Generation of Gene Targeted Knockout Pig Fetuses and Evaluation of Off-Target Activity
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Generation of Gene Targeted Knockout Pig Fetuses and Evaluation of Off-Target Activity


The use of genome engineering to manipulate biological systems and organisms has enabled a broad range of research for applications in basic science, medicine, biotechnology, and agriculture. The following research used CRISPR/Cas9 technology to generate pigs with nonfunctional genes of interest. Clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 is an efficient, RNA-guided endonuclease technology that specifically targets and mutates a selected gene of interest. Androgen receptor (AR) is a gene of interest targeted for mutation in this research. Androgens are steroid hormones that regulate the development and maintenance of the male reproductive system via androgen receptors. This research aims to create AR knockout pigs to render male pigs sterile and be a novel approach to genetic containment, a major concern of genetically modified animals. Although containment looks different depending on the species, effective containment strategies are necessary to ensure modified genes are not introduced into free-ranging animals. When using CRISPR/Cas9 technology for genome engineering, off-target mutations are a challenge. Off-target activity could lead to mutations at unintended sites, potentially resulting in loss of gene function and unintended phenotypes. High frequency of off-target activity for RNA-guided endonuclease (RGEN)-induced mutations has been reported; however, reported off-target detection used in silico and in vitro methods, which cannot precisely predict mutations that occur in vivo. This research aims to generate steroid 5 alpha reductase 2 (SRD5A2) and JUNO knockout pigs to evaluate gRNA/Cas9 off-target activity in two unrelated genes of interest, as well as further understand the mechanism behind boar taint and the mechanism behind mammalian fertilization. SRD5A2 is the enzyme that synthesizes a compound responsible for boar taint, a stale urine and fecal odor and flavor in meat from boars. Animal welfare concerns surrounding castration, the current method to remove boar taint, are the driving force to develop practical methods to eliminate boar taint. Juno, an oocyte plasma membrane protein, and Izumo1, a sperm cell surface protein, have been identified as the first cell-surface receptor pair essential for fertilization in the mouse. While Juno has been shown to be conserved across multiple species, conservation of its function has not been confirmed. For each gene of interest, guide RNAs (gRNAs) were designed and tested for mutation efficiency in pig blastocysts. Once confirmed as efficient, gRNA or a combination of gRNAs and Cas9 protein were introduced into in vitro fertilized (IVF) zygotes via electroporation. Electroporated embryos were then transferred via embryo transfer into recipient sows. Twenty-two- to 24-day old knockout fetuses were collected for all three genes of interest, demonstrating fetal viability to 24 days. For the AR locus, guide RNAs targeting exon 2 and exon 5 were compared. Although proportion of conceptuses collected as fetuses did not differ for conceptuses with edits at exon 2 compared with edits at exon 5 in co-transfer experiments, survival tended to be higher for conceptuses with edits at exon 2 (P=0.07). All AR knockout fetuses obtained, were determined to be female (XX), which was significantly different from an expected 50:50 ratio (P<0.001). Blastocyst sexing was also evaluated for AR knockouts, showing no significant difference when compared to wild type (WT) frequencies. For SRD5A2 and JUNO 22- to 24-day knockout fetuses, potential off-target sites were identified for each gRNA and evaluated via Sanger sequencing for off-target activity. All potential off-target sites demonstrated no off-target activity in both knockout fetuses and knockout blastocysts. These studies demonstrated fetal viability to 24 days for conceptuses with edits to three genes of interest. The gRNAs used to target two genes of interest also demonstrated no off-target activity at multiple predicted off-target sites.

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