LincRNA#1 knockout alone does not affect polled phenotype in cattle heterozygous for the celtic POLLED allele

Abstract

A long intergenic non-coding RNA (lincRNA#1) is overexpressed in the horn bud region of polled (hornless) bovine fetuses, suggesting a potential role in horn bud suppression. Genome editing was used to test whether the absence of this sequence was associated with the horned phenotype. Two gRNAs with high mutation efficiencies targeting the 5' and the 3' regions flanking the lincRNA#1 sequence were co-injected with Cas9 as ribonucleoprotein complexes into bovine zygotes (n = 121) 6 h post insemination. Of the resulting blastocysts (n = 31), 84% had the expected 3.7 kb deletion; of these embryos with the 3.7 kb deletions, 88% were biallelic knockouts. Thirty-nine presumptive edited 7-day blastocysts were transferred to 13 synchronized recipient cows resulting in ten pregnancies, five with embryos heterozygous for the dominant P_C POLLED allele at the POLLED locus, and five with the recessive pp genotype. Eight (80%) of the resulting fetuses were biallelic lincRNA#1 knockouts, with the remaining two being mosaic. RT-qPCR analysis was used to confirm the absence of lincRNA#1 expression in knockout fetuses. Phenotypic and histological analysis of the genotypically (P_Cp) POLLED, lincRNA#1 knockout fetuses revealed similar morphology to non-edited, control polled fetuses, indicating the absence of lincRNA#1 alone does not result in a horned phenotype.

Figure 1

Comparison of uninjected and microinjected zygote development rates and mutation efficiencies. Zygotes were microinjected with gRNA/Cas9 ribonucleoproteins targeting the 5′ and 3′ regions surrounding lincRNA#1 at 6 h post insemination. Blastocyst development rate of uninjected control (green) and microinjected embryos when targeting the (a) 5′ and (b) 3′ regions surrounding lincRNA#1. Percentage of Cas9-induced mutations in blastocysts when injected with ribonucleoproteins targeting the (c) 5′ and (d) 3′ regions surrounding lincRNA#1. Error bars = SEM. **P < 0.01.

Figure 2

Comparison of uninjected and microinjected zygote development rates and mutation and knockout efficiencies. Zygotes were microinjected with Cas9 protein and either linc 5′g1 and linc 3′g1 (Co1), or linc 5′g2 and linc 3′g1 (Co2), at 6 h post insemination. (a) Percentage of uninjected control (green) and microinjected zygotes that reached the blastocyst stage of development. (b) Mutation rates and (c) knockout rates in embryos injected with gRNA/Cas9 ribonucleoproteins for Co1 (blue) and Co2 (yellow) injection groups. Blastocysts were classified as mutated if a mutation occurred in at least one target site. (d) Type of lincRNA#1 knockout (%) in injected embryos. Bi = biallelic (aqua); Mosaic (purple). Error bars = SEM. *P < 0.05.

Figure 3

Fetal genotypic analysis from embryo transfers (ETs) and their corresponding recipients (recip.). Gels visualizing the genotypes of fetuses from (a) ET1 and (b) ET2. DNA was extracted from fetal and recipient tissue and PCR amplified. Gel electrophoresis was done to visualize the lincRNA#1 targeted deletion. lincRNA#1 amplicon is 4189 bp and expected knockout size is 456 bp. Recipient DNA follows the respective fetuses they carried.

Figure 4

Phenotypic analysis of horn bud development in edited P_Cp fetuses and age-matched unedited horned (pp) and polled (P_Cp) controls. Fetuses were harvested at 90 days of gestation. Black arrow indicates cranial indent indicative of horn bud development. Edited fetuses were #mosaic or *biallelic lincRNA#1 knockout.

Figure 5

Histological analysis of a representative fetus harvested from embryo transfer (ET) 2 alongside horned and polled controls. (a–c) Fontal skin and (d–f) horn bud region of age matched horned and polled control fetuses alongside fetus 2, a representative biallelic lincRNA#1 knockout fetus from ET2, at 90 days of gestation. Multiple layers of vacuolated keratinocytes and nerve bundles (black stars) can be seen in the horn bud region of the horned control fetus, and hair follicles can be seen in the frontal skin of all fetuses as well as the horn bud regions of the polled control and the knockout fetus (yellow arrows).

Figure 6

Relative expression of lincRNA#1 and OLIG1 in the horn bud region of lincRNA#1 knockout fetuses (KO F1-5), and one horned (HCL1) and two polled (PCL1 & PCL2) age matched controls. lincRNA#1 (black) and OLIG1 (salmon) were normalized relative to three reference genes (GAPDH, RPLP0 and HPRT1). lincRNA#1 KO genotypes are as follows: mosaic KO (F1), biallelic KO (F2), mosaic KO (F3), biallelic KO (F4) and biallelic KO (F5).