A hypomorphic mutation in the mouse Csn1s1 gene generated by CRISPR/Cas9 pronuclear microinjection

Caseins are major milk proteins that have an evolutionarily conserved role in nutrition. Sequence variations in the casein genes affect milk composition in livestock species. Regulatory elements of the casein genes could be used to direct the expression of desired transgenes into the milk of transgenic animals. Dozens of casein alleles have been identified for goats, cows, sheep, camels and horses, and these sequence variants are associated with altered gene expression and milk protein content. Most of the known mutations affecting casein genes’ expression are located in the promoter and 3’-untranslated regions. We performed pronuclear microinjections with Cas9 mRNA and sgRNA against the first coding exon of the mouse Csn1s1 gene to introduce random mutations in the α-casein (Csn1s1) signal peptide sequence at the beginning of the mouse gene. Sanger sequencing of the founder mice identified 40 mutations. As expected, mutations clustered around the sgRNA cut site (3 bp from PAM). Most of the mutations represented small deletions (1–10 bp), but we detected several larger deletions as well (100–300 bp). Functionally most mutations led to gene knockout due to a frameshift or a start codon loss. Some of the mutations represented in-frame indels in the first coding exon. Of these, we describe a novel hypomorphic Csn1s1 (Csn1s1c.4-5insTCC) allele. We measured Csn1s1 protein levels and confirmed that the mutation has a negative effect on milk composition, which shows a 50 % reduction in gene expression and a 40–80 % decrease in Csn1s1 protein amount, compared to the wild-type allele. We assumed that mutation affected transcript stability or splicing by an unknown mechanism. This mutation can potentially serve as a genetic marker for low Csn1s1 expression.

1. An L.Y., Yuan Y.G., Yu B.L., Yang T.J., Cheng Y. Generation of human lactoferrin transgenic cloned goats using donor cells with dual markers and a modified selection procedure. Theriogenology. 2012; 78:1303-1311. DOI 10.1016/j.theriogenology.2012.05.027.

2. Burkov I.A., Serova I.A., Battulin N.R., Smirnov A.V., Babkin I.V., Andreeva L.E., Dvoryanchikov G.A., Serov O.L. Expression of the human granulocyte-macrophage colony stimulating factor (hGMCSF) gene under control of the 5′-regulatory sequence of the goat alpha-S1-casein gene with and without a MAR element in transgenic mice. Transgenic Res. 2013;22:949-964. DOI 10.1007/s11248-013-9697-4.

3. Cartegni L., Chew S.L., Krainer A.R. Listening to silence and understanding nonsense: exonic mutations that affect splicing. Nat. Rev. Genet. 2002;3(4):285-298. DOI 10.1038/nrg775. PMID 11967553.

4. Cieslak J., Wodas L., Borowska A., Pawlak P., Czyzak-Runowska G., Wojtowski J., Puppel K., Kuczynska B., Mackowski M. 5′-flanking variants of equine casein genes (CSN1S1, CSN1S2, CSN2, CSN3) and their relationship with gene expression and milk composition. J. Appl. Genet. 2019;60(1):71-78. DOI 10.1007/s13353-018-0473-2.

5. Dos Santos C.O., Dolzhenko E., Hodges E., Smith A.D., Hannon G.J. An epigenetic memory of pregnancy in the mouse mammary gland. Cell Rep. 2015;11:1102-1109. DOI 10.1016/j.celrep.2015.04.015.

6. Fomichev K.A., Sazanova A.L., Malewski T., Kaminski S., Sazanov A.A. Associations between two novel rSNPs in 5′-flanking region of the bovine casein gene cluster and milk performance traits. Gene. 2012;496:49-54. DOI 10.1016/j.gene.2011.12.038.

7. Guan D., Mármol-Sánchez E., Cardoso T.F., Such X., Landi V., Tawari N.R., Amills M. Genomic analysis of the origins of extant casein variation in goats. J. Dairy Sci. 2019;102:5230-5241. DOI 10.3168/jds.2018-15281.

8. Hogg D.R., Harries L.W. Human genetic variation and its effect on miRNA biogenesis, activity and function. Biochem. Soc. Trans. 2014; 42(4):1184-1189. DOI 10.1042/BST20140055. PMID 25110023.

9. Houdebine L.M. Production of pharmaceutical proteins by transgenic animals. Comp. Immunol. Microbiol. Infect. Dis. 2009;32:107-121. DOI 10.1016/j.cimid.2007.11.005.

10. Huang W., Peñagaricano F., Ahmad K.R., Lucey J.A., Weigel K.A., Khatib H. Association between milk protein gene variants and protein composition traits in dairy cattle. J. Dairy Sci. 2012;95:440-449. DOI 10.3168/jds.2011-4757.

11. Kalds P., Zhou S., Cai B., Liu J., Wang Y., Petersen B., Sonstegard T., Wang X., Chen Y. Sheep and goat genome engineering: from random transgenesis to the CRISPR Era. Front. Genet. 2019;10:750. DOI 10.3389/fgene.2019.00750. eCollection2019.

12. Kim J.J., Yu J., Bag J., Bakovic M., Cant J.P. Translation attenuation via 3′ terminal codon usage in bovine csn1s2 is responsible for the difference in αs2- and β-casein profile in milk. RNA Biol. 2015;12: 354-367. DOI 10.1080/15476286.2015.1017231.

13. Kolb A.F., Huber R.C., Lillico S.G., Carlisle A., Robinson C.J., Neil C., Petrie L., Sorensen D.B., Olsson I.A., Whitelaw C.B. Milk lacking α-casein leads to permanent reduction in body size in mice. PLoS One. 2011;6:e21775. DOI 10.1371/journal.pone.0021775.

14. Lee H.K., Willi M., Wang C., Yang C.M., Smith H.E., Liu C., Hennighausen L. Functional assessment of CTCF sites at cytokinesensing mammary enhancers using CRISPR/Cas9 gene editing in mice. Nucleic Acids Res. 2017;45:4606-4618. DOI 10.1093/nar/gkx185.

15. Li W.R., Liu C.X., Zhang X.M., Chen L., Peng X.R., He S.G., Lin J.P., Han B., Wang L.Q., Huang J.C., Liu M.J. CRISPR/Cas9-mediated loss of FGF5 function increases wool staple length in sheep. FEBS J. 2017;284:2764-2773. DOI 10.1111/febs.14144.

16. Lindeboom R.G.H., Vermeulen M., Lehner B., Supek F. The impact of nonsense-mediated mRNA decay on genetic disease, gene editing and cancer immunotherapy. Nat. Genet. 2019;51(11):1645- 1651. DOI 10.1038/s41588-019-0517-5. Epub 2019 Oct 28. PMID 31659324. PMCID PMC6858879.

17. Liu H.C., Pai S.Y., Chen H.L., Lai C.W., Tsai T.C., Cheng W.T., Yang S.H., Chen C.M. Recombinant Derp5 allergen with αS1-casein signal peptide secreted in murine milk protects against dust mite allergen-induced airway inflammation. J. Dairy Sci. 2014;97:6792- 6803. DOI 10.3168/jds.2014-8484.

18. Lu R., Zhang T., Song S., Zhou M., Jiang L., He Z., Yuan Y., Yuan T., Lu Y., Yan K., Cheng Y. Accurately cleavable goat β-lactoglobulin signal peptide efficiently guided translation of a recombinant human plasminogen activator in transgenic rabbit mammary gland. Biosci. Rep. 2019;39:6. DOI 10.1042/BSR20190596.

19. Mucaki E.J., Shirley B.C., Rogan P.K. Expression changes confirm genomic variants predicted to result in allele-specific, alternative mRNA splicing. Front. Genet. 2020;11:109. DOI 10.3389/fgene.2020.00109. PMID 32211018. PMCID PMC7066660.

20. Noce A., Pazzola M., Dettori M.L., Amills M., Castelló A., Cecchinato A., Bittante G., Vacca G.M. Variations at regulatory regions of the milk protein genes are associated with milk traits and coagulation properties in the Sarda sheep. Anim. Genet. 2016;47:717-726. DOI 10.1111/age.12474.

21. Park K.E., Powell A., Sandmaier S.E., Kim C.M., Mileham A., Donovan D.M., Telugu B.P. Targeted gene knock-in by CRISPR/Cas ribonucleoproteins in porcine zygotes. Sci. Rep. 2017;7:42458. DOI 10.1038/srep42458.

22. Popp M.W., Maquat L.E. Leveraging rules of nonsense-mediated mRNA decay for genome engineering and personalized medicine. Cell. 2016;165(6):1319-1322. DOI 10.1016/j.cell.2016.05.053. PMID 27259145. PMCID PMC4924582.

23. Qian X., Kraft J., Ni Y., Zhao F.Q. Production of recombinant human proinsulin in the milk of transgenic mice. Sci. Rep. 2014;4:6465. DOI 10.1038/srep06465.

24. Rijnkels M., Elnitski L., Miller W., Rosen J.M. Multispecies comparative analysis of a mammalian-specific genomic domain encoding secretory proteins. Genomics. 2003;82:417-432. DOI 10.1016/s0888-7543(03)00114-9.

25. Rijnkels M., Freeman-Zadrowski C., Hernandez J., Potluri V., Wang L., Li W., Lemay D.G. Epigenetic modifications unlock the milk protein gene loci during mouse mammary gland development and differentiation. PLoS One. 2013;8:e53270. DOI 10.1371/journal.pone.0053270.

26. Sanchez M.P., Wolf V., El Jabri M., Beuvier E., Rolet-Répécaud O., Gaüzère Y., Minéry S., Brochard M., Michenet A., Taussat S., Barbat-Leterrier A., Delacroix-Buchet A., Laithier C., Fritz S., Boichard D. Short communication: Confirmation of candidate causative variants on milk composition and cheesemaking properties in Montbéliarde cows. J. Dairy Sci. 2018;101:10076-10081. DOI 10.3168/jds.2018-14986.

27. Sterne-Weiler T., Sanford J.R. Exon identity crisis: disease-causing mutations that disrupt the splicing code. Genome Biol. 2014;15(1):201. DOI 10.1186/gb4150. PMID 24456648. PMCID PMC4053859.

28. Thompson B.A., Walters R., Parsons M.T., Dumenil T., Drost M., Tiersma Y., Lindor N.M., Tavtigian S.V., de Wind N., Spurdle A.B., InSiGHT Variant Interpretation Committee. Contribution of mRNA splicing to mismatch repair gene sequence variant interpretation. Front. Genet. 2020;11:798. DOI 10.3389/fgene.2020.00798. PMID 32849802. PMCID PMC7398121.

29. Tuladhar R., Yeu Y., Tyler Piazza J., Tan Z., Rene Clemenceau J., Wu X., Barrett Q., Herbert J., Mathews D.H., Kim J., Hyun Hwang T., Lum L. CRISPR-Cas9-based mutagenesis frequently provokes on-target mRNA misregulation. Nat. Commun. 2019;10(1):4056. DOI 10.1038/s41467-019-12028-5. PMID 31492834. PMCID PMC6731291.

30. Uusi-Oukari M., Hyttinen J.M., Korhonen V.P., Västi A., Alhonen L., Jänne O.A., Jänne J. Bovine alpha s1-casein gene sequences direct high level expression of human granulocyte-macrophage colonystimulating factor in the milk of transgenic mice. Transgenic Res. 1997;6:75-84. DOI 10.1023/a:1018461201385.

31. Wan Y.J., Zhang Y.L., Zhou Z.R., Jia R.X., Li M., Song H., Wang H.Z., Wang L.Z., Zhang G.M., You J.H., Wang F. Efficiency of donor cell preparation and recipient oocyte source for production of transgenic cloned dairy goats harboring human lactoferrin. Theriogenology. 2012;78:583-592. DOI 10.1016/j.theriogenology.2012.03.004.

32. Wang K., Yan H., Xu H., Yang Q., Zhang S., Pan C., Chen H., Zhu H., Liu J., Qu L., Lan X. A novel indel within goat casein alpha S1 gene is significantly associated with litter size. Gene. 2018;671:161-169. DOI 10.1016/j.gene.2018.05.119.

33. Wu X., Lin Y., Xiong F., Zhou Y., Yu F., Deng J., Huang P., Chen H. The extremely high level expression of human serum albumin in the milk of transgenic mice. Transgenic Res. 2012;21:1359-1366. DOI 10.1007/s11248-012-9612-4.

34. Yamaji D., Kang K., Robinson G.W., Hennighausen L. Sequential activation of genetic programs in mouse mammary epithelium during pregnancy depends on STAT5A/B concentration. Nucleic Acids Res. 2013;41:1622-1636. DOI 10.1093/nar/gks1310.

35. Yu Z., Meng Q., Yu H., Fan B., Yu S., Fei J., Wang L., Dai Y., Li N. Expression and bioactivity of recombinant human lysozyme in the milk of transgenic mice. J. Dairy Sci. 2006;89:2911-2918. DOI 10.3168/jds.S0022-0302(06)72563-2.

36. Yuan Y.G., An L., Yu B., Song S., Zhou F., Zhang L., Gu Y., Yu M., Cheng Y. Expression of recombinant human alpha-lactalbumin in the milk of transgenic goats using a hybrid pomoter/enhancer. J. Anal. Method. Chem. 2014;281031. DOI 10.1155/2014/281031.

37. Yue X.P., Zhang X.M., Wang W., Ma R.N., Deng C.J., Lan X.Y., Chen H., Li F., Xu X.R., Ma Y., Lei C.Z. The CSN1S1 N and F alleles identified by PCR-SSCP and their associations with milk yield and composition in Chinese dairy goats. Mol. Biol. Rep. 2011;38: 2821-2825. DOI 10.1007/s11033-010-0428-0.

38. Zhou S., Cai B., He C., Wang Y., Ding Q., Liu J., Liu Y., Ding Y., Zhao X., Li G., Li C., Yu H., Kou Q., Niu W., Petersen B., Sonstegard T., Ma B., Chen Y., Wang X. Programmable base editing of the sheep genome revealed no genome-wide off-target mutations. Front. Genet. 2019;10:215. DOI 10.3389/fgene.2019.00215.