Abstract
Genetic engineering in livestock was greatly enhanced by the emergence of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9), which can be programmed with a single-guide RNA (sgRNA) to generate site-specific DNA breaks. However, the uncertainties caused by wide variations in sgRNA activity impede the utility of this system in generating genetically modified pigs. Here, we described a single blastocyst genoty** system to provide a simple and rapid solution to evaluate and compare the sgRNA efficiency at inducing indel mutations for a given gene locus. Assessment of sgRNA mutagenesis efficiencies can be achieved within 10 days from the design of the sgRNA. The most effective sgRNA selected by this system was successfully used to induce site-specific insertion through homology-directed repair at a frequency exceeding 13%. Additionally, the highly efficient gene deletion via the selected sgRNA was confirmed in pig fibroblast cells, which could serve as donor cells for somatic cell nuclear transfer. We further showed that direct cytoplasmic injection of Cas9 mRNA and the favorable sgRNA into zygotes could generate biallelic knockout piglets with an efficiency of up to 100%. Thus, our method considerably reduces the uncertainties and expands the practical possibilities of CRISPR/Cas9-mediated genome engineering in pigs.
Similar content being viewed by others
Introduction
Pigs are an important source of food and nutrition in humans and are widely used to study a variety of human diseases. The efficient and precise genetic modification of pigs would facilitate the generation of tailored disease models and strains with valuable agricultural traits1,2. However, despite the large number of available techniques, such as pronuclear injection3, sperm-mediated transfection4,5, oocyte transduction6 and intracytoplasmic sperm injection (ICSI)-mediated transgenesis7, the generation of a genetically engineered pig by homologous recombination remains a relatively time-consuming procedure. Somatic cell nuclear transfer (SCNT) has facilitated the ability to make genome modified pigs by circumventing most of the shortcomings of above techniques. However, the SCNT has low efficiency and has been hampered by establishment of cell lines with the desired genetic modification due to a lack of available germ line-competent pluripotent stem cells8,9. Several genome-engineering techniques have been developed for guiding nucleases to induce site-specific double-strand breaks (DSBs) in the genome, making it possible to efficiently generate genetically modified pigs10,11,12,13,14,15,16.
The recently developed Type II bacterial clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system has been recently developed and adapted to genome editing10,11. This system requires a 20-nucleotide guide sequence contained within an associated CRISPR RNA (crRNA) transcript, a trans-activating crRNA (tracrRNA) partially complementary to the crRNA and a Cas endonuclease to catalyze DNA cleavage17. The Cas9 endonuclease from the Streptococcus pyogenes type II CRISPR/Cas system can be engineered to produce targeted genome modifications in a sequence-specific manner by providing a synthetic single-guide RNA (sgRNA) consisting of a fusion of crRNA and tracrRNA18. This CRISPR/Cas9 system has been successfully adapted to generate genetically modified animals, including mice19, rats20, zebrafish21, frogs22, fruit flies23, monkeys24 and livestock25,26,27,28.
Recently, the CRISPR/Cas9 system was demonstrated to efficiently generate biallelic knockout pigs through a direct cytoplasmic injection of Cas9 mRNA and sgRNA into pig zygotes25. This indicated that the CRISPR/Cas9 system shows potential in complex pig genome engineering. However, given the lengthy gestation period and the high cost of housing, it is a challenge in pigs to confirm the presence of the indel mutation in the target sequence of modified pig genomes using chromatin samples from fetuses or newborn piglets after the completion of an actual experiment. Moreover, intensive labor and numerous sows are required to obtain a sufficient number of in vivo-derived zygotes. Therefore, an optimized CRISPR/Cas9-based genome engineering pig system can maximize the efficiency of genetic modifications.
Although sgRNA activity can be quite high, there is significant variability among sgRNAs in their ability to produce null alleles and sgRNA targeting efficiency varies significantly between loci and even between target sites within the same locus29,30,31. For precise genetic modification (knockin or base substitution), the targeting efficiency of the sgRNA is the most critical factor than general gene deletion32. Thus, selecting the most effective sgRNAs for a particular gene locus would greatly expand the utility of a porcine CRISPR/Cas9 system. In the present study, we rapidly estimated the sgRNA efficiency at inducing indel mutations by single blastocyst genoty**. Then, the most favorable sgRNA was verified by mediating knock-in in embryos and generating knockout pigs. Our method considerably reduces the uncertainties and expands the practical possibilities of genome engineering in livestock.
Results
Design and construction of CRISPR
MITF protein is a master regulator of melanocyte development and an important oncogene in melanoma33. Mutations in the human mitf gene have been found in patients with the hypopigmentation and deafness syndromes, Waardenburg (WS) and Tietz (TS)34. Recently, numerous pig models of human diseases have been developed using gene targeting approach owing to pig sharing more physiological similarities with humans. It prompts us to generate mitf genes knockout pigs to model human WS and TS syndromes. We designed four different sgRNAs (F1, F2, R1 and R2) that target 47 bp regions of exon 8 of the pig mitf gene (Fig. 1A), which is a part of the basic helix-loop-helix leucine zipper (bHLH-Zip) domain sequence and is essential for MITF DNA-binding activity35. The sgRNA target sequence (20 nt) did not cross-react with any other sites in the pig genome and was followed by an NGG protospacer adjacent motif (PAM), which is necessary for Cas9 cleavage.
Cas9 mRNA was generated by the in vitro transcription of a linearized and T7 promoter-driven pXT7-hCas9 plasmid template, which included a human-codon-optimized version of Cas9 cDNA and nuclear localization signals (NLSs) at both ends of Cas9 (Fig. 1B). The T7-sgRNA PCR products for the in vitro transcription of sgRNA were obtained as described in the Material & Methods section. Only good-quality, purified sgRNAs and Cas9 mRNA (as assessed by gel electrophoresis) were used for oocyte injections (Fig. 1B).
Rapidly selecting the most effective sgRNAs by single blastocyst genoty**
The lack of a simple platform to unbiasedly evaluate the efficacies of sgRNA creates uncertainties and restricts the ability to modify the pig genome. Toward this end, we developed an experimental system to rapidly select the most favorable sgRNA for a specific gene locus based on single-blastocyst genoty**. An overview of the experimental process with approximate timings is shown in Fig. 2A.
To compare the mutagenesis efficiencies of different sgRNAs, approximately 2–10 pL of RNA mixture (containing 125 ng/μL of Cas9 mRNA and 12.5 ng/μL of individual sgRNA) were microinjected into the cytoplasm of mature MII pig oocytes. After parthenogenetic activation, oocytes were cultured to the blastocyst stage. The in vitro blastocyst rate of oocytes injected with Cas9 mRNA/sgRNA (F1, 27.68%; F2, 25.99%; R1, 26.61%; R2, 25.44%; respectively) and oocytes injected with water (28.16%) were normal and comparable with each other (Table 1), suggesting that the Cas9 mRNA and sgRNA had low or no toxicity for early pig embryonic development. A single blastocyst was randomly selected and lysed for genoty** analysis.
PCR products, including the target site, were amplified and analyzed by restriction fragment length polymorphism (RFLP) for identification of the mutations (Fig. 2B). A failure of the restriction enzyme digestion suggested the occurrence of DNA sequence mutations in the target regions. Some of the non-digested PCR products were sequenced and aligned to reference sequences, which confirmed that the losses of the respective restriction sites were due to mutations at the target sites (Fig. 2B).
The RFLP analysis showed that all four sgRNAs could induce indel mutations in the target region but with different mutagenesis efficiencies. As summarized in Table 1, the sgRNAs generated the mutant embryos at approximately 50–80% efficiencies. Among the tested sgRNAs, the R1 sgRNA produced approximately 12% monoallelic and 69% biallelic mutant embryos, suggesting that R1 was the most favorable sgRNA for the CRISPR/Cas9 system in this target region.
Highly efficient R1 sgRNA-mediated knock-in in porcine embryo
The DSBs mediated by CRISPR/Cas9 can stimulate a homologous recombination in the presence of a DNA donor with the appropriate homology arms. Recent works have demonstrated that single-stranded DNA oligonucleotides (ssODNs) can be used as substitutes for conventional plasmid-based targeting vectors as donor templates for homology-directed repair (HDR)36. Because of its high mutagenesis efficiency, we hypothesized that R1 sgRNA could assist in HDR. Hence, we co-injected Cas9 mRNA, sgRNA and ssODN containing 6 bp KpnI restriction site flanked by 26 bps homologous sequences on each side into mature MII pig oocytes (Fig. 3A). After parthenogenetic activation, oocytes were cultured to the blastocyst stage and single blastocysts were picked for genoty**. In these experiments, the RFLP assays, as shown in Fig. 3B, identified 3 out of 23 R1 sgRNA-injected blastocysts carrying the KpnI site at the target locus, indicating R1 sgRNA yielded an HDR efficiency as high as 13.04% with ssODN at a concentration of 80 ng/μL. Subsequent sequence analyses indicated two precise KpnI site insertions, which demonstrated successful targeted restriction site insertions by R1 sgRNA-mediated HDR in pig embryos (Fig. 3B). Another insertion showed a precise addition at the 3′ end, whereas 117 bps indels were noted at the 5′ side of the modification site (Fig. 3C). In addition, we failed to detect HDR induced by F2 sgRNA or R1 sgRNA with 10 ng/μL ssODN (Table 2), suggesting that CRISPR/Cas9 and ssODN-mediated HDR were highly dependent on the targeting efficiency and the ssODN concentration.
Highly efficient R1 sgRNA-mediated gene targeting in porcine fibroblasts
The dominant strategy for generating transgenic pigs is to first genetically modify fibroblasts and then conduct SCNT. To determine whether the selected sgRNA by mRNA injection into PA-derived blastocysts can induce highly efficient mutations in somatic cells, we investigated the mutagenesis efficiencies of different sgRNAs in porcine primary fibroblasts. The plasmids expressing Cas9 and R1 or R2 sgRNA were transfected into fibroblasts and transfected single cells were sorted and cultured in 96-well plates. Of 17 colonies obtained by R1 sgRNA transfections, eight carried mutations in the target sequence and seven had biallelic mutations. However, of the 10 colonies obtained by R2 sgRNA transfection, only two colonies carried mutations in the target sequence and none had biallelic mutations (Fig. 4). The results (summarized in Table 3) demonstrated that the mutagenesis efficiencies of sgRNAs in fibroblasts were comparative with those in single blastocyst assays, suggesting that the mutagenesis efficiencies of a given sgRNA were consistent in both embryo and somatic cells.
Generation of Mitf knockout pigs by zygote injection of R1 sgRNA and Cas9 mRNA
The ultimate aim of this study was to efficiently generate genetically modified pigs through the direct cytoplasmic injections of Cas9 mRNA and sgRNA into zygotes. Thus, we next transferred the Cas9 mRNA and R1 sgRNA-injected zygotes into surrogate pigs to produce piglets. A total of 40 embryos were delivered to 3 surrogates and one pregnancy was established (Table 4). Two live-born piglets were obtained and showed the white coat-color phenotype over its entire body (Fig. 5A); the wild-type pigs exhibited pigment deposition at the two ends of the body (Fig. 5A). RFLP (Fig. 5B) and sequence analysis (Fig. 5C) assays showed that the piglets were all genotyped as bi-allelic mutations, suggesting that R1 sgRNA could efficiently facilitate the CRISPR/Cas9 system to generate Mitf knockout pigs. The skin tissues of the tail were dissected from the mutant piglets and Western blot analysis was performed to confirm the disruption of MITF in these pigs. Compared with wild-type piglet, MITF was completely absent in the two mutant piglets (Fig. 5D). Moreover, the genomic DNA isolated from the two mutant piglets were used to perform off-target analyses. The fragments around the potential off-target loci were amplified and sequenced. No unwanted mutations occurred at these genomic sites of the two mutant piglets (Supplemental Table S2 and Figure S1).
References
Prather, R. S. Pig genomics for biomedicine. Nat Biotechnol 31, 122–124 (2013).
Prather, R. S., Lorson, M., Ross, J. W., Whyte, J. J. & Walters, E. Genetically engineered pig models for human diseases. Annu Rev Anim Biosci 1, 203–219 (2013).
Uchida, M. et al. Production of transgenic miniature pigs by pronuclear microinjection. Transgenic Res 10, 577–582 (2001).
Lavitrano, M. et al. Efficient production by sperm-mediated gene transfer of human decay accelerating factor (hDAF) transgenic pigs for xenotransplantation. Proc Natl Acad Sci USA 99, 14230–14235 (2002).
Lavitrano, M. et al. Sperm-mediated gene transfer: production of pigs transgenic for a human regulator of complement activation. Transplant Proc 29, 3508–3509 (1997).
Cabot, R. A. et al. Transgenic pigs produced using in vitro matured oocytes infected with a retroviral vector. Anim Biotechnol 12, 205–214 (2001).
Pereyra-Bonnet, F. et al. A unique method to produce transgenic embryos in ovine, porcine, feline, bovine and equine species. Reprod Fertil Dev 20, 741–749 (2008).
Brevini, T. A., Antonini, S., Cillo, F., Crestan, M. & Gandolfi, F. Porcine embryonic stem cells: Facts, challenges and hopes. Theriogenology 68 Suppl 1, S206–213 (2007).
Keefer, C. L., Pant, D., Blomberg, L. & Talbot, N. C. Challenges and prospects for the establishment of embryonic stem cell lines of domesticated ungulates. Anim Reprod Sci 98, 147–168 (2007).
Cong, L. et al. Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819–823 (2013).
Mali, P. et al. RNA-guided human genome engineering via Cas9. Science 339, 823–826 (2013).
Miller, J. C. et al. An improved zinc-finger nuclease architecture for highly specific genome editing. Nat Biotechnol 25, 778–785 (2007).
Miller, J. C. et al. A TALE nuclease architecture for efficient genome editing. Nat Biotechnol 29, 143–148 (2011).
Moscou, M. J. & Bogdanove, A. J. A simple cipher governs DNA recognition by TAL effectors. Science 326, 1501 (2009).
Smith, J. et al. A combinatorial approach to create artificial homing endonucleases cleaving chosen sequences. Nucleic Acids Res 34, e149 (2006).
Urnov, F. D. et al. Highly efficient endogenous human gene correction using designed zinc-finger nucleases. Nature 435, 646–651 (2005).
Sapranauskas, R. et al. The Streptococcus thermophilus CRISPR/Cas system provides immunity in Escherichia coli. Nucleic Acids Res 39, 9275–9282 (2011).
**ek, M. et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816–821 (2012).
Wang, H. et al. One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering. Cell 153, 910–918 (2013).
Li, W., Teng, F., Li, T. & Zhou, Q. Simultaneous generation and germline transmission of multiple gene mutations in rat using CRISPR-Cas systems. Nat Biotechnol 31, 684–686 (2013).
Jao, L. E., Wente, S. R. & Chen, W. Efficient multiplex biallelic zebrafish genome editing using a CRISPR nuclease system. Proc Natl Acad Sci USA 110, 13904–13909 (2013).
Nakayama, T. et al. Simple and efficient CRISPR/Cas9-mediated targeted mutagenesis in Xenopus tropicalis. Genesis 51, 835–843 (2013).
Yu, Z. et al. Highly efficient genome modifications mediated by CRISPR/Cas9 in Drosophila. Genetics 195, 289–291 (2013).
Niu, Y. et al. Generation of gene-modified cynomolgus monkey via Cas9/RNA-mediated gene targeting in one-cell embryos. Cell 156, 836–843 (2014).
Hai, T., Teng, F., Guo, R., Li, W. & Zhou, Q. One-step generation of knockout pigs by zygote injection of CRISPR/Cas system. Cell Res 24, 372–375 (2014).
Ni, W. et al. Efficient gene knockout in goats using CRISPR/Cas9 system. PloS one 9, e106718 (2014).
Whitworth, K. M. et al. Use of the CRISPR/Cas9 system to produce genetically engineered pigs from in vitro-derived oocytes and embryos. Biol Reprod 91, 78 (2014).
Zhou, X. et al. Generation of CRISPR/Cas9-mediated gene-targeted pigs via somatic cell nuclear transfer. Cell Mol Life Sci 72, 1175–1184 (2015).
Doench, J. G. et al. Rational design of highly active sgRNAs for CRISPR-Cas9-mediated gene inactivation. Nat Biotechnol 32, 1262–1267 (2014).
Fu, Y., Sander, J. D., Reyon, D., Cascio, V. M. & Joung, J. K. Improving CRISPR-Cas nuclease specificity using truncated guide RNAs. Nat Biotechnol 32, 279–284 (2014).
Koike-Yusa, H., Li, Y., Tan, E. P., Velasco-Herrera Mdel, C. & Yusa, K. Genome-wide recessive genetic screening in mammalian cells with a lentiviral CRISPR-guide RNA library. Nat Biotechnol 32, 267–273 (2014).
Port, F., Chen, H. M., Lee, T. & Bullock, S. L. Optimized CRISPR/Cas tools for efficient germline and somatic genome engineering in Drosophila. Proc Natl Acad Sci USA 111, E2967–2976 (2014).
Levy, C., Khaled, M. & Fisher, D. E. MITF: master regulator of melanocyte development and melanoma oncogene. Trends Mol Med 12, 406–414 (2006).
**ault, V. et al. Review and update of mutations causing Waardenburg syndrome. Hum Mutat 31, 391–406 (2010).
Grill, C. et al. MITF mutations associated with pigment deficiency syndromes and melanoma have different effects on protein function. Hum Mol Genet 22, 4357–4367 (2013).
Inui, M. et al. Rapid generation of mouse models with defined point mutations by the CRISPR/Cas9 system. Sci Rep 4, 5396 (2014).
Kuscu, C., Arslan, S., Singh, R., Thorpe, J. & Adli, M. Genome-wide analysis reveals characteristics of off-target sites bound by the Cas9 endonuclease. Nat Biotechnol 32, 677–683 (2014).
Wu, X. et al. Genome-wide binding of the CRISPR endonuclease Cas9 in mammalian cells. Nat Biotechnol 32, 670–676 (2014).
Sakurai, T., Watanabe, S., Kamiyoshi, A., Sato, M. & Shindo, T. A single blastocyst assay optimized for detecting CRISPR/Cas9 system-induced indel mutations in mice. BMC Biotechnol 14, 69 (2014).
Zhang, J. H. et al. Improving the specificity and efficacy of CRISPR/CAS9 and gRNA through target specific DNA reporter. J Biotechnol 189C, 1–8 (2014).
Zhou, J. et al. Dual sgRNAs facilitate CRISPR/Cas9-mediated mouse genome targeting. FEBS J 281, 1717–1725 (2014).
Cui, X. S., Li, X. Y. & Kim, N. H. Global gene transcription patterns in in vitro-cultured fertilized embryos and diploid and haploid murine parthenotes. Biochem Biophys Res Commun 352, 709–715 (2007).
Deshmukh, R. S. et al. DNA methylation in porcine preimplantation embryos developed in vivo and produced by in vitro fertilization, parthenogenetic activation and somatic cell nuclear transfer. Epigenetics 6, 177–187 (2011).
Kono, T. et al. Birth of parthenogenetic mice that can develop to adulthood. Nature 428, 860–864 (2004).
Wang, S., Sengel, C., Emerson, M. M. & Cepko, C. L. A gene regulatory network controls the binary fate decision of rod and bipolar cells in the vertebrate retina. Dev Cell 30, 513–527 (2014).
Yao, J. et al. Efficient bi-allelic gene knockout and site-specific knock-in mediated by TALENs in pigs. Sci Rep 4, 6926 (2014).
Acknowledgements
The authors thank all members of Zhao’s lab for their help in this project. This study was supported by the National Basic Research Program of China (2011CBA0100, 2011CB944100 and 2011BAI15B02), the National High Technology Research and Development Program of China (2012AA020602) and National Natural Science Foundation of China (31172281 and 31272440).
Author information
Authors and Affiliations
Contributions
S.C., Q.Z. and J.G.Z. conceived and designed the study. X.W., J.W.Z., C.C., J.H., T.H., Y.W., Q.T.Z., H.Z., G.Q., X.M. and H.W. performed the study. X.W., J.W.Z., S.C. and J.G.Z. analyzed and prepared the manuscript. All authors reviewed the manuscript.
Ethics declarations
Competing interests
The authors declare no competing financial interests.
Electronic supplementary material
Rights and permissions
This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/
About this article
Cite this article
Wang, X., Zhou, J., Cao, C. et al. Efficient CRISPR/Cas9-mediated biallelic gene disruption and site-specific knockin after rapid selection of highly active sgRNAs in pigs. Sci Rep 5, 13348 (2015). https://doi.org/10.1038/srep13348
Received:
Accepted:
Published:
DOI: https://doi.org/10.1038/srep13348
- Springer Nature Limited
This article is cited by
-
CRISPR-based genome editing of a diurnal rodent, Nile grass rat (Arvicanthis niloticus)
BMC Biology (2024)
-
One-step base editing in multiple genes by direct embryo injection for pig trait improvement
Science China Life Sciences (2022)
-
Use of gene-editing technology to introduce targeted modifications in pigs
Journal of Animal Science and Biotechnology (2018)
-
Cattle with a precise, zygote-mediated deletion safely eliminate the major milk allergen beta-lactoglobulin
Scientific Reports (2018)
-
Mosaicism diminishes the value of pre-implantation embryo biopsies for detecting CRISPR/Cas9 induced mutations in sheep
Transgenic Research (2018)