. Next, CHANGE-Seq was carried out with AR guides g2 and g3 using genomic DNA from three unrelated wild-type pigs, generating paired 150 bp reads using both the Illumina MiniSeq and NextSeq platforms. The total number of off-target sites detected across the three replicates was 132 (12 sites in common in all replicates) for g2 and 335 (68 common across replicates) for g3. Of the 20 off-target sites tested for each guide with the in vitro assay (Aim 3.1), CHANGE-Seq identified 3 of them for g2 (2 of which cut in vitro) and 6 for g3 (4 cut in vitro). The results of CHANGE-Seq were used to investigate these putative off-target sites in gene edited blastocysts and collected fetuses to determine if this can be a screening option to test for off-target events prior to generating animals. We used the rhAmp-Seq technique which allows for multiplex PCR resulting in hundreds of amplicons in a single reaction that can be used to create a sequencing library for the Illumina platform. We generated rhampSeq CRISPR panels of 100 off-target sites for each guide (g2 and g3) including off-target sites that were common to all 3 wild-type animals, sites present at high frequency(read count) and off-target sites that were not observed in the CHANGE-seq but cut amplicon DNA in the in vitro experiments. A total of 8 blastocysts with mutation efficiency of greater than 93% were analyzed for each guide as were 8 gene edited fetuses. Two different approaches for alignment of reads were compared and both found only three off-target mutations (1 with g2, 2 with g3) in both the blastocysts and fetuses. This data is currently being validated.
Publications
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2022
Citation:
Jennifer L. Jankovitz, Xintong Li and Elizabeth A. Maga. Androgen Receptor Developmental Timeline and Expression Patterns in Fetal Pigs. Abstract and poster presentation at the 2022 SSR annual meeting.
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Progress 09/01/20 to 08/31/21
Outputs
Target Audience:Graduate students being trained in the area of gene editing via laboratory instruction. Attendees of the 2021 virtual BRAG PD meeting. Changes/Problems:Due to campus restrictions regarding Covid-19, not as many embryo transfer surgeries as planned were able to be carried out this year. With easing of restrictions in June of this year, we anticipate being able to perform a full schedule of surgeries in the coming year. What opportunities for training and professional development has the project provided?Three graduate students were mentored on both embryo production and manipulation as well as on the analysis of gene editing events. 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?More embryo transfers will be carried out with edited zygotes to complete Aim 1.2 with the generation of live-born gene edited pigs. Once animals are born, characterization of them will begin (Aims 2.1 and 2.2). Work on detecting off-target events continues as planned.
Impacts
What was accomplished under these goals?
This project addresses two concerns associated with genetic engineering/gene editing in animals- the containment of genetically engineered animals and the specificity of gene editing events. To address these concerns, we are working on a platform to render males sterile and methodologies to assess off-target cutting by the gene editing system CRISPR/Cas9. Using CRISPR/Cas9, we are working in the pig to disrupt a gene responsible for the production of male sex hormones, thereby rendering males infertile as a means of containment (as they would be unable to pass along any other genetic modifications in the animals). Transfer of androgen receptor (AR) CRISPR-Cas9 targeted zygotes to recipient gilts has failed to establish pregnancies to term while transfer of non-manipulated zygotes has. We are working to determine if this is due to embryonic lethality of AR knockout in pigs or embryo culture conditions in order to determine if AR knockout will be a viable option for genetic containment. Off-target events have been assessed in vitro and in vivo (blastocysts and recovered fetuses) at both predicted sites and genome-wide. Together, we are making progress towards producing sterile male pigs using gene editing and assessing if there are any unwanted editing events that occur during the process with the goals of providing a novel approach to genetic containment that would also alleviate the need for castration of male pigs and providing information to regulators regarding the safety of this technology. A more detailed description of the progress this year is outlined below. Progress has been made towards Objectives 1 and 3. Objective 1: Generation of pigs lacking the ability to make male reproductive hormones Aim 1.2: Generation of pigs lacking a functional androgen receptor. Four embryo transfer surgeries with a total of 195 electroporated zygotes have been carried out with a total of 15 fetuses recovered between days 22-24 of gestation. Of these 15 fetuses, 14 were edited at the AR locus and 1 was wild-type for a 93% efficiency of AR gene disruption. However, when additional embryo transfers were left to go to term, all aborted at approximately day 28 of gestation. The failure to generate live-born edited pigs could either be due to necessity of AR gene expression during early development or to the culture and media conditions used. We have detected presence of AR transcript in day 18 wild-type fetuses, hence there is a possibility that unlike in mice, AR knockout is embryonic lethal. However, transfer of zygotes with other unrelated edits have also failed to result in the birth of live offspring, pointing to a systemic issue. The current pregnancy with non-edited zygotes was achieved using different culture media (FLI) and low oxygen maturation. These conditions will now be used for AR-edited embryos. Objective 3: Assessment of the fidelity of the gene editing tools used Aim 3.1: Screen most likely off-target sites by PCR. We had previously screened 20 potential off-target sites for each of two AR guide RNAs (g2 and g3) using an in vitro assay and found that 5 of the 20 sites cut for g2 and 4 of the 20 for g3. When these sites were screened in blastocysts with on-target mutations, no cutting at off-target sites was evident (n=4 for g2, n=5 for g3). The same was true for the 6 knockout fetuses that have been examined to date. We are currently screening more fetuses and analyzing the in vitro data for a poorly designed gRNA. This data tells us that while guides can cut off-target amplicons in vitro, it is more difficult for them to cut off-target in the context of genomic DNA (blastocysts and fetuses). Aim 3.3: Whole genome sequencing of parthenote-derived blastocysts. Rather than investigating off-target events by just looking at predicted off-target cutting sites, a genome wide approach is being used in non-edited pig genomic DNA and edited and non-edited blastocysts and fetuses using CHANGE-Seq (circularization for high-throughput analysis of nuclease genome-wide effects by sequencing). This method enriches for genomic sites cut by guide RNAs by first tagmenting and circularizing genomic DNA followed by treating the circularized DNA with Ca9 protein and guide RNA. Any molecules cut by the guide are now linear and substrates for next generation sequencing. CHANGE-Seq has been carried out on non-edited pig genomic DNA and results are currently being analyzed. Preliminary results show that off-target cutting does occur, including at sites predicted by the in vitro work. We are currently scaling the technique for use on genomic DNA of blastocysts (less DNA than from a somatic cell) to determine if this can be a screening option to test for off-target events prior to generating animals.
Publications
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Progress 09/01/19 to 08/31/20
Outputs
Target Audience:Graduate students being trained in the area of gene editing via laboratory instruction. Undergraduate students conducting research internships in the area of gene editing. Scientists and researchers working in the area of gene editing and reproduction attending the virtual poster session at the 2020 Society for the Study of Reproduction (SSR) annual meeting. Changes/Problems:Due to campus restrictions regarding Covid-19, not as many embryo transfer surgeries as planned were able to be carried out this year. Our limited surgery schedule will resume in September. What opportunities for training and professional development has the project provided?Three graduate students were mentored on both embryo production and manipulation as well as on the analysis of gene editing events. How have the results been disseminated to communities of interest?Work was presented in poster form at the 2020 Society for the Study of Reproduction (SSR) annual meeting (virtual). What do you plan to do during the next reporting period to accomplish the goals?More embryo transfers will be carried out with edited zygotes to complete Aim 1.2 with the generation of live-born gene edited pigs. Once animals are born, characterization of them will begin (Aims 2.1 and 2.2). Work on detecting off-target events continues as planned.
Impacts
What was accomplished under these goals?
This project addresses two concerns associated with genetic engineering/gene editing in animals- the containment of genetically engineered animals and the specificity of gene editing events. To address these concerns, we are working on a platform to render males sterile and methodologies to assess off-target cutting by the gene editing system CRISPR/Cas9. Using CRISPR/Cas9, we are working in the pig to disrupt a gene responsible for the production of male sex hormones, thereby rendering males infertile as a means of containment (as they would be unable to pass along any other genetic modifications in the animals). We have shown that disruption of the target gene is compatible with development until at least 24 days of gestation and have optimized the system for creating the gene edited pigs. Once born they will be fully characterized to determine is this is a viable approach to genetic containment. To assess the specificity of making these gene edits, we have tested over 40 potential off-target sites and found that the gene editing tools are able to cut at 5 of them. A more in depth look at off target cutting is underway using whole genome sequencing. Together, we are making progress towards producing sterile male pigs using gene editing and assessing if there are any unwanted editing events that occur during the process with the goals of providing a novel approach to genetic containment that would also alleviate the need for castration of male pigs and providing information to regulators regarding the safety of this technology. A more detailed description of the progress this year is outlined below. Progress has been made towards Objectives 1 and 3. Objective 1: Generation of pigs lacking the ability to make male reproductive hormones Aim 1.1: Design and validation of gRNAs targeting the pig androgen receptor gene. This aim has been completed as we have characterized three guide RNAs (gRNAs) in parthenotes. Two of the guides (g2 and g3) were located in exon 2 (previously referred to as exon 1, disruption should eliminate both classical and non-classical signaling pathways and one (g1) in exon 5 (previously referred to as exon 4, disruption should eliminate only the classical signaling pathway). The mutation efficiency of individual gRNAs or combinations of gRNAs (g2 and g3) was greater than 80%. Aim 1.2: Generation of pigs lacking a functional androgen receptor. To test viability of androgen receptor knockout pigs, zygotes generated by in vitro fertilization of in vitro matured oocytes were electroporated with a combination of both guides targeting exon 2 (g2+3) or the single guide targeting exon 5 (g1). Equal numbers of 2- to 4-cell stage embryos targeting exons 2 and 5 were surgically transfered to the oviduct of recipient sows. Fetuses were collected 22-24 days post embryo transfer and genotyped for mutation at the target loci by sequencing. A total of 108 embryos electroporated with g2+3 and 87 with g1 were transfered and resulted in the recovery of and 18 fetuses, seven of which were mutated in exon 2 (g2+3) and 7 were mutated in exon 5. Partial deletion of exon 2 or exon 5 was compatible with fetal viability until at least 24 days. One pregnancy with androgen receptor edited embryos is currently underway and slated to go to term for the birth of live gene edited pigs. Objective 3: Assessment of the fidelity of the gene editing tools used Aim 3.1: Screen most likely off-target sites by PCR. Potential off-target cutting sites in the pig genome were analyzed using three different open source software programs, CRISPOR, CCTOP and CASOFFinder, to identify the top 20 probable off-target sites for g2 and g3 and for a badly designed guide (g6) in exon 2. The number of putative off-target sites ranged from 91-894 for g2, 143-1,138 for g3 and 2,789-5,052 for g6 across the three programs. For each guide, up to 15 sites present in all three approaches with a high score for cutting probability in intronic/exon regions were selected for analysis along with 5 sites unique to CRISPOR and CASOFFinder and one for CCTOP. Primers were designed for 24 potential off-target sites for g2 and 25 for g3. After optimizing all primer pairs using the DNA of two different wild-type pigs, each site was amplified in three different animals and the amplicons from 2 of the three animals sent for Sanger sequencing to confirm presence of the putative guide binding site. If the sequence was inconclusive or indicated a potential single nucleotide polymorphism (SNP), the third sample was sequenced. Three sites for g2 and five sites for g3 either had numerous mismatches or confirmed SNPs and were excluded from in vitro testing. The in vitro test consisted of incubating each potential off-target amplicon with the guide and Cas9 protein followed by gel electrophoresis to determine if the guide could indeed cut the predicted off-target sites. Five of the 20 sites tested for g2 were cut and four of 20 sites for g3 were cut indicating that these guides can cut elsewhere in the genome. Aim 3.3: Whole genome sequencing of parthenote-derived blastocysts. To optimize sequencing protocols to detect off-target events genome-wide, work is being carried out on genomic DNA. We will use the CHANGE-Seq technique to observe genome-wide off-target events. Reagents have been obtained and work is underway.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2020
Citation:
Kelly A. Zacanti, Insung Park, Joan D. Rowe, Bret R. McNabb, Pablo J. Ross, Elizabeth A. Maga and Trish J. Berger. Pig Androgen Receptor Knockout Fetuses via CRISPR/Cas9 Technology. Abstract #2208 and virtual poster presentation at the 2020 SSR annual meeting.
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Progress 09/01/18 to 08/31/19
Outputs
Target Audience:Scientists and researchers working in the area of biotechnology risk assessment. Federal regulators. Graduate students being trained in the area of gene editing. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Two graduate students were mentored on both embryo production and manipulation as well as on the analysis of gene editing events. One undergraduate student assited with embryo collection. How have the results been disseminated to communities of interest?Work was presented at the annual BRAG PD meeting. What do you plan to do during the next reporting period to accomplish the goals?More embryo transfers will be carried out with zygotes microinjected with guide 2 to complete Aim 1.2 with the generation of live-born gene edited pigs. Once animals are born, characterization of them will begin (Aims 2.1 and 2.2). Work on detecting off-target events will continue.
Impacts
What was accomplished under these goals?
Progress has been made toward both Objectives 1 and 3. Aim 1.1 has been completed with the evaluation of three candidate guide RNAs specific to the pig androgen receptor gene. Two guides targeting exon 1 of the gene and one guide targeting exon 4 were assessed in parthenotes. Combinations of the guides were also assessed. Guides and Cas9 protein (1:2 ratio) were microinjected or electroporated into porcine zygotes and mutation rates determined by DNA sequencing after development to the blastocyst stage. Mutation rate at the desired site ranged from 57-100% for individual guides. One guide for exon 1 (guide 2) resulted in a 93% rate of biallelic mutations and was chosen for further use. Aim 1.2: One embryo transfer was carried out with microinjected zygotes using the chosen guide, but the sow did not become pregnant. Aim 3.1: A list of the top 20 off-target cleavage sites has been compiled and primers designed. Work is ongoing to analyze the blastocysts generated in Aim 1.1 for off-target cleavage at these sites.
Publications
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