Efficient CRISPR/Cas9-mediated biallelic gene disruption and site-specific knockin after rapid selection of highly active sgRNAs in pigs

www.nature.com/scientificreports OPEN received: 11 February 2015 accepted: 22 July 2015 Published: 21 August 2015 Efficient CRISPR/Cas9-mediated biallelic gene disruption and site-specific knockin after rapid selection of highly active sgRNAs in pigs Xianlong Wang1,*, Jinwei Zhou2,*, Chunwei Cao1,*, Jiaojiao Huang1,3, Tang Hai1, Yanfang Wang4, Qiantao Zheng1,3, Hongyong Zhang1,3, Guosong Qin1, Xiangnan Miao1, Hongmei Wang1,3, Suizhong Cao2, Qi Zhou1,3 & Jianguo Zhao1,3 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 genotyping 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. 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 1 State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China. 2College of Veterinary Medicine, Sichuan Agriculture University, Ya’an, Sichuan 625014, China. 3University of Chinese Academy of Sciences, Beijing 100049, China. 4Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing 100193, China. *These authors contributed equally to this work. Correspondence and requests for materials should be addressed to S.C. (email: ) or Q.Z. (email: qzhou@ ioz.ac.cn) or J.Z. (email: ) Scientific Reports | 5:13348 | DOI: 10.1038/srep13348 1 www.nature.com/scientificreports/ 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 pigs10–16. 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 livestock25–28. 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 locus29–31. 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 genotyping. 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), whic (...truncated)