Frontiers of Agricultural Science and Engineering

One-step generation of myostatin gene knockout sheep via the CRISPR/Cas9 system
One-step generation of myostatin gene knockout sheep via the CRISPR/Cas9 system
Hongbing HAN1, Yonghe MA2, Tao WANG2, Ling LIAN1, Xiuzhi TIAN1, Rui HU1, Shoulong DENG3, Kongpan LI2, Feng WANG1, Ning LI2, Guoshi LIU1, Yaofeng ZHAO2, Zhengxing LIAN1
1. Key Laboratory of Animal Genetics and Breeding of the Ministry of Agriculture, Beijing Key Laboratory for Animal Genetic Improvement, College of Animal Science and Technology, China Agricultural University, Beijing 100193, China
2. State Key Laboratory of Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing 100193, China
3. State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China
lianzhx@cau.edu.cn

The CRISPR/Cas (clustered regularly interspaced short palindromic repeats, CRISPR-associated) adaptive immune system, which was discovered in bacteria and archaea, can specifically degrade invasive viral and plasmid DNA by base pairing between crRNAs (CRISPR RNAs) and the target DNA [ 1, 2]. Recently, the Streptococcus pyogenes type II CRISPR system was shown to be able to perform efficient targeted gene disruption by employing three fundamental components: Cas9 endonuclease, which catalyzes DNA cleavage, and crRNAs and tracrRNAs ( trans-activating crRNAs), which are both crucial for directing Cas9 to target sites and for transforming Cas9 from an inhibited conformation into an active state [ 3, 4, 5]. However, a comparable level of gene targeting can be mediated by a chimeric single-guide RNAs (sgRNAs), which results from the fusion of a crRNA and a tracrRNA, and this system is easier to operate both in vivo and in vitro [ 4]. Compared with the complicated design and assembly of ZFNs (zinc finger nucleases) and TALENs (transcription activator-like effector nucleases), redirecting Cas9 to a new target site requires only the alteration of a gene-specific 20 nt DNA sequence in sgRNAs, which can be synthesized on a large scale [ 6, 7]. RNA-guided Cas9 has recently been demonstrated to be a robust tool for genome engineering in many cell lines and organisms [ 8, 9, 10, 11, 12, 13].

Conventional gene targeting in large domestic animals has commonly been considered an intractable task involving screening for gene-targeted cells, the deletion of selective markers, and somatic cell nuclear transfer (SCNT), and it usually results in abortion or unhealthy newborns due to abnormal epigenetic modifications [ 14, 15]. Recently, Zhou and colleagues reported the first gene-knockout pigs generated using a one-step zygote injection of the CRISPR/Cas9 system, demonstrating a highly promising rapid method to create large domestic gene-knockout animals [ 16]. Here, we report the first successful one-step generation of gene-knockout sheep using the same method.

To test the feasibility of gene targeting in sheep using the CRISPR/Cas9 system, we designed sgRNAs targeting the myostatin ( MSTN) gene (Supplementary information, Table S1). Myostatin is a transforming growth factor-βfamily member that negatively regulates muscle mass. Naturally occurring MSTN mutations in dogs and Belgian Blue cattle have been found to result in similar double-muscled phenotypes [ 17, 18]. The disruption of the MSTN gene in mice has been shown to also cause a pronounced increase in skeletal muscle mass [ 19]. Thus, animal breeding scientists are highly interested in modifying the MSTN gene in large domestic animals, such as pigs and sheep. Further research should be performed to determine whether such genetic modifications could improve meat production in these animals.

We designed sgRNAs targeting the third exon of MSTN (Fig. 1a), resulting in out-of-frame indels (insertions or deletions) predicted to abolish normal MSTN function. To determine the working efficiency of the Cas9 system in vitro, sheep codon-optimized Cas9 and sgRNA expression plasmids were cotransfected into sheep fibroblast cells. Genomic DNA isolated from the cells 48 h after transfection was subjected to PCR amplification and the Surveyor assay to confirm cleavage. The two expected cleavage bands were observed, suggesting that the MSTN gene was mutated in a proportion of the transfected cells. The average gene targeting efficiency was estimated to be 19.3% (Supplementary information, Fig. S1).

Fig.1 Generation of MSTN-knockout sheep and analysis of the mutant alleles. (a) A schematic of the sgRNAs targeting the third exon of the MSTN gene. The PAM motif is shown in green. The target site is underlined, and the 12 bp seed sequence is highlighted in blue. The primers for the PCR analysis are indicated by arrows; (b) Indel mutations in the ear and muscle were detected using the Surveyor assay. Ear and muscle tissues from the control group with a wild-type MSTN gene produced a 556 bp band. Monoallelic mutant sheep produced multiple bands with lengths of 556 bp, 331 bp, and 225 bp; (c) Sequence analysis of the mutations detected in the two lambs. Deletions are indicated by a dashed line, and insertions are shaded in cyan. The numbers following the sequences indicate the specific type of mutation, and the clone numbers are surrounded by brackets

After confirming that the Cas9 system worked in sheep fibroblast cells in vitro, we examined whether it also worked in vivo. Briefly, Cas9 mRNA and the sgRNAs were in vitro transcribed using T7 RNA polymerase (Supplementary information, Table S2). A mixture of the Cas9 mRNA and the sgRNAs was microinjected into 213 embryos in two independent experiments. The injected embryos were immediately transferred into 55 surrogate females, 31 of which became pregnant, suggesting a low toxicity of the Cas9:sgRNA mixture. Between 140 and 152 days after the uterine transfer, 35 lambs were successfully delivered, resulting in a live birth rate of 16.4% (Supplementary information, Table S3). Tissue samples from the hind leg muscles and ears of all the lambs were dissected for MSTN genotyping. We PCR amplified the regions surrounding the target site in the MSTN gene and performed the Surveyor assay to assess cleavage (Supplementary information, Table S4). Apparent cleavage bands were detected in two lambs (one male and one female) (Fig. 1b), suggesting gene disruption by Cas9. To confirm this gene disruption, we cloned the PCR products and randomly selected more than 80 clones derived from each lamb for Sanger sequencing. Consistent with the cleavage assays, five different mutant alleles were found in the two animals, with indels ranging from 0 to -18 bp (Fig. 1c). The female lamb was shown to have the wild-type allele and two mutant alleles, and the male lamb harbored the wild-type allele and five mutant alleles. We thus regarded these two animals as monoallelic mutants. Among the five mutant alleles, three caused out-of-frame mutations that disrupted the coding region; the other two mutant alleles, an in-frame deletion (-18 bp) and a substitution of five nucleotides (0 bp), may have had little effect on the function of MSTN.

Previous reports have indicated that a small number of mismatches between sgRNAs and the complementary target DNA are easily tolerated, resulting in a high frequency of off-target mutagenesis in human cells [ 20, 21]. To determine whether there were off-target mutations in the two lambs, we searched the entire sheep genome. We found six potential off-target loci containing a maximum of five mismatches compared with the specific sgRNAs designed for the MSTN gene (Supplementary information, Table S5). The genomic regions flanking the putative off-target sites were amplified and examined using both Surveyor assays and the direct sequencing of PCR products. The cleavage bands of off-target1 were found to result from nearby SNPs, but no mutations in any intended loci were revealed (Supplementary information, Fig. S2 and Table S5). However, we cannot exclude the possibility that an extremely low level of some off-target mutations beyond the sensitivity of the method we used may have occurred. Taken together, the results indicate that we have successfully generated MSTN genetically modified sheep through the one-step microinjection of a Cas9 RNA:sgRNA mixture into fertilized eggs, although the production efficiency (2/35) of mutant lambs was not as high as those previously reported for other species [ 11, 16].

At least two mutant alleles were identified in each of the two MSTN-knockout lambs. The presence of multiple mutant alleles is a common phenomenon, and it has also been observed in other genetically modified species generated by the microinjection of a Cas9 RNA:sgRNA mixture into zygotes [ 11, 13, 16]. These observations strongly suggest that when using this method, Cas9-mediated double-stranded DNA breaks (DSBs) could occur many times independently after the one-cell embryo stage, leading to multiple modified alleles and, thus, mosaic animals. Considering the time and efficiency issues, a better method to create gene-targeted large animals might be the injection of a preassembled Cas9:sgRNA protein complex directly into the nucleus of a one-cell-stage embryo.

In summary, we have successfully obtained MSTN gene-mutated sheep, demonstrating that the direct injection of Cas9:sgRNA into zygotes can be widely used to create gene knockouts in large domestic animals. This method may greatly facilitate improvements in animal breeding and the application of these animals in biomedical studies.

References
1 Bhaya D, Davison M, Barrangou R. CRISPR-Cas systems in bacteria and archaea: versatile small RNAs for adaptive defense and regulation. Annual Review of Genetics, 2011, 45(1): 273-297. DOI:10.1146/annurev-genet-110410-132430 . PMID:22060043 [Cited within: 1] [JCR: 17.436]
2 Terns M P, Terns R M. CRISPR-based adaptive immune systems. Current Opinion in Microbiology, 2011, 14(3): 321-327. DOI:10.1016/j.mib.2011.03.005 . PMID:21531607 [Cited within: 1] [JCR: 8.23]
3 Gasiunas G, Barrangou R, Horvath P, Siksnys V. Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria. Proceedings of the National Academy of Sciences of the United States of America, 2012, 109(39): E2579-E2586. DOI:10.1073/pnas.120850710. PMID:22949671 [Cited within: 1] [JCR: 9.737]
4 Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna J A, Charpentier E. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science, 2012, 337(6096): 816-821. DOI:10.1126/science.1225829 . PMID:22745249 [Cited within: 1]
5 Jinek M, Jiang F G, Taylor D W, Sternberg S H, Kaya E, Ma E B, Anders C, Hauer M, Zhou KH, Lin S, Kaplan M, Iavarone A T, Charpentier E, Nogales E, Doudna J A. Structures of Cas9 endonucleases reveal RNA-mediated conformational activation. Science, 2014, 343(6176). . DOI:10.1126/science.1247997 (first published online) [Cited within: 1]
6 Deltcheva E, Chylinski K, Sharma C M, Gonzales K, Chao Y, Pirzada Z A, Eckert M R, Vogel J, Charpentier E. CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III. Nature, 2011, 471(7340): 602-607. DOI:10.1038/nature09886 . PMID:21455174 [Cited within: 1] [JCR: 38.597]
7 Deveau H, Garneau J E, Moineau S. CRISPR/Cas system and its role in phage-bacteria interactions. Annual Review of Microbiology, 2010, 64(1): 475-493. DOI:10.1146/annurev.micro.112408.134123 . PMID:20528693 [Cited within: 1] [JCR: 12.9]
8 Mali P, Yang L, Esvelt K M, Aach J, Guell M, DiCarlo J E, Norville J E, Church G M. RNA-guided human genome engineering via Cas9. Science, 2013, 339(6121): 823-826. DOI:10.1126/science.1232033 . PMID:23287722 [Cited within: 1]
9 Cong L, Ran F A, Cox D, Lin S, Barretto R, Habib N, Hsu P D, Wu X, Jiang W, Marraffini L A, Zhang F. Multiplex genome engineering using CRISPR/Cas systems. Science, 2013, 339(6121): 819-823. DOI:10.1126/science.1231143 . PMID:23287718 [Cited within: 1]
10 Hwang W Y, Fu Y, Reyon D, Maeder M L, Tsai S Q, Sand er J D, Peterson R T, Yeh J R, Joung J K. Efficient genome editing in zebrafish using a CRISPR-Cas system. Nature Biotechnology, 2013, 31(3): 227-229. DOI:10.1038/nbt.2501 . PMID:23360964 [Cited within: 1] [JCR: 32.438]
11 Wang H, Yang H, Shivalila C S, Dawlaty M M, Cheng A W, Zhang F, Jaenisch R. One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering. Cell, 2013, 153(4): 910-918. DOI:10.1016/j.cell.2013.04.025 . PMID:23643243 [Cited within: 2] [JCR: 31.957]
12 Li W, Teng F, Li T, Zhou Q. Simultaneous generation and germline transmission of multiple gene mutations in rat using CRISPR-Cas systems. Nature Biotechnology, 2013, 31(8): 684-686. DOI:10.1038/nbt.2652 . PMID:23929337 [Cited within: 1] [JCR: 32.438]
13 Niu Y, Shen B, Cui Y, Chen Y, Wang J, Wang L, Kang Y, Zhao X, Si W, Li W, Xiang A P, Zhou J, Guo X, Bi Y, Si C, Hu B, Dong G, Wang H, Zhou Z, Li T, Tan T, Pu X, Wang F, Ji S, Zhou Q, Huang X, Ji W, Sha J. Generation of gene-modified cynomolgus monkey via Cas9/RNA-mediated gene targeting in one-cell embryos. Cell, 2014, 156(4): 836-843. DOI:10.1016/j.cell.2014.01.027 . PMID:2448610 [Cited within: 2] [JCR: 31.957]
14 Hauschild J, Petersen B, Santiago Y, Queisser A L, Carnwath J W, Lucas-Hahn A, Zhang L, Meng X, Gregory P D, Schwinzer R, Cost G J, Niemann H. Efficient generation of a biallelic knockout in pigs using zinc-finger nucleases. Proceedings of the National Academy of Sciences of the United States of America, 2011, 108(29): 12013-12017. DOI:10.1073/pnas.110642210. PMID:21730124 [Cited within: 1] [JCR: 9.737]
15 Carlson D F, Tan W, Lillico S G, Stverakova D, Proudfoot C, Christian M, Voytas D F, Long C R, Whitelaw C B, Fahrenkrug S C. Efficient TALEN-mediated gene knockout in livestock. Proceedings of the National Academy of Sciences of the United States of America, 2012, 109(43): 17382-17387. DOI:10.1073/pnas.121144610. PMID:23027955 [Cited within: 1] [JCR: 9.737]
16 Hai T, Teng F, Guo R, Li W, Zhou Q. One-step generation of knockout pigs by zygote injection of CRISPR/Cas system. Cell Research, 2014, 24(3): 372-375. DOI:10.1038/cr.2014.11 . PMID:24481528 [Cited within: 3] [JCR: 10.526] [CJCR: 1.1032]
17 Grobet L, Royo Martin L J, Poncelet D, Pirottin D, Brouwers B, Riquet J, Schoeberlein A, Dunner S, Ménissier F, Massaband a J, Fries R, Hanset R, Georges M. A deletion in the bovine myostatin gene causes the double-muscled phenotype in cattle. Nature Genetics, 1997, 17(1): 71-74. DOI:10.1038/ng0997-71 . PMID:928810 [Cited within: 1] [JCR: 35.209]
18 Mosher D S, Quignon P, Bustamante C D, Sutter N B, Mellersh C S, Parker H G, Ostrand er E A. A mutation in the myostatin gene increases muscle mass and enhances racing performance in heterozygote dogs. PLoS Genetics, 2007, 3(5): e79. DOI:10.1371/journal.pgen.0030079 . PMID:17530926 [Cited within: 1] [JCR: 8.517]
19 McPherron A C, Lawler A M, Lee S J. Regulation of skeletal muscle mass in mice by a new TGF-beta superfamily member. Nature, 1997, 387(6628): 83-90. DOI:10.1038/387083a0 . PMID:9139826 [Cited within: 1] [JCR: 38.597]
20 Fu Y, Foden J A, Khayter C, Maeder M L, Reyon D, Joung J K, Sand er J D. High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells. Nature Biotechnology, 2013, 31(9): 822-826. DOI:10.1038/nbt.2623 . PMID:23792628 [Cited within: 1] [JCR: 32.438]
21 Hsu P D, Scott D A, Weinstein J A, Ran F A, Konermann S, Agarwala V, Li Y, Fine E J, Wu X, Shalem O, Cradick T J, Marraffini L A, Bao G, Zhang F. DNA targeting specificity of RNA-guided Cas9 nucleases. Nature Biotechnology, 2013, 31(9): 827-832. DOI:10.1038/nbt.2647 . PMID:23873081 [Cited within: 1] [JCR: 32.438]