Elsevier

Theriogenology

Volume 86, Issue 8, November 2016, Pages 1886-1896.e1
Theriogenology

Research article
Efficient edition of the bovine PRNP prion gene in somatic cells and IVF embryos using the CRISPR/Cas9 system

https://doi.org/10.1016/j.theriogenology.2016.06.010Get rights and content

Abstract

The recently developed engineered nucleases, such as zinc-finger nucleases, transcription activator-like effector nucleases, and clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated nuclease (Cas) 9, provide new opportunities for gene editing in a straightforward manner. However, few reports are available regarding CRISPR application and efficiency in cattle. Here, the CRISPR/Cas9 system was used with the aim of inducing knockout and knock-in alleles of the bovine PRNP gene, responsible for mad cow disease, both in bovine fetal fibroblasts and in IVF embryos. Five single-guide RNAs were designed to target 875 bp of PRNP exon 3, and all five were codelivered with Cas9. The feasibility of inducing homologous recombination (HR) was evaluated with a reporter vector carrying EGFP flanked by 1 kbp PRNP regions (pHRegfp). For somatic cells, plasmids coding for Cas9 and for each of the five single-guide RNAs (pCMVCas9 and pSPgRNAs) were transfected under two different conditions (1X and 2X). For IVF zygotes, cytoplasmic injection was conducted with either plasmids or mRNA. For plasmid injection groups, 1 pg pCMVCas9 + 0.1 pg of each pSPgRNA (DNA2X) was used per zygote. In the case of RNA, two amounts (RNA1X and RNA2X) were compared. To assess the occurrence of HR, a group additionally cotransfected or coinjected with pHRegfp plasmid was included. Somatic cell lysates were analyzed by polymerase chain reaction and surveyor assay. In the case of embryos, the in vitro development and the genotype of blastocysts were evaluated by polymerase chain reaction and sequencing. In somatic cells, 2X transfection resulted in indels and large deletions of the targeted PRNP region. Regarding embryo injection, higher blastocyst rates were obtained for RNA injected groups (46/103 [44.6%] and 55/116 [47.4%] for RNA1X and RNA2X) than for the DNA2X group (26/140 [18.6%], P < 0.05). In 46% (26/56) of the total sequenced blastocysts, specific gene editing was detected. The total number of genetic modifications (29) was higher than the total number of gene-edited embryos, as three blastocysts from the group RNA2X reported more than one type of modification. The modifications included indels (10/56; 17.9%) and large deletions (19/56; 33.9%). Moreover, it was possible to detect HR in 1/8 (12.5%) embryos treated with RNA2X. These results report that the CRISPR/Cas9 system can be applied for site-specific edition of the bovine genome, which could have a great impact on the development of large animals resistant to important zoonotic diseases.

Introduction

Site-specific genetic engineering is a valuable tool for pharmaceutical research, development of biomedical models, and also for accelerated breeding. However, until a few years ago, knockout and knock-in in mammal cells and embryos comprised a complex challenge, especially when applied to large domestic species.

The recent advent of engineered nucleases has enabled the precise modification of genomes of different species, through simple introduction of site-specific double-strand breaks, which can be repaired either by the non-homologous end joining machinery or by homology-directed repair, in the presence of a homologous template [1]. Although the first reports on the use of engineered nucleases for precise genetic engineering of domestic species relied on zinc-finger nucleases [2], [3], [4], [5], [6] and transcription activator-like effector nucleases [7], [8], [9], [10]; more recently, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated nuclease (Cas) 9 emerged as the tool of choice, mainly due to its simple design and construction [11], [12], [13], [14], [15], [16], [17], [18], [19].

Clustered regularly interspaced short palindromic repeat/Cas is a simple and effective tool for genome edition on the basis of the defense mechanism against viruses used by bacteria and archea [20], [21]. The main advantage of CRISPRs is that a single-guide RNA can direct the Cas to the target sequence in the genome by base complementarity, at sites demarcated by conserved sequences called proto-spacer adjacent motifs [22]. To form a functional DNA-targeting complex, Cas9 requires two distinct RNA transcripts: CRISPR RNA and trans-acting CRISPR RNA [22], [23]. Jinek et al. [22] reconfigured this dual RNA as a single-guide RNA (sgRNA), including sequences that are sufficient to program Cas9 to introduce double-stranded breaks in target DNAs of 20 nucleotides. Initial reports with this system were promising [24], [25], and it was rapidly adapted for the genome edition of cells of many different species, including large animals [26], [27]. Soon thereafter, gene-edited pigs and goats were efficiently produced by somatic cell nuclear transfer, using CRISPR/Cas9 edited cells as donors [28], [29], [30], [31]. More recently, a more straightforward approach, consisting on cytoplasmic injection of one-cell embryos, resulted in genome-edited mice, rat, sheep, monkeys, pigs, goats, and rabbits [12], [16], [18], [19], [32], [33], [34]. Efficiency rates obtained so far were variable, ranging from 63% in pigs [14] to 15%–21% in goats [18]. In addition, CRISPR/Cas9 RNA injection in zygotes can result in mosaicism [17], [35], [36], [37].

Despite the potential that the CRISPR technology could have in cattle, only few reports are available so far [26], [38], [39]. Here, we tested the feasibility of inducing genetic modifications on Bos taurus prion gene (PRNP), responsible for mad cow disease via CRISPR/Cas9 application. The PRNP gene encodes the PrPC glycoprotein; however, a misfolded isomer (PrPBSE) of the normal cellular prion protein is accumulated in affected brains [40]. Prion diseases include transmissible spongiform encephalopathies such as Creutzfeldt-Jakob disease in humans, scrapie in sheep, and bovine spongiform encephalopathy in cattle. Although nowadays, the bovine spongiform encephalopathy epidemics is contained through a ban on feeding cattle with ruminant derived bone meal, spontaneous misfolding of the PrPc protein could originate some PrPBSE strains [41], [42], [43]. In mice, PRNP homozygous (−/−) knockout were healthy and resistant to scrapie, and PRNP heterozygous (−/+) mice expressed PrPC at about half of the normal level [44], [45], [46], [47], [48]. In addition, in cattle, PRNP knockdown animals, generated by RNAi [49], [50], and PRNP knockouts, produced by SCNT with donor cell lines subjected to two rounds of traditional cell modifications, were described [51]. However, with inefficiencies of traditional systems, the introgression of PRNP knockout genetics into cattle comprises a significant and costly challenge.

This report takes advantage of the CRISPR–Cas9 system adaptability to specifically modify bovine PRNP coding exon 3 both in bovine fetal fibroblasts and in early embryos. In particular, sgRNAs were designed not only to induce indels, but also to delete 875 bp of exon 3. The feasibility of inducing homologous recombination (HR) was also evaluated. Our results reported that this strategy could be efficiently applied to provoke deletions in bovine cell lines and embryos. However, most embryos were mosaic, and HR of large constructs was achieved at low efficiencies.

Section snippets

Chemicals

Except where otherwise indicated, all chemicals were obtained from Sigma Chemical Company (St. Louis, MO, USA).

Cas9/sgRNA design

Mammalian codon-optimized recombinant human Cas9 under transcriptional control of the CMV promoter pST1374-NLS-flag-linker-rhCas9 (pCMVCas9) was a gift from Xingxu Huang (Addgene plasmid 44758) [52]. The five sgRNAs were designed to target both ends of a 875 bp sequence on PRNP exon 3 (Fig. 1C). All possible sgRNAs (5′-N20NGG-3′) were identified and blasted to detect possible

Evaluation of the CRISPR/Cas9 system for genomic editing of PRNP exon 3 in bovine fetal fibroblasts

To evaluate the efficiency of the CRISPR/Cas9 system to target PRNP exon 3, five sgRNA plasmids (pSPgRNA) were cotransfected with the Cas9 plasmid (pCMVCas9) with or without the plasmid pHRegfp. When pHRegfp was also transfected, high EGFP expression rates were detected (Fig. 2A, B). For the 2X concentration, shorter PCR products were also identified in 10/15 (66.6%) PCRs performed on lysates from two independent transfection events (Fig. 2D). By surveyor assay, it was possible to identify

Discussion

Until recently, specific gene modification was very difficult to achieve in livestock species. Since the introduction of engineered nucleases, a revolution in gene targeting begun. In this report, we found that Bos taurus PRNP gene can be efficiently mutated with the CRISPR system both in somatic cells and in zygotes. The design of five sgRNAs targeting 875 bp on PRNP exon 3, contiguous to sequences homologous to a HR donor vector, is a simple strategy for the simultaneous detection of

Acknowledgments

The authors would like to thank slaughterhouses COCARSA S.A. (San Fernando) and I.F.S.S.A (Loma Hermosa) for providing biological material. In addition, CONICET, DAAD, and Fulbright are gratefully acknowledged.

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