Human 8-cell embryos enable efficient induction of disease-preventive mutations without off-target effect by cytosine base editor

Abstract

Approximately 140 million people worldwide are homozygous carriers of APOE4 (ϵ4), a strong genetic risk factor for late onset familial and sporadic Alzheimer’s disease (AD), 91% of whom will develop AD at earlier age than heterozygous carriers and noncarriers. Susceptibility to AD could be reduced by targeted editing of APOE4, but a technical basis for controlling the off-target effects of base editors is necessary to develop low-risk personalized gene therapies. Here, we first screened eight cytosine base editor variants at four injection stages (from 1- to 8-cell stage), and found that FNLS-YE1 variant in 8-cell embryos achieved the comparable base conversion rate (up to 100%) with the lowest bystander effects. In particular, 80% of AD-susceptible ϵ4 allele copies were converted to the AD-neutral ϵ3 allele in human ϵ4-carrying embryos. Stringent control measures combined with targeted deep sequencing, whole genome sequencing, and RNA sequencing showed no DNA or RNA off-target events in FNLS-YE1-treated human embryos or their derived stem cells. Furthermore, base editing with FNLS-YE1 showed no effects on embryo development to the blastocyst stage. Finally, we also demonstrated FNLS-YE1 could introduce known protective variants in human embryos to potentially reduce human susceptivity to systemic lupus erythematosus and familial hypercholesterolemia. Our study therefore suggests that base editing with FNLS-YE1 can efficiently and safely introduce known preventive variants in 8-cell human embryos, a potential approach for reducing human susceptibility to AD or other genetic diseases.

Key words： human embryo; APOE4; disease-preventive mutations; base editor

References

1 Akioyamen LE, Genest J, Shan SD et al. Estimating the prevalence of heterozygous familial hypercholesterolaemia: a systematic review and meta-analysis. BMJ Open 2017;7:e016461.

2 Alvin Huang YW, Zhou B, Nabet AM et al. Differential signaling mediated by ApoE2, ApoE3, and ApoE4 in human neurons parallels Alzheimer’s disease risk. J Neurosci 2019;39:7408–7427.

3 Anzalone AV, Randolph PB, Davis JR et al. Search-and-replace genome editing without double-strand breaks or donor DNA. Nature 2019;576:149–157.

4 Arnett FC, Shulman LE. Studies in familial systemic lupus erythematosus. Medicine 1976;55:313–322.

5 Bae S, Park J, Kim JS. Cas-OFFinder: a fast and versatile algorithm that searches for potential off-target sites of Cas9 RNA-guided endonucleases. Bioinformatics 2014;30:1473–1475.

6 Castellano JM, Kim J, Stewart FR et al. Human apoE isoforms differentially regulate brain amyloid-β peptide clearance. Sci Transl Med 2011;3:3002156.

7 Chen AE, Egli D, Niakan K et al. Optimal timing of inner cell mass isolation increases the efficiency of human embryonic stem cell derivation and allows generation of sibling cell lines. Cell Stem Cell 2009;4:103–106.

8 Cibulskis K, Lawrence MS, Carter SL et al. Sensitive detection of somatic point mutations in impure and heterogeneous cancer samples. Nat Biotechnol 2013;31:213–219.

9 Cohen JC, Boerwinkle E, Thomas H et al. Sequence variations in PCSK9, low LDL, and protection against coronary heart disease. N Engl J Med 2006;354:1264–1272.

10 Corder EH, Saunders AM, Strittmatter WJ et al. Gene dose of apolipoprotein E type 4 allele and the risk of Alzheimer’s disease in late onset families. Science 1993;261:921–923.

11 Dang L, Zhou X, Zhong X et al. Correction of the pathogenic mutation in TGM1 gene by adenine base editing in mutant embryos. Mol Ther 2022;30:175–183.

12 Diogo D, Bastarache L, Liao KP et al. TYK2 protein-coding variants protect against rheumatoid arthritis and autoimmunity, with no evidence of major pleiotropic effects on non-autoimmune complex traits. PLoS One 2015;10:e0122271.

13 Doman JL, Raguram A, Newby GA et al. Evaluation and minimization of Cas9-independent off-target DNA editing by cytosine base editors. Nat Biotechnol 2020;38:620–628.

14 Fang H, Bergmann EA, Arora K et al. Indel variant analysis of short-read sequencing data with Scalpel. Nat Protoc 2016;11:2529–2548.

15 Farrer LA, Cupples LA, Haines JL et al. Effects of age, sex, and ethnicity on the association between apolipoprotein E genotype and Alzheimer disease: a meta-analysis. JAMA 1997;278:1349–1356.

16 Fu Y, Foden JA, Khayter C et al. High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells. Nat Biotechnol 2013;31:822–826.

17 Gaudelli NM, Komor AC, Rees HA et al. Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage. Nature 2017;551:464–471.

18 Gehrke JM, Cervantes O, Clement MK et al. An APOBEC3A-Cas9 base editor with minimized bystander and off-target activities. Nat Biotechnol 2018;36:977–982.

19 Grunewald J, Zhou R, Garcia SP et al. Transcriptome-wide off-target RNA editing induced by CRISPR-guided DNA base editors. Nature 2019a;569:433–437.

20 Grunewald J, Zhou R, Iyer S et al. CRISPR DNA base editors with reduced RNA off-target and self-editing activities. Nat Biotechnol 2019b;37:1041–1048.

21 Halliday MR, Rege SV, Ma Q et al. Accelerated pericyte degeneration and blood-brain barrier breakdown in apolipoprotein E4 carriers with Alzheimer’s disease. J Cereb Blood Flow Metab 2016;36:216–227.

22 Harper AR, Nayee S, Topol EJ. Protective alleles and modifier variants in human health and disease. Nat Rev Genet 2015;16:689–701.

23 Hsu PD, Scott DA, Weinstein JA et al. DNA targeting specificity of RNA-guided Cas9 nucleases. Nat Biotechnol 2013;31:827–832.

24 Huang Y, Mahley RW. Apolipoprotein E: structure and function in lipid metabolism, neurobiology, and Alzheimer’s diseases. Neurobiol Dis 2014;72 Pt A:3–12.

25 Huang Y, Mucke L. Alzheimer mechanisms and therapeutic strategies. Cell 2012;148:1204–1222.

26 Iannucci J, Sen A, Grammas P. Isoform-specific effects of apolipoprotein E on markers of Inflammation and toxicity in brain glia and neuronal cells in vitro. Curr Issues Mol Biol 2021;43:215–225.

27 Jonsson T, Atwal JK, Steinberg S et al. A mutation in APP protects against Alzheimer’s disease and age-related cognitive decline. Nature 2012;488:96–99.

28 Komor AC, Kim YB, Packer MS et al. Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. Nature 2016;533:420–424.

29 López-Isac E, Campillo-Davo D, Bossini-Castillo L et al.; Spanish Scleroderma Group. Influence of TYK2 in systemic sclerosis susceptibility: a new locus in the IL-12 pathway. Ann Rheum Dis 2016;75:1521–1526.

30 Lee MT, Bonneau AR, Giraldez AJ. Zygotic genome activation during the maternal-to-zygotic transition. Annu Rev Cell Dev Biol 2014;30:581–613.

31 Liang P, Ding C, Sun H et al. Correction of beta-thalassemia mutant by base editor in human embryos. Protein Cell 2017;8:811–822.

32 Lin YT, Seo J, Gao F et al. APOE4 causes widespread molecular and cellular alterations associated with Alzheimer’s disease phenotypes in human iPSC-derived brain cell types. Neuron 2018;98:1141–1154.e7.

33 Liu CC, Kanekiyo T, Xu H et al. Apolipoprotein E and Alzheimer disease: risk, mechanisms and therapy. Nat Rev Neurol 2013a;9:106–118.

34 Liu CC, Kanekiyo T, Xu H et al. Apolipoprotein E and Alzheimer disease: risk, mechanisms and therapy. Nat Rev Neurol 2013b;9:106–118.

35 Mahley RW, Weisgraber KH, Huang Y. Apolipoprotein E4: a causative factor and therapeutic target in neuropathology, including Alzheimer’s disease. Proc Natl Acad Sci USA 2006;103:5644–5651.

36 Miname MH, Santos RD. Reducing cardiovascular risk in patients with familial hypercholesterolemia: risk prediction and lipid management. Prog Cardiovasc Dis 2019;62:414–422.

37 Montagne A, Nation DA, Sagare AP et al. APOE4 leads to blood-brain barrier dysfunction predicting cognitive decline. Nature 2020;581:71–76.

38 Nordestgaard BG, Chapman MJ, Humphries SE et al.; European Atherosclerosis Society Consensus Panel. Familial hypercholesterolaemia is underdiagnosed and undertreated in the general population: guidance for clinicians to prevent coronary heart disease: consensus statement of the European Atherosclerosis Society. Eur Heart J 2013;34:3478–390a.

39 Ossenkoppele R, Jansen WJ, Rabinovici GD et al.; Amyloid PET Study Group. Prevalence of amyloid PET positivity in dementia syndromes: a meta-analysis. JAMA 2015;313:1939–1949.

40 Ostrowitzki S, Deptula D, Thurfjell L et al. Mechanism of amyloid removal in patients with Alzheimer disease treated with gantenerumab. Arch Neurol 2012;69:198–207.

41 Petrushina I, Davtyan H, Hovakimyan A et al. Comparison of efficacy of preventive and therapeutic vaccines targeting the N terminus of β-amyloid in an animal model of Alzheimer’s disease. Mol Ther 2017;25:153–164.

42 Raber J, Huang Y, Ashford JW. ApoE genotype accounts for the vast majority of AD risk and AD pathology. Neurobiol Aging 2004;25:641–650.

43 Ramos-Niembro F, Alarcon-Segovia D. Familial aspects of mixed connective tissue disease (MCTD). I. Occurrence of systemic lupus erythematosus in another member in two families and aggregation of MCTD in another family. J Rheumatol 1978;5:433–440.

44 Rebeck GW, Reiter JS, Strickland DK et al. Apolipoprotein E in sporadic Alzheimer’s disease: allelic variation and receptor interactions. Neuron 1993;11:575–580.

45 Reiman EM, Arboleda-Velasquez JF, Quiroz YT et al.; Alzheimer’s Disease Genetics Consortium. Exceptionally low likelihood of Alzheimer’s dementia in APOE2 homozygotes from a 5,000-person neuropathological study. Nat Commun 2020;11:667.

46 Rinne JO, Brooks DJ, Rossor MN et al. 11C-PiB PET assessment of change in fibrillar amyloid-beta load in patients with Alzheimer’s disease treated with bapineuzumab: a phase 2, double-blind, placebo-controlled, ascending-dose study. Lancet Neurol 2010;9:363–372.

47 Saunders CT, Wong WS, Swamy S et al. Strelka: accurate somatic small-variant calling from sequenced tumor-normal sample pairs. Bioinformatics 2012;28:1811–1817.

48 Selkoe DJ. The molecular pathology of Alzheimer’s disease. Neuron 1991;6:487–498.

49 Seripa D, D’Onofrio G, Panza F et al. The genetics of the human APOE polymorphism. Rejuvenation Res 2011;14:491–500.

50 Seripa D, Matera MG, Daniele A et al. The missing ApoE allele. Ann Hum Genet 2007;71:496–500.

51 Serrano-Pozo A, Qian J, Monsell SE et al. APOEϵ2 is associated with milder clinical and pathological Alzheimer disease. Ann Neurol 2015;77:917–929.

52 Sestak AL, Shaver TS, Moser KL et al. Familial aggregation of lupus and autoimmunity in an unusual multiplex pedigree. J Rheumatol 1999;26:1495–1499.

53 Sevigny J, Chiao P, Bussière T et al. The antibody aducanumab reduces Aβ plaques in Alzheimer’s disease. Nature 2016;537:50–56.

54 Shi Y, Yamada K, Liddelow SA et al.; Alzheimer’s Disease Neuroimaging Initiative. ApoE4 markedly exacerbates tau-mediated neurodegeneration in a mouse model of tauopathy. Nature 2017;549:523–527.

55 Smith CJ, Ashford JW, Perfetti TA. Putative survival advantages in young apolipoprotein ϵ4 carriers are associated with increased neural stress. J Alzheimers Dis 2019a;68:885–923.

56 Smith EMD, Lythgoe H, Midgley A et al. Juvenile-onset systemic lupus erythematosus: update on clinical presentation, pathophysiology and treatment options. Clin Immunol 2019b;209:108274.

57 Stitziel NO, Stirrups KE, Masca NG et al.; Myocardial Infarction Genetics and CARDIoGRAM Exome Consortia Investigators. Coding variation in ANGPTL4, LPL, and SVEP1 and the risk of coronary disease. N Engl J Med 2016;374:1134–1144.

58 Sturm AC, Knowles JW, Gidding SS et al.; Convened by the Familial Hypercholesterolemia Foundation. Clinical genetic testing for familial hypercholesterolemia: JACC scientific expert panel. J Am Coll Cardiol 2018;72:662–680.

59 Tanzi RE. The genetics of Alzheimer disease. Cold Spring Harb Perspect Med 2012;2:a006296.

60 van Dyck CH. Anti-amyloid-β monoclonal antibodies for Alzheimer’s disease: pitfalls and promise. Biol Psychiatry 2018;83:311–319.

61 Verghese PB, Castellano JM, Garai K et al. ApoE influences amyloid-β (Aβ) clearance despite minimal apoE/Aβ association in physiological conditions. Proc Natl Acad Sci USA 2013;110:25.

62 Walton RT, Christie KA, Whittaker MN et al. Unconstrained genome targeting with near-PAMless engineered CRISPR-Cas9 variants. Science 2020;368:290–296.

63 Wang X, Li J, Wang Y et al. Efficient base editing in methylated regions with a human APOBEC3A-Cas9 fusion. Nat Biotechnol 2018;36:946–949.

64 Wennberg AM, Tosakulwong N, Lesnick TG et al. Association of apolipoprotein E ϵ4 with transactive response DNA-binding protein 43. JAMA Neurol 2018;75:1347–1354.

65 Wilm A, Aw PP, Bertrand D et al. LoFreq: a sequence-quality aware, ultra-sensitive variant caller for uncovering cell-population heterogeneity from high-throughput sequencing datasets. Nucleic Acids Res 2012;40:11189–11201.

66 Yamada M, Johannesson B, Sagi I et al. Human oocytes reprogram adult somatic nuclei of a type 1 diabetic to diploid pluripotent stem cells. Nature 2014;510:533–536.

67 Yang WL, Shen N, Ye DQ et al. Genome-wide association study in Asian populations identifies variants in ETS1 and WDFY4 associated with systemic lupus erythematosus. PLoS Genet 2010;6:e1000841.

68 Yin J, Liu M, Liu Y et al. Optimizing genome editing strategy by primer-extension-mediated sequencing. Cell Discov 2019; 5:18.

69 Zeng Y, Li J, Li G et al. Correction of the marfan syndrome pathogenic FBN1 mutation by base editing in human cells and heterozygous embryos. Mol Ther 2018;26:2631–2637.

70 Zhang M, Zhou C, Wei Y et al. Human cleaving embryos enable robust homozygotic nucleotide substitutions by base editors. Genome Biol 2019;20:101.

71 Zhang Y, Bo L, Zhang H et al. E26 transformation-specific-1 (ETS1) and WDFY family member 4 (WDFY4) polymorphisms in Chinese patients with rheumatoid arthritis. Int J Mol Sci 2014;15:2712–2721.

72 Zhou C, Sun Y, Yan R et al. Off-target RNA mutation induced by DNA base editing and its elimination by mutagenesis. Nature 2019;571:275–278.

73 Zlokovic BV. Cerebrovascular effects of apolipoprotein E: implications for Alzheimer disease. JAMA Neurol 2013;70:440–444.

74 Zuccaro MV, Xu J, Mitchell C et al. (2020) Allele-specific chromosome removal after Cas9 cleavage in human embryos. Cell 183:1650–1664.e15 e1615.

75 Zuo E, Sun Y, Wei W et al. Cytosine base editor generates substantial off-target single-nucleotide variants in mouse embryos. Science 2019;364:289–292.

76 Zuo E, Sun Y, Yuan T et al. A rationally engineered cytosine base editor retains high on-target activity while reducing both DNA and RNA off-target effects. Nat Methods 2020;17:600–604.