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• W3134615468 endingPage "100497" @default.
• W3134615468 startingPage "100497" @default.
• W3134615468 abstract "The CRISPR/Cas9 system has been used in a wide range of applications in the production of gene-edited animals and plants. Most efforts to insert genes have relied on homology-directed repair (HDR)-mediated integration, but this strategy remains inefficient for the production of gene-edited livestock, especially monotocous species such as cattle. Although efforts have been made to improve HDR efficiency, other strategies have also been proposed to circumvent these challenges. Here we demonstrate that a homology-mediated end-joining (HMEJ)-based method can be used to create gene-edited cattle that displays precise integration of a functional gene at the ROSA26 locus. We found that the HMEJ-based method increased the knock-in efficiency of reporter genes by eightfold relative to the traditional HDR-based method in bovine fetal fibroblasts. Moreover, we identified the bovine homology of the mouse Rosa26 locus that is an accepted genomic safe harbor and produced three live-born gene-edited cattle with higher rates of pregnancy and birth, compared with previous work. These gene-edited cattle exhibited predictable expression of the functional gene natural resistance-associated macrophage protein-1 (NRAMP1), a metal ion transporter that should and, in our experiments does, increase resistance to bovine tuberculosis, one of the most detrimental zoonotic diseases. This research contributes to the establishment of a safe and efficient genome editing system and provides insights for gene-edited animal breeding. The CRISPR/Cas9 system has been used in a wide range of applications in the production of gene-edited animals and plants. Most efforts to insert genes have relied on homology-directed repair (HDR)-mediated integration, but this strategy remains inefficient for the production of gene-edited livestock, especially monotocous species such as cattle. Although efforts have been made to improve HDR efficiency, other strategies have also been proposed to circumvent these challenges. Here we demonstrate that a homology-mediated end-joining (HMEJ)-based method can be used to create gene-edited cattle that displays precise integration of a functional gene at the ROSA26 locus. We found that the HMEJ-based method increased the knock-in efficiency of reporter genes by eightfold relative to the traditional HDR-based method in bovine fetal fibroblasts. Moreover, we identified the bovine homology of the mouse Rosa26 locus that is an accepted genomic safe harbor and produced three live-born gene-edited cattle with higher rates of pregnancy and birth, compared with previous work. These gene-edited cattle exhibited predictable expression of the functional gene natural resistance-associated macrophage protein-1 (NRAMP1), a metal ion transporter that should and, in our experiments does, increase resistance to bovine tuberculosis, one of the most detrimental zoonotic diseases. This research contributes to the establishment of a safe and efficient genome editing system and provides insights for gene-edited animal breeding. Gene-edited livestock that relied on site-specific engineered endonucleases, especially, CRISPR/Cas9, has become an important resource for animal breeding and biomedical research (1Carlson D.F. Lancto C.A. Zang B. Kim E.S. Walton M. Oldeschulte D. Seabury C. Sonstegard T.S. Fahrenkrug S.C. Production of hornless dairy cattle from genome-edited cell lines.Nat. Biotechnol. 2016; 34: 479-481Crossref PubMed Scopus (186) Google Scholar, 2Gao Y. Wu H. Wang Y. Liu X. Chen L. Li Q. Cui C. Liu X. Zhang J. Zhang Y. Single Cas9 nickase induced generation of NRAMP1 knockin cattle with reduced off-target effects.Genome Biol. 2017; 18: 13Crossref PubMed Scopus (115) Google Scholar, 3Miao X. Recent advances in the development of new transgenic animal technology.Cell Mol. Life Sci. 2013; 70: 815-828Crossref PubMed Scopus (27) Google Scholar, 4Niu D. Wei H.J. Lin L. George H. Wang T. Lee H. Zhao H.Y. Wang Y. Kan Y.N. Shrock E. Lesha E. Wang G. Luo Y.L. Qing Y.B. Jiao D.L. et al.Inactivation of porcine endogenous retrovirus in pigs using CRISPR-Cas9.Science. 2017; 357: 1303-1307Crossref PubMed Scopus (424) Google Scholar). A considerable part of the applications for the enhancement of disease resistance and the production of biomedical materials rely on functional gene knock-in (KI) (5Ma T. Tao J.L. Yang M.H. He C.J. Tian X.Z. Zhang X.S. Zhang J.L. Deng S.L. Feng J.Z. Zhang Z.Z. Wang J. Ji P.Y. Song Y.K. He P.L. Han H.B. et al.An AANAT/ASMT transgenic animal model constructed with CRISPR/Cas9 system serving as the mammary gland bioreactor to produce melatonin-enriched milk in sheep.J. Pineal Res. 2017; 63Crossref Scopus (26) Google Scholar, 6Shanthalingam S. Tibary A. Beever J.E. Kasinathan P. Brown W.C. Srikumaran S. Precise gene editing paves the way for derivation of Mannheimia haemolytica leukotoxin-resistant cattle.Proc. Natl. Acad. Sci. U. S. A. 2016; 113: 13186-13190Crossref PubMed Scopus (24) Google Scholar). Safe and efficient insertion and expression of functional gene are crucial for the practical application of genome editing technology in livestock. CRISPR/Cas9-triggered DNA double-strand breaks (DSBs) at target sites (7Gaj T. Gersbach C.A. Barbas C.F. ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering.Trends Biotechnol. 2013; 31: 397-405Abstract Full Text Full Text PDF PubMed Scopus (2513) Google Scholar) can be typically repaired by nonhomologous end-joining (NHEJ) pathway and the competing homologous recombination (HR) pathway (8Filippo J.S. Sung P. Klein H. Mechanism of eukaryotic homologous recombination.Annu. Rev. Biochem. 2008; 77: 229-257Crossref PubMed Scopus (1167) Google Scholar). Moreover, microhomology-mediated end-joining (MMEJ) pathway has also been reported to be an alternative NHEJ pathway to repair DSBs (9McVey M. Lee S.E. MMEJ repair of double-strand breaks (director's cut): Deleted sequences and alternative endings.Trends Genet. 2008; 24: 529-538Abstract Full Text Full Text PDF PubMed Scopus (673) Google Scholar, 10Nussenzweig A. Nussenzweig M.C. A backup DNA repair pathway moves to the forefront.Cell. 2007; 131: 223-225Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar). In general, NHEJ repair pathway introducing small inserts and/or deletions (indels) at the DSB sites is often applied to endogenous gene knockout, while HR repair pathway contributes to the integration of exogenous DNA fragments flanked by homology arms (HAs) into host genome. However, since the HR pathway is mainly restricted to the S and G2 phases of the cell cycle and has a lower frequency than NHEJ pathway (11Jackson S.P. Bartek J. The DNA-damage response in human biology and disease.Nature. 2009; 461: 1071-1078Crossref PubMed Scopus (3853) Google Scholar, 12Shrivastav M. De Haro L.P. Nickoloff J.A. Regulation of DNA double-strand break repair pathway choice.Cell Res. 2008; 18: 134-147Crossref PubMed Scopus (979) Google Scholar), the inefficiency of homology-directed repair HDR-mediated precise integration of a large DNA fragment limits the generation of gene-edited livestock. Currently, most studies have focused on enhancing the efficiency of HDR, such as optimizing parameters for targeting constructs (13Jung C.J. Zhang J.L. Trenchard E. Lloyd K.C. West D.B. Rosen B. de Jong P.J. Efficient gene targeting in mouse zygotes mediated by CRISPR/Cas9-protein.Transgenic Res. 2017; 26: 263-277Crossref PubMed Scopus (14) Google Scholar), suppressing NHEJ repair pathway (14Chu V.T. Weber T. Wefers B. Wurst W. Sander S. Rajewsky K. Kuhn R. Increasing the efficiency of homology-directed repair for CRISPR-Cas9-induced precise gene editing in mammalian cells.Nat. Biotechnol. 2015; 33: 543-548Crossref PubMed Scopus (812) Google Scholar), or enhancing HR repair pathway (15Song J. Yang D.S. Xu J. Zhu T.Q. Chen Y.E. Zhang J.F. RS-1 enhances CRISPR/Cas9-and TALEN-mediated knock-in efficiency.Nat. Commun. 2016; 710548Crossref PubMed Scopus (275) Google Scholar). However, the efficiency of HDR remains low and its increase is only available for certain cell types. Three accessible strategies, HMEJ-, NHEJ-, and MMEJ-based methods, were proposed to mediate efficient exogenous gene KI at the expected locus in human cells (16He X.J. Tan C.L. Wang F. Wang Y.F. Zhou R. Cui D.X. You W.X. Zhao H. Ren J.W. Feng B. Knock-in of large reporter genes in human cells via CRISPR/Cas9-induced homology-dependent and independent DNA repair.Nucleic Acids Res. 2016; 44e85Crossref PubMed Scopus (196) Google Scholar, 17Zhang J.P. Li X.L. Li G.H. Chen W.Q. Arakaki C. Botimer G.D. Baylink D. Zhang L. Wen W. Fu Y.W. Xu J. Chun N. Yuan W.P. Cheng T. Zhang X.B. Efficient precise knockin with a double cut HDR donor after CRISPR/Cas9-mediated double-stranded DNA cleavage.Genome Biol. 2017; 1835Crossref PubMed Scopus (245) Google Scholar), mouse cells (18Bressan R.B. Dewari P.S. Kalantzaki M. Gangoso E. Matjusaitis M. Garcia-Diaz C. Blin C. Grant V. Bulstrode H. Gogolok S. Skarnes W.C. Pollard S.M. Efficient CRISPR/Cas9-assisted gene targeting enables rapid and precise genetic manipulation of mammalian neural stem cells.Development. 2017; 144: 635-648Crossref PubMed Scopus (62) Google Scholar), monkey embryos (19Yao X. Liu Z. Wang X. Wang Y. Nie Y.H. Lai L. Sun R.L. Shi L.Y. Sun Q. Yang H. Generation of knock-in cynomolgus monkey via CRISPR/Cas9 editing.Cell Res. 2018; 28: 379-382Crossref PubMed Scopus (31) Google Scholar, 20Yao X. Wang X. Hu X.D. Liu Z. Liu J.L. Zhou H.B. Shen X.W. Wei Y. Huang Z.J. Ying W.Q. Wang Y. Nie Y.H. Zhang C.C. Li S.L. Cheng L.P. et al.Homology-mediated end joining-based targeted integration using CRISPR/Cas9.Cell Res. 2017; 27: 801-814Crossref PubMed Scopus (174) Google Scholar), and model organisms (21Shi Z.Y. Wang F.Q. Cui Y. Liu Z.Z. Guo X.G. Zhang Y.Q. Deng Y. Zhao H. Chen Y.L. Heritable CRISPR/Cas9-mediated targeted integration in Xenopus tropicalis.FASEB J. 2015; 29: 4914-4923Crossref PubMed Scopus (47) Google Scholar, 22Auer T.O. Duroure K. De Cian A. Concordet J.P. Del Bene F. Highly efficient CRISPR/Cas9-mediated knock-in in zebrafish by homology-independent DNA repair.Genome Res. 2014; 24: 142-153Crossref PubMed Scopus (456) Google Scholar, 23Ochiai H. Sakamoto N. Fujita K. Nishikawa M. Suzuki K.I. Matsuura S. Miyamoto T. Sakuma T. Shibata T. Yamamoto T. Zinc-finger nuclease-mediated targeted insertion of reporter genes for quantitative imaging of gene expression in sea urchin embryos.Proc. Natl. Acad. Sci. U. S. A. 2012; 109: 10915-10920Crossref PubMed Scopus (37) Google Scholar). By comparing the gene integration efficiency between the HDR-, HMEJ-, NHEJ-, and MMEJ-based methods, interestingly, different results were observed in different cell types or species (16He X.J. Tan C.L. Wang F. Wang Y.F. Zhou R. Cui D.X. You W.X. Zhao H. Ren J.W. Feng B. Knock-in of large reporter genes in human cells via CRISPR/Cas9-induced homology-dependent and independent DNA repair.Nucleic Acids Res. 2016; 44e85Crossref PubMed Scopus (196) Google Scholar, 20Yao X. Wang X. Hu X.D. Liu Z. Liu J.L. Zhou H.B. Shen X.W. Wei Y. Huang Z.J. Ying W.Q. Wang Y. Nie Y.H. Zhang C.C. Li S.L. Cheng L.P. et al.Homology-mediated end joining-based targeted integration using CRISPR/Cas9.Cell Res. 2017; 27: 801-814Crossref PubMed Scopus (174) Google Scholar). To date, apart from the HDR-based method, it still remains unclear whether the other three methods can be employed to mediate high-efficiency KI in livestock. Genomic safe harbors (GSHs) are intragenic or extragenic regions of the genome permitting sufficient expression of the inserted genes without adverse effects on the host cell or organism (24Sadelain M. Papapetrou E.P. Bushman F.D. Safe harbours for the integration of new DNA in the human genome.Nat. Rev. Cancer. 2012; 12: 51-58Crossref Scopus (148) Google Scholar, 25Papapetrou E.P. Schambach A. Gene insertion into genomic safe harbors for human gene therapy.Mol. Ther. 2016; 24: 678-684Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). They are preferred genomic acceptor sites for genome editing. ROSA26 locus, an accepted GSH in mouse, has been targeted for the exogenous gene addition in human cells (26Irion S. Luche H. Gadue P. Fehling H.J. Kennedy M. Keller G. Identification and targeting of the ROSA26 locus in human embryonic stem cells.Nat. Biotechnol. 2007; 25: 1477-1482Crossref PubMed Scopus (224) Google Scholar) and in mouse (27Zambrowicz B.P. Imamoto A. Fiering S. Herzenberg L.A. Kerr W.G. Soriano P. Disruption of overlapping transcripts in the ROSA beta geo 26 gene trap strain leads to widespread expression of beta-galactosidase in mouse embryos and hematopoietic cells.Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3789-3794Crossref PubMed Scopus (704) Google Scholar), rat (28Kobayashi T. Kato-Itoh M. Yamaguchi T. Tamura C. Sanbo M. Hirabayashi M. Nakauchi H. Identification of rat Rosa26 locus enables generation of knock-in rat lines ubiquitously expressing tdTomato.Stem Cells Dev. 2012; 21: 2981-2986Crossref PubMed Scopus (45) Google Scholar), rabbit (29Yang D.S. Song J. Zhang J.F. Xu J. Zhu T.Q. Wang Z. Lai L.X. Chen Y.E. Identification and characterization of rabbit ROSA26 for gene knock-in and stable reporter gene expression.Sci. Rep. 2016; 625161Crossref PubMed Scopus (36) Google Scholar) and even sheep (30Wu M. Wei C. Lian Z. Liu R. Zhu C. Wang H. Cao J. Shen Y. Zhao F. Zhang L. Mu Z. Wang Y. Wang X. Du L. Wang C. Rosa26-targeted sheep gene knock-in via CRISPR-Cas9 system.Sci. Rep. 2016; 6: 24360Crossref PubMed Scopus (40) Google Scholar), and pig (31Li X.P. Yang Y. Bu L. Guo X.G. Tang C.C. Song J. Fan N.N. Zhao B.T. Ouyang Z. Liu Z.M. Zhao Y. Yi X.L. Quan L.Q. Liu S.C. Yang Z.G. et al.Rosa26-targeted swine models for stable gene over-expression and Cre-mediated lineage tracing.Cell Res. 2014; 24: 501-504Crossref PubMed Scopus (67) Google Scholar). It is ubiquitously expressed in adult tissues of above species and supports efficient integration of target sequences. Gene-edited cells and individuals showed strong and ubiquitous expression of inserted genes without apparent defects. Furthermore, the bovine ROSA26 (bROSA26) locus has been already identified and its locus tagged with enhanced green fluorescent protein (EGFP) using TALENs (32Wang M. Sun Z.L. Zou Z.Y. Ding F.R. Li L. Wang H.P. Zhao C.J. Li N. Dai Y.P. Efficient targeted integration into the bovine Rosa26 locus using TALENs.Sci. Rep. 2018; 810385Crossref PubMed Scopus (10) Google Scholar). However, major previous studies focused on insertion of reporter genes instead of functional genes. Bovine tuberculosis caused by Mycobacterium bovis (M. bovis) is one of the most detrimental zoonotic diseases (33Grange J.M. Mycobacterium bovis infection in human beings.Tuberculosis. 2001; 81: 71-77Crossref PubMed Scopus (185) Google Scholar, 34Thoen C. LoBue P. de Kantor I. The importance of Mycobacterium bovis as a zoonosis.Vet. Microbiol. 2006; 112: 339-345Crossref PubMed Scopus (204) Google Scholar), which leads to serious threat to global public health and agriculture (35Ayele W.Y. Neill S.D. Zinsstag J. Weiss M.G. Pavlik I. Bovine tuberculosis: An old disease but a new threat to Africa.Int. J. Tuberc. Lung Dis. 2004; 8: 924-937PubMed Google Scholar). At present, the disease remains widespread and is not effectively controlled or eliminated in some less developed areas (36Waters W.R. Palmer M.V. Buddle B.M. Vordermeier H.M. Bovine tuberculosis vaccine research: Historical perspectives and recent advances.Vaccine. 2012; 30: 2611-2622Crossref PubMed Scopus (148) Google Scholar). We have reported Cas9 nuclease-mediated NRAMP1 gene KI cattle. The overexpression of bovine NRAMP1 gene provides the gene-edited cattle with increased resistance to tuberculosis (2Gao Y. Wu H. Wang Y. Liu X. Chen L. Li Q. Cui C. Liu X. Zhang J. Zhang Y. Single Cas9 nickase induced generation of NRAMP1 knockin cattle with reduced off-target effects.Genome Biol. 2017; 18: 13Crossref PubMed Scopus (115) Google Scholar). However, the low rates of pregnancy and birth limited the mass production of gene-edited cattle. In this study, we firstly identified bROSA26 locus and the optimal promoter that supported selected markers expression in bovine fetal fibroblasts (BFFs) for screening targeted colonies to perform somatic cell nuclear transfer (SCNT). Then we detected that the HMEJ-based method facilitated DNA integration and showed higher efficiency than the HDR-, MMEJ-, NHEJ-based methods in BFFs. Using the HMEJ-based method, we targeted to the bROSA26 locus to stimulate functional NRAMP1 gene KI and ultimately more effectively produced gene-edited cattle. These gene-edited cattle showed predictable expression and the ability to respond to M. bovis infection without off-target modification at potential off-target sites and without disturbance to nearby endogenous genes. Therefore, bROSA26 locus was identified as a potential GSH, allowing efficient HMEJ-based insertion of functional genes to produce cattle with increased resistance to tuberculosis, which will greatly accelerate the efficient production of gene-edited livestock. Mouse, human, rat, porcine, sheep, and rabbit data indicate that Rosa26 promoter region and exon 1 contained highly conserved sequences (29Yang D.S. Song J. Zhang J.F. Xu J. Zhu T.Q. Wang Z. Lai L.X. Chen Y.E. Identification and characterization of rabbit ROSA26 for gene knock-in and stable reporter gene expression.Sci. Rep. 2016; 625161Crossref PubMed Scopus (36) Google Scholar, 30Wu M. Wei C. Lian Z. Liu R. Zhu C. Wang H. Cao J. Shen Y. Zhao F. Zhang L. Mu Z. Wang Y. Wang X. Du L. Wang C. Rosa26-targeted sheep gene knock-in via CRISPR-Cas9 system.Sci. Rep. 2016; 6: 24360Crossref PubMed Scopus (40) Google Scholar, 31Li X.P. Yang Y. Bu L. Guo X.G. Tang C.C. Song J. Fan N.N. Zhao B.T. Ouyang Z. Liu Z.M. Zhao Y. Yi X.L. Quan L.Q. Liu S.C. Yang Z.G. et al.Rosa26-targeted swine models for stable gene over-expression and Cre-mediated lineage tracing.Cell Res. 2014; 24: 501-504Crossref PubMed Scopus (67) Google Scholar). The sequence of exon 1 of mouse Rosa26 transcript variant 2 plus putative promoter region was blasted against Bos taurus reference genomic sequence (taxid: 9913) in NCBI database, a highly conserved region (the highest degree of sequence similarity >84%) on bovine chromosome 22 was identified (Fig. S1). The sequence alignments of porcine ROSA26 promoter (1 kb upstream of exon 1) and exon 1 showed high sequence conservation (the highest degree of sequence similarity >92%) (Fig. S1). Sequences flanking this region contain the same genes to those in the Rosa26 locus of mouse and porcine (Lhfpl4, Setd5, and Thumpd3, Fig. 1A). We predicted the sequence of bROSA26 exon 1 from mouse Rosa26 exon 1 and designed a primer to perform 3ʹ rapid amplification of cDNA ends (RACE) analysis. One noncoding RNA product of at least 853 bp transcribed from the bROSA26 locus was identified (Fig. S2). Quantitative real-time PCR (qPCR) analysis reaction for exon 1 and exon 2 demonstrated that the noncoding RNA was expressed in various adult tissues (Fig. 1B). Similar expression patterns were observed using oligonucleotides that amplify a 363 bp product across the intron between exon 1 and exon 2 in a conventional RT-PCR reaction (Fig. 1C). Figure S1 shows high sequence conservation of Rosa26 promoter region among mouse, bovine, and pig. Mouse and pig share the same 5ʹ start of the Rosa26 transcript. Therefore, we assumed the corresponding site as the 5ʹ start of the bROSA26 transcript. Firstly, we amplified the proximal sequence from 2007 bp upstream to 505 bp downstream (relative to the putative start) using Holstein cattle genomic DNA as template. Then a series of eight reporter constructs with progressively larger deletions from the 5ʹ end of the promoter were generated. The effects of these modifications were evaluated upon transfection of the corresponding luciferase reporter plasmids into BFFs, and the results of these analyses were shown in Figure 1D. Luciferase assays revealed that pGL4.10-1007/+505 showed the highest transcriptional activity but lower than two common strong promoters (pGL4.10-CMV and pGL4.10-EF1α) (Fig. 1D). Subsequently, five reporter constructs with progressively larger deletions from the 3ʹ end of the promoter were generated. We observed that pGL4.10-1007/+105 showed the highest promoter activities (Fig. 1E). Taken together, these results indicated that the region from –1007 to +105 relative to the putative TSS acts as an optimal promoter with a moderate level for endogenous gene expression. According to the result of 3ʹ RACE analysis, we designed five sgRNAs specific to the bROSA26 locus intron 1 (1512-bp) region between exon 1 and exon 2 on chromosome 22 (Fig. 2A). We constructed five SSA reporter plasmids containing designed target sites and five Cas9 expression plasmids containing 20-nt guide sequence and then cotransfected the corresponding SSA reporter plasmids and Cas9 expression plasmids into 293T cells. The activity of sgRNAs was screened with the luciferase assay as previously described (37Bhakta M.S. Segal D.J. The generation of zinc finger proteins by modular assembly.Methods Mol. Biol. 2010; 649: 3-30Crossref PubMed Scopus (56) Google Scholar). All the sgRNAs except the sgRNA 45 showed extremely significant activity and the sgRNA 11 showed the highest activity (Fig. 2B). Therefore, we chose target site 11 to achieve the insertion of the exogenous gene in subsequent experiments. Given this broad expression of Rosa26 in adult tissues and the moderate activity of endogenous promoter, we were next interested in determining whether this locus could be targeted for selection of individual colonies. To evaluate whether bovine endogenous ROSA26 promoter can drive reporter genes expression in BFFs, a reporter vector pROSA26-SA-EGFP-Puro-HDR, expressing selected markers, was constructed as shown in Figures 2A and S3A. The vector contains a 5ʹ arm and a 3ʹ arm of homology, which together span 1578 bp of the bROSA26 locus. The vector overlaps with sequences of the intron 1 and the exon 2 of the bROSA26 locus. A splice acceptor (SA) sequence and a promoterless selected markers cassette separate the HAs and two LoxP sites. The selected markers cassette consists of the EGFP and puromycin resistance gene, which were fused by the porcine teschovirus-1 2A peptide sequence. The transcription of the selected markers was expected to mimic that of endogenous ROSA26 by SA sequence. The LoxP sites are positioned such that after expression of Cre recombinase (Cre), the selected markers cassette is removed after subsequent exogenous gene target for the production of marker-free gene-edited cattle. Plasmids encoding Cas9 protein, Cas9/sgRNA11, were cotransfected with pROSA26-SA-EGFP-Puro-HDR (Fig. S3A) into BFFs to achieve stable genetic modification of cells that were targeted to bROSA26 locus through HDR. After screening with puromycin, drug-resistant colonies (Fig. 2C) were picked and analyzed by 5ʹ junction PCR for evidence of correct targeting (Fig. S3B). To rule out potential false-positives, we performed 3ʹ junction PCR on genomic DNA from 5ʹ junction PCR-positive colonies (Fig. S3B). Sequence analysis of the resulting 1824-bp (left homology arm) and 2833-bp (right homology arm) fragments of 5ʹ and 3ʹ junction PCR confirmed site-specific integration of the targeting vector into the bROSA26 locus (Fig. 3A). These results clearly demonstrated the ability of the endogenous bROSA26 promoter to drive the reporter genes expression for selecting individual colonies in BFFs. Efficient KI of exogenous DNA is the key to generating a sufficient number of targeted colonies for SCNT. To test the feasibility and efficiency of HMEJ-, NHEJ-, or MMEJ-based methods in cattle, we constructed another three types of donors: an HMEJ donor (sgRNA target sites plus long ∼800 bp HAs), an MMEJ donor (sgRNA target sites plus short ∼20 bp HAs), and an NHEJ donor (only sgRNA target sites) (Fig. 2A). These donors can be cleaved at the sgRNA11 target site by Cas9/sgRNA11, which would cleave both genome and donor plasmid, to provide linear templates carrying HAs. At 7 days after cotransfecting each of the four types of donors, respectively, with Cas9/sgRNA11 in BFFs, we detected that the KI efficiency of the HMEJ-based method was significantly higher than that of the other methods by FACS (Fig. 2D). To further clarify whether the HMEJ-based method facilitated DNA integration at a higher efficiency than other three strategies, we cotransfected each of the four types of donors, respectively, with Cas9/sgRNA11 into BFFs. Stably transfected colonies were identified following 10–12 days of puromycin selection. The HMEJ-based method had an approximate eightfold increase in the number of target colonies compared with the HDR-based method (Fig. 2E). Junction PCR and sequencing confirmed the correct joining between genome and donor plasmids in HMEJ and HDR groups (Figs. 2A and S4). These results suggested that the HMEJ-based method, which simultaneously introduced DSBs in genome and donors, was also able to induce precise integration of reporter genes at target sites in BFFs and showed the higher KI efficiency, compared with the HDR-based method. However, no target colonies were observed in NHEJ groups and MMEJ groups (Fig. 2E), which suggested that the NHEJ-based method and the MMEJ-based method may be inefficient for precise integration of reporter genes at bROSA26 locus in BFFs. Collectively, these data were consistent with the results observed by FACS analyses, and they clearly showed that HMEJ-based method was a highly desirable strategy for efficient KI of exogenous DNA. We constructed the gene-targeting vector, pROSA26-SA-EGFP-Puro-HMEJ-NRAMP1, by inserting the NRAMP1 gene and its original promoter sequence into pROSA26-SA-EGFP-Puro-HMEJ, directing NRAMP1 expression only in bovine macrophages and other dedicated phagocytes, as previously described (38Hedges J.F. Kimmel E. Snyder D.T. Jerome M. Jutila M.A. Solute carrier 11A1 is expressed by innate lymphocytes and augments their activation.J. Immunol. 2013; 190: 4263-4273Crossref PubMed Scopus (26) Google Scholar). Subsequently, we introduced this targeting vector along with Cas9/sgRNA11 into BFFs and achieved the insertion of NRAMP1 gene (Fig. 3A). Stably transfected cells (Fig. S5A), after selection with puromycin, were screened by 5ʹ -junction (1.824-bp) PCR, 3ʹ -junction (2261-bp) PCR and sequence analysis to confirm that gene-edited cassette was inserted into the intended specific site (Fig. S5, B and C and Fig. 3A). Then these targeted colonies were used for Southern blot analyses to further evaluate the insertion of gene-edited cassette. As expected, the integration of a single copy of the exogenous gene was confirmed by using an external of the genome homology region probe1 by showing a 6.2-kb band from the endogenous ROSA26 allele and a 3.8-kb band characteristic of the insertion (Fig. 3B). None of these targeted colonies showed random integration of the exogenous gene by the appearance of an expected single 6.7-kb band by using a probe specific for the NRAMP1 gene (Fig. 3B). SCNT was carried out to reconstruct bovine embryos by using the randomly picked seven heterozygous targeted colonies (Table 1). Then embryos were successfully reconstructed, and some reconstructed embryos were developed to blastocyst stage. Gene-edited blastocysts were transferred into the oviducts of 34 recipient heifers. Eleven (32.3%) surrogates were confirmed pregnant by ultrasound examination 1 month after the embryo transfer. Finally, three live calves were produced (Fig. 4A). For Cas9 nuclease-mediated KI cattle, HMEJ-based safe-harbor genome editing led to a dramatic increase in the rates of pregnancy and birth (32.3% and 8.8%), as compared with previous studies (12.7% and 2.3%) (2Gao Y. Wu H. Wang Y. Liu X. Chen L. Li Q. Cui C. Liu X. Zhang J. Zhang Y. Single Cas9 nickase induced generation of NRAMP1 knockin cattle with reduced off-target effects.Genome Biol. 2017; 18: 13Crossref PubMed Scopus (115) Google Scholar).Table 1Animal production statisticsCell lineEmbryos/recipientsPregnant at day 30Pregnant at day 90Liveborn08185/522208282/210011234/411119153/310019323/3100237912/12400231215/5100Total34/3411/34 (32.3%)3/34 (8.8%)3/34 (8.8%) Open table in a new tab To determine whether the exogenous NRAMP1 gene was precisely integrated at the target site, we performed 5ʹ junction PCR, 3ʹ junction PCR and Southern blot analyses to check the three gene-targeted calves. As expected, the gene-edited cattle were heterozygous for site-specific NRAMP1 KI at the target site (Fig. 4, B and C). Subsequently, we cloned eight main sgRNA11 potential off-target sites that were predicted based on sequence similarity to the target sequence from all gene-edited cattle genome. We did not detect any typical indels in all of the analyzed off-target sites (Fig. S6). These results demonstrated precise integration of the exogenous gene at bROSA26 locus without detected off-target modification. We isolated mononuclear cells from the peripheral blood of gene-edited cattle and wild-type cattle and induced them into macrophages. The monocyte-derived macrophages (MDMs) were separated from each animal individually and mixed for subsequent studies. To test whether bROSA26 locus can support predictable exogenous gene expression while minimizing the impact on the expression of nearby endogenous genes in gene-edited cattle. We extracted mRNA from the MDMs of gene-edited cattle and wild-type cattle and performed qPCR analysis. No significant difference was detected in the relative levels of expression of the nearby endogenous genes (SETD5 and THUMPD3) between the gene-edited and wild-type cattle (Fig. 5A). Note that no expression of LHFPL4 was detected between" @default.
• W3134615468 created "2021-03-15" @default.
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• W3134615468 date "2021-01-01" @default.
• W3134615468 modified "2023-10-06" @default.
• W3134615468 title "HMEJ-based safe-harbor genome editing enables efficient generation of cattle with increased resistance to tuberculosis" @default.
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