A deletion at the polled PC locus alone is not sufficient to cause a polled phenotype in cattle

Animal care

All experiments carried out utilizing animals were conducted in compliance with the ARRIVE guidelines and approved and completed in compliance with the Institutional Animal Care and Use Committee (IACUC) protocol #20746 at the University of California, Davis. Housing and maintenance of recipient cattle was conducted at the University of California, Davis Beef Barn and Feedlot.

Guide-RNA design and construction

Guide sequences were designed using the online tools sgRNA Scorer 2.0^31,32 and Cas-OFFinder³³ targeting the 5′ and 3′ regions flanking the 10 bp target at the polled locus. Guides were selected with no less than three mismatches in the guide sequence for off-target sites using the UMD3.1.1 bovine reference genome³⁴, and at least one mismatch in the seed region (8–11 bp upstream of the PAM sequence). Oligonucleotides were ordered from Eurofins USA (Louisville, KY) for the top four guides for construction of the gRNAs (two targeting upstream and two targeting downstream of the 10 bp target). In vitro transcription of the oligonucleotides was done using the AmpliScribe T7-Flash Transcription kit (Lucigen, Palo Alto, CA) and purified using the MEGAclear Transcription Clean-Up kit (Thermo Fisher, Chicago, IL) as described by Vilarino et al³⁵. Synthetic guides targeting the same sequences were also ordered from Synthego (Menlo Park, CA) with the option of no modifications being done to the gRNAs. Cleavage efficiency was tested using an in vitro cleavage assay by combining 60 ng of PCR amplified product, 100 ng of gRNA, 150 ng of Cas9 protein (PNA Bio, Inc., Newbury Park, CA), 1 μL of 10X BSA, 1 μL of NEB Buffer 3.1 and water bringing the total volume to 10 μL in a 0.2 μL tube and incubated at 37 °C for 1 h. The incubated product was then run on a 2% agarose gel with 5 μL of SYBR Gold (Invitrogen, Waltham, MA) at 100 V for 1 h and visualized using a ChemiDoc-ItTS2 Imager (UVP, LLC, Upland, CA).

Embryo production

Bovine ovaries were collected from a local processing plant and transported to the laboratory at 35–37 °C in sterile saline. Cumulus-oocyte complexes (COCs) were aspirated from follicles and groups of 50 COCs were transferred to 4-well dishes containing 500 μL of maturation media (BO-IVM, IVF Bioscience, Falmouth, United Kingdom). COCs were incubated for 20–22 h at 38.5 °C in a humidified 5% CO₂ incubator. Approximately 25 oocytes per drop were fertilized in 60 μL drops of SOF-IVF with either 2 × 10⁶ sperm per mL for an incubation period of 6 or 8 h or 1 × 10⁶ sperm per mL for an incubation period of 18 h at 38.5 °C in a humidified 5% CO₂ incubator using the protocol described in Bakhtari et al³⁶. Sperm used was from a known genotypically horned sire. Previous preliminary experiments we conducted revealed the majority of ovaries obtain from the processing plant were from horned cattle, allowing for the creation of genotypically horned embryos. Presumptive zygotes were denuded by light vortex in SOF-HEPES medium³⁶ for 5 min. 25 zygotes per drop were incubated in 50 μL drops of culture media (Bo-IVC, IVF Bioscience, Falmouth, United Kingdom) at 38.5 °C in a humidified atmosphere of 5% CO₂, 5% O₂ and 90% N₂ for 7–8 days from insemination.

Guide-RNA testing

Mutation rates for each guide were determined by laser-assisted cytoplasmic injection³⁷ of in vitro fertilized embryos with 6pL of a solution containing 67 ng/μL of gRNA and 167 ng/μL of Cas9 protein (PNA Bio, Inc., Newbury Park, CA) incubated at room temperature for 30 min prior to injection to allow for the formation of RNP complexes. Injected embryos were incubated for 7–8 days from insemination. Embryos that reached blastocyst stage were lysed in 10 μL of Epicenter DNA extraction buffer (Lucigen, Palo Alto, CA) using a Simpli-Amp Thermal Cycler (Applied Biosystems, Foster City, CA) at 65 °C for 6 min, 98 °C for 2 min and held at 4 °C. The target region was amplified by two rounds of the polymerase chain reaction (PCR) using primers developed with Primer3 (Supplementary Table S6)^38,39. The first round of PCR was performed on a SimpliAmp Thermal Cycler (Applied Biosystems, Foster City, CA) with 10 μL GoTAQ Green Master Mix (Promega Biosciences LLC, San Luis Obispo, CA), 0.4 μL of each primer at 10 mM and 9.2 μL of DNA in lysis buffer for 5 min at 95 °C, 35 cycles of 30 s at 95 °C, 30 s at 62 °C, and 2 min at 72 °C, followed by 5 min at 72 °C. The second round of PCR was run with 10 μL GoTAQ Green Master Mix (Promega Biosciences LLC, San Luis Obispo, CA), 4.2 μL of water, 0.4 μL of each primer at 10 mM and 5 μL of first round PCR for 3 min at 95 °C, 35 cycles of 30 s at 95 °C, 30 s at 60 °C, and 1 min at 72 °C, followed by 5 min at 72 °C. Products were visualized on a 1.3% agarose gel using a ChemiDoc-ItTS2 Imager (UVP, LLC, Upland, CA), purified using the QIAquick Gel Extraction Kit (Qiagen, Valencia, CA), Sanger sequenced (GENEWIZ, LLC, South Plainfield, NJ) and analyzed with CRISP-ID⁴⁰ and Synthego’s ICE analysis. Mutation rates for co-injected in vitro fertilized embryos were determined using the same methods described above—injecting RNP complexes of the most efficient 5′ and 3′ IVT or synthetic gRNAs (67 ng/μL each) and 167 ng/μL of Cas9 protein (PNA Bio, Inc., Newbury Park, CA).

Embryo transfers

Estrus synchronization of recipient cattle began 16 days prior to the embryo transfer with the use of an intravaginal progesterone releasing device (1.38 g; Eazi-Breed CIDR; Zoetis) and the administration of gonadorelin (100 mcg; Factrel; Zoetis) done on day 0. On day 7, the CIDR was removed and prostaglandin (25 mg; Lutalyse; Zoetis) was administered. A second dose of gonadorelin (100 mcg; Factrel; Zoetis) was given on day 9 and recipients were monitored for signs of estrus. Confirmation of recipient synchronization was done on day 15 via corpus luteum detection using a transrectal ultrasound. On day 16, embryo transfers were performed. Recipients received a caudal epidural of 100 mg 2% lidocaine (Xylocaine; Fresenius) prior to embryo transfer. Two to three blastocysts were loaded into 0.25 cc straws and transferred using the non-surgical transcervical technique into the uterine horn ipsilateral to the corpus luteum. On day 35 of gestation, transrectal ultrasonography (5.0 MHz linear probe; EVO Ibex, E.I. Medical Imaging) was done to confirm pregnancies, and reconfirmed on day 80. A total of four embryo transfers were performed, and recipients were resynchronized for subsequent embryo transfers if they did not become pregnant from prior embryo transfers.

Phenotypic and genotypic analysis of fetuses

Recipient cattle were slaughtered between 95 and 151 days of gestation via penetrating captive bolt and subsequent exsanguination. The fetuses were retrieved from the uterine horns, and horn bud phenotyping was performed by the large animal veterinarian onsite. Fetal liver and tail samples were taken for later genotypic analysis, and the frontal skin and horn bud regions were taken for histological analysis. Recipient muscle tissue was also taken for experimental controls. All samples were washed three times in PBS before collection.

To determine fetal genotypes, DNA was extracted from tissue samples using the DNeasy Blood & Tissue Kit (Qiagen, Valencia, CA) and PCR amplified with the 2nd round primers used for gRNA testing. PCR was done using a SimpliAmp Thermal Cycler (Applied Biosystems, Foster City, CA) with 12.5 μL GoTAQ Green Master Mix (Promega Biosciences LLC, San Luis Obispo, CA), 1 μL of each primer at 10 mM, 9.5 μL of water and 100 ng of DNA for 5 min at 95 °C, 35 cycles of 30 s at 95 °C, 30 s at 60 °C, and 1 min at 72 °C, followed by 5 min at 72 °C. Products were visualized on a 1.3% agarose gel using a ChemiDoc-ItTS2 Imager (UVP, LLC, Upland, CA), purified using the QIAquick Gel Extraction Kit (Qiagen, Valencia, CA), Sanger sequenced (GENEWIZ, LLC, South Plainfield, NJ) and analyzed with CRISP-ID [44] and Synthego’s ICE analysis. Fetuses were also tested for the P_F allele using the same PCR protocol with a modified anneal and extension temp for the P_F primers given in Supplementary Table S6. The P_M and P_G alleles were not tested for as they were not applicable based on the breeds of cattle used to produce the fetuses.

Histological analysis of fetuses

Fetal horn bud and frontal skin tissue samples were fixed in 4% paraformaldehyde at 4 °C for 18 h, washed 3 × in phosphate-buffered saline (PBS) on a rocker for 30 min and placed in 70% ethanol. They were subsequently processed in a vacuum infiltration processor (Sakura Tissue-Tek VIP 5, Torrance, CA) where they were dehydrated in a graded ethanol series and cleared with xylene. Samples were then embedded in paraffin blocks and 5 µm microtome sections were cut (Leica RM2255, Leica Biosystems, Buffalo Grove, IL) and stained with hematoxylin and eosin. Digital images were obtained with an Echo Revolve (Discover Echo Inc., San Diego, CA) microscope.

Statistical analysis

Comparison between blastocyst and fetal development, mutation and deletion rates were evaluated using logistic regression models created with the glm “general linear model” function in R with gRNA, gRNA type, and time of injection modeled as fixed effects. Differences were considered significant when P < 0.05.