Research progress of synthetic mammalian genomics

doi:10.12211/2096-8280.2021-006

Abstract

Abstract:

Synthetic genomics aims at genome-scale engineering or de novo synthesis through the design and chemical synthesis of large DNA sequences, which contributes to the revealing of connections between genotype and phenotype to construct organisms with expected functions. With the advances of synthetic genomics in lower model organisms, such as Escherichia coli and Saccharomyces cerevisiae, designing and rebuilding the large DNA fragments for mammalians could enhance the functional remodeling of their genomes. Designing higher mammalian genomes based on principles developed with the lower organism genome design can lead to understanding of more complex mammalian genomes, and in the meantime improve the design principles. In spite of multiple challenges, design and synthesis of mammalian large DNA fragments would provide promising methods and solutions. Overcoming the technical difficulties of manipulating large fragments of DNA in mammalian cells shows unique potentials in a variety of applications. For example, it can customize chromosomes for the construction of chromosomal disease models, build more complete humanized immune systems, etc. However, the current design and manipulation of large DNA fragments in mammalian cells are faced with many unsolved bottlenecks. Although the whole genome sequencing of several higher mammals has been completed, the annotation for the genomes of higher mammals is still far from complete. The existing assembly technology is difficult to accurately assemble complex repetitive sequences, and the vector presents poor versatility for shuttling between different cells. Moreover, the lower efficiency in transferring large fragments of DNA is a major bottleneck hindering the manipulation of large fragments in mammalian cells. This article systematically reviews recent progress in synthetic mammalian genomics by focusing on the breakthroughs in design-assembly-transfer technical route, and highlights further applications in human medicines and healthcare field.

Key words: synthetic genomics, genome writing, synthetic mammalian chromosome, genome engineering, mammalian cells, manipulation of large DNA

摘要：

合成基因组学通过设计与合成大片段DNA序列，开展基因组尺度的工程化改造或从头合成，从而揭示基因型和表型的关联，并构建预定功能的生物。正如在大肠杆菌、酿酒酵母等低等模式生物中合成基因组学的实践，对哺乳动物基因组的大片段DNA设计再造也必然会加深对更为复杂的动物基因组的理解并加强对基因组的功能重塑。面对生命健康领域存在的重大挑战，设计合成哺乳动物大片段DNA为其提供了新的思路和解决方案，特别是在染色体疾病模型构建、人源化免疫系统等方面展示出独特的应用潜力。然而，目前大片段DNA在哺乳动物细胞中的设计和操纵仍是一个巨大的挑战，面临着高等哺乳动物基因组注释不完善、复杂序列组装困难、穿梭载体通用性差、大片段DNA转移低效等问题。本文围绕设计-组装-转移的技术路线，评述哺乳动物合成基因组学领域的重要研究进展，详细介绍重要的技术突破，并展望哺乳动物合成基因组学在医药健康领域的应用。

关键词: 合成基因组学, 基因组编写, 合成型哺乳动物染色体, 基因组工程, 哺乳动物细胞, 大片段DNA操纵技术

CLC Number:

Bo HE, Zongheng FU, Yi WU, Guangrong ZHAO. Research progress of synthetic mammalian genomics[J]. Synthetic Biology Journal, 2022, 3(1): 78-97.

何博, 付宗恒, 吴毅, 赵广荣. 哺乳动物合成基因组学研究进展[J]. 合成生物学, 2022, 3(1): 78-97.

Figures/Tables 4

Fig. 1 Design principles for genomes

Fig. 2 Centromeric regions of synthetic mammalian chromosomes(a) Bottom-up centromere region based on satellite repeat sequence; (b) Bottom-up centromere region based on non-natural repetitive sequences; (c) Top-down artificial chromosomes from truncated natural chromosomes

Tab. 1 Method for delivering large fragments of DNA to mammalian cells

方法	转移介质	最大承载量	目的细胞	存在缺点	文献
脂质体转染	FuGENE 6	200 kb	HT1080	脂质材料对细胞有毒性	[46]
脂质体转染	Lipofection	400 kb	HT1080	脂质材料对细胞有毒性	[86]
电转	缓冲溶液	200 kb	mESc	需要昂贵细胞电转仪	[63, 87]
显微注射	缓冲溶液	590 kb	CHO	显微操作通量低	[88-89]
病毒载体	HSV-1	152 kb	hESc	操作步骤烦琐、存在生物安全问题	[90-91]
病毒载体	Epstein-barr virus	330 kb	Raji	操作步骤烦琐、存在生物安全问题	[92]
MMCT	微细胞	Mb级	HT1080	操作步骤烦琐	[93]
MMCT	微细胞	Mb级	Hela
Eco-MMCT	微细胞	Mb级	mESc
酵母原生质体-细胞融合	酵母原生质体	1.1 Mb	HEK293	操作步骤烦琐	[94]
		100 kb	HeLa		[95]
		1.4 Mb	mES

Fig. 3 Method for delivering large fragments of DNA to mammalian cells(a) DNA molecules assembled by S.cerevisiae are enriched in E. coli, extracted and purified in vitro and transferred to host cells by microinjection, lipofection, electroporation, or virus-mediated transduction; (b) DNA molecules assembled by S.cerevisiae are transferred to host cells through the PEG-mediated fusion of yeast protoplasts and mammalian cells; (c) Top-down artificial chromosomes constructed in donor cell are transferred to host cell through the microcell-mediated chromosome transfer

References 128

1	丁明珠, 李炳志, 王颖, 等. 合成生物学重要研究方向进展[J]. 合成生物学, 2020, 1(1): 7-28.
	DING M Z, LI B Z, WANG Y, et al. Significant research progress in synthetic biology[J]. Synthetic Biology Journal, 2020, 1(1): 7-28.
2	BOEKE J D, CHURCH G, HESSEL A, et al. The genome project-write[J]. Science, 2016, 353(6295): 126-127.
3	BAKER M. Synthetic genomes: the next step for the synthetic genome[J]. Nature, 2011, 473(7347): 403, 405-403, 408.
4	GIBSON D G, GLASS J I, LARTIGUE C, et al. Creation of a bacterial cell controlled by a chemically synthesized genome[J]. Science, 2010, 329(5987): 52-56.
5	HUTCHISON C A, CHUANG R Y, NOSKOV V N, et al. Design and synthesis of a minimal bacterial genome[J]. Science, 2016, 351(6280): aad6253.
6	WANG K H, FREDENS J, BRUNNER S F, et al. Defining synonymous codon compression schemes by genome recoding[J]. Nature, 2016, 539(7627): 59-64.
7	FREDENS J, WANG K H, DE LA TORRE D, et al. Total synthesis of Escherichia coli with a recoded genome[J]. Nature, 2019, 569(7757): 514-518.
8	SHEN Y, WANG Y, CHEN T, et al. Deep functional analysis of synII, a 770-kilobase synthetic yeast chromosome[J]. Science, 2017, 355(6329): eaaf4791.
9	MERCY G, MOZZICONACCI J, SCOLARI V F, et al. 3D organization of synthetic and scrambled chromosomes[J]. Science, 2017, 355(6329): eaaf4597.
10	XIE Z X, LI B Z, MITCHELL L A, et al. “Perfect” designer chromosome V and behavior of a ring derivative[J]. Science, 2017, 355(6329): eaaf4704.
11	MITCHELL L A, WANG A, STRACQUADANIO G, et al. Synthesis, debugging, and effects of synthetic chromosome consolidation: synVI and beyond[J]. Science, 2017, 355(6329): eaaf4831.
12	WU Y, LI B Z, ZHAO M, et al. Bug mapping and fitness testing of chemically synthesized chromosome X[J]. Science, 2017, 355(6329): eaaf4706.
13	ZHANG W M, ZHAO G H, LUO Z Q, et al. Engineering the ribosomal DNA in a megabase synthetic chromosome[J]. Science, 2017, 355(6329): eaaf3981.
14	OSTROV N, BEAL J, ELLIS T, et al. Technological challenges and milestones for writing genomes[J]. Science, 2019, 366(6463): 310-312.
15	HOSHIKA S, LEAL N A, KIM M J, et al. Hachimoji DNA and RNA: a genetic system with eight building blocks[J]. Science, 2019, 363(6429): 884-887.
16	OSTROV N, LANDON M, GUELL M, et al. Design, synthesis, and testing toward a 57-codon genome[J]. Science, 2016, 353(6301): 819-822.
17	HOCHREIN L, MITCHELL L A, SCHULZ K, et al. L-SCRaMbLE as a tool for light-controlled Cre-mediated recombination in yeast[J]. Nature Communications, 2018, 9: 1931.doi:10.1038/s41467-017-02208-6 .
18	LUO Z Q, WANG L H, WANG Y, et al. Identifying and characterizing SCRaMbLEd synthetic yeast using ReSCuES[J]. Nature Communications, 2018, 9: 1930.doi:10.1038/s41467-017-00866-y .
19	LIU W, LUO Z Q, WANG Y, et al. Rapid pathway prototyping and engineering using in vitro and in vivo synthetic genome SCRaMbLE-in methods[J]. Nature Communications, 2018, 9: 1936.doi:10.1038/s41467-018-04254-0 .
20	WU Y, ZHU R Y, MITCHELL L A, et al. In vitro DNA SCRaMbLE[J]. Nature Communications, 2018, 9(1): 1935.doi:10.1038/s41467-018-03743-6 .
21	SHEN M J, WU Y, YANG K, et al. Heterozygous diploid and inter species SCRaMbLEing[J]. Nature Communications, 2018, 9(1): 1934.doi:10.1038/s41467-018-04157-0 .
22	JIA B, WU Y, LI B Z, et al. Precise control of SCRaMbLE in synthetic haploid and diploid yeast[J]. Nature Communications, 2018, 9: 1933.doi:10.1038/s41467-018-03084-4 .
23	BLOUNT B A, GOWERS G O F, HO J C H, et al. Rapid host strain improvement by in vivo rearrangement of a synthetic yeast chromosome[J]. Nature Communications, 2018, 9: 1932.doi:10.1038/s41467-018-03143-w .
24	JIANG S, LI H, HONG H, et al. Spatial density of open chromatin: an effective metric for the functional characterization of topologically associated domains[J]. Briefings in Bioinformatics, 2020, 22(3): bbaa210.
25	MALYSHEV D A, DHAMI K, LAVERGNE T, et al. A semi-synthetic organism with an expanded genetic alphabet[J]. Nature, 2014, 509(7500): 385-388.
26	LUO X Z, REITER M A, D'ESPAUX L, et al. Complete biosynthesis of cannabinoids and their unnatural analogues in yeast[J]. Nature, 2019, 567(7746): 123-126.
27	ROBERTSON W E, FUNKE L F H, DE LA TORRE D, et al. Creating custom synthetic genomes in Escherichia coli with REXER and GENESIS[J]. Nature Protocols, 2021, 16(5): 2345-2380.
28	ROBERTSON W E, FUNKE L F H, DE LA TORRE D, et al. Sense codon reassignment enables viral resistance and encoded polymer synthesis[J]. Science, 2021, 372(6546): 1057-1062.
29	RICHARDSON S M, MITCHELL L A, STRACQUADANIO G, et al. Design of a synthetic yeast genome[J]. Science, 2017, 355(6329): 1040-1044.
30	KANNAN K, GIBSON D G. Yeast genome, by design[J]. Science, 2017, 355(6329): 1024-1025.
31	CONSORTIUM E P. The ENCODE (ENCyclopedia Of DNA Elements) Project[J]. Science, 2004, 306(5696): 636-640.
32	QU H Z, FANG X D. A brief review on the human encyclopedia of DNA elements (ENCODE) project[J]. Genomics, Proteomics & Bioinformatics, 2013, 11(3): 135-141.
33	CONSORTIUM E P, SNYDER M P, GINGERAS T R, et al. Perspectives on encode[J]. Nature, 2020, 583(7818): 693-698.
34	LEE C M, BARBER G P, CASPER J, et al. UCSC Genome Browser enters 20th year[J]. Nucleic Acids Research, 2020, 48(D1): D756-D761.
35	KARCZEWSKI K J, FRANCIOLI L C, TIAO G, et al. The mutational constraint spectrum quantified from variation in 141, 456 humans[J]. Nature, 2020, 581(7809): 434-443.
36	TAM V, PATEL N, TURCOTTE M, et al. Benefits and limitations of genome-wide association studies[J]. Nature Reviews Genetics, 2019, 20(8): 467-484.
37	LANDER E S, LINTON L M, BIRREN B, et al. Initial sequencing and analysis of the human genome[J]. Nature, 2001, 409(6822): 860-921.
38	VENTER J C, ADAMS M D, MYERS E W, et al. The sequence of the human genome[J]. Science, 2001, 291(5507): 1304-1351.
39	International Human Genome Sequencing Consortium. Finishing the euchromatic sequence of the human genome[J]. Nature, 2004, 431(7011): 931-945.
40	LOGSDON G A, VOLLGER M R, HSIEH P, et al. The structure, function and evolution of a complete human chromosome 8[J]. Nature, 2021, 593(7857): 101-107.
41	CARNINCI P, KASUKAWA T, KATAYAMA S, et al. The transcriptional landscape of the mammalian genome[J]. Science, 2005, 309(5740): 1559-1563.
42	JUMPER J, EVANS R, PRITZEL A, et al. Highly accurate protein structure prediction with AlphaFold[J]. Nature, 2021, 596(7873): 583-589.
43	TUNYASUVUNAKOOL K, ADLER J, WU Z, et al. Highly accurate protein structure prediction for the human proteome[J]. Nature, 2021, 596(7873): 590-596.
44	HAYDEN K E. Human centromere genomics: now it’s personal[J]. Chromosome Research, 2012, 20(5): 621-633.
45	FUKAGAWA T, EARNSHAW W C. The centromere: chromatin foundation for the kinetochore machinery[J]. Developmental Cell, 2014, 30(5): 496-508.
46	LOGSDON G A, GAMBOGI C W, LISKOVYKH M A, et al. Human artificial chromosomes that bypass centromeric DNA[J]. Cell, 2019, 178(3): 624-639.
47	EBERSOLE T A, ROSS A, CLARK E, et al. Mammalian artificial chromosome formation from circular alphoid input DNA does not require telomere repeats[J]. Human Molecular Genetics, 2000, 9(11): 1623-1631.
48	GRIMES B R, RHOADES A A, WILLARD H F. α-Satellite DNA and vector composition influence rates of human artificial chromosome formation[J]. Molecular Therapy, 2002, 5(6): 798-805.
49	IKENO M, GRIMES B, OKAZAKI T, et al. Construction of YAC-based mammalian artificial chromosomes[J]. Nature Biotechnology, 1998, 16(5): 431-439.
50	MEJÍA J E, ALAZAMI A, WILLMOTT A, et al. Efficiency of de novo centromere formation in human artificial chromosomes[J]. Genomics, 2002, 79(3): 297-304.
51	OHZEKI J I, BERGMANN J H, KOUPRINA N, et al. Breaking the HAC Barrier: Histone H3K9 acetyl/methyl balance regulates CENP-A assembly[J]. The EMBO Journal, 2012, 31(10): 2391-2402.
52	OKADA T, OHZEKI J I, NAKANO M, et al. CENP-B controls centromere formation depending on the chromatin context[J]. Cell, 2007, 131(7): 1287-1300.
53	HARRINGTON J J, BOKKELEN G VAN, MAYS R W, et al. Formation of de novo centromeres and construction of first-generation human artificial microchromosomes[J]. Nature Genetics, 1997, 15(4): 345-355.
54	IIDA Y, KIM J H, KAZUKI Y, et al. Human artificial chromosome with a conditional centromere for gene delivery and gene expression[J]. DNA Research, 2010, 17(5): 293-301.
55	KOUPRINA N, PETROV N, MOLINA O, et al. Human artificial chromosome with regulated centromere: a tool for genome and cancer studies[J]. ACS Synthetic Biology, 2018, 7(9): 1974-1989.
56	KOUPRINA N, SAMOSHKIN A, ERLIANDRI I, et al. Organization of synthetic alphoid DNA array in human artificial chromosome (HAC) with a conditional centromere[J]. ACS Synthetic Biology, 2012, 1(12): 590-601.
57	KIM J H, KONONENKO A, ERLIANDRI I, et al. Human artificial chromosome (HAC) vector with a conditional centromere for correction of genetic deficiencies in human cells[J]. Proceedings of the National Academy of Sciences of the United States of America, 2011, 108(50): 20048-20053.
58	PESENTI E, LISKOVYKH M, OKAZAKI K, et al. Analysis of complex DNA rearrangements during early stages of HAC formation[J]. ACS Synthetic Biology, 2020, 9(12): 3267-3287.
59	BARNETT M A, BUCKLE V J, EVANS E P, et al. Telomere directed fragmentation of mammalian chromosomes[J]. Nucleic Acids Research, 1993, 21(1): 27-36.
60	BROWN D M, GLASS J I. Technology used to build and transfer mammalian chromosomes[J]. Experimental Cell Research, 2020, 388(2): 111851.
61	LAJOIE M J, ROVNER A J, GOODMAN D B, et al. Genomically recoded organisms expand biological functions[J]. Science, 2013, 342(6156): 357-360.
62	KLAWITTER S, FUCHS N V, UPTON K R, et al. Reprogramming triggers endogenous L1 and Alu retrotransposition in human induced pluripotent stem cells[J]. Nature Communications, 2016, 7: 10286.
63	LEE E C, LIANG Q, ALI H, et al. Complete humanization of the mouse immunoglobulin loci enables efficient therapeutic antibody discovery[J]. Nature Biotechnology, 2014, 32(4): 356-363.
64	MULLER H, ANNALURU N, SCHWERZMANN J W, et al. Assembling large DNA segments in yeast[J]. Methods in Molecular Biology, 2012, 852: 133-150.
65	JUHAS M, AJIOKA J W. High molecular weight DNA assembly in vivo for synthetic biology applications[J]. Critical Reviews in Biotechnology, 2017, 37(3): 277-286.
66	ITAYA M, TSUGE K, KOIZUMI M, et al. Combining two genomes in one cell: stable cloning of the Synechocystis PCC6803 genome in the Bacillus subtilis 168 genome[J]. Proceedings of the National Academy of Sciences of the United States of America, 2005, 102(44): 15971-15976.
67	ITAYA M, FUJITA K, KUROKI A, et al. Bottom-up genome assembly using the Bacillus subtilis genome vector[J]. Nature Methods, 2008, 5(1): 41-43.
68	ZHANG Y M, BUCHHOLZ F, MUYRERS J P P, et al. A new logic for DNA engineering using recombination in Escherichia coli [J]. Nature Genetics, 1998, 20(2): 123-128.
69	FU J, BIAN X Y, HU S, et al. Full-length RecE enhances linear-linear homologous recombination and facilitates direct cloning for bioprospecting[J]. Nature Biotechnology, 2012, 30(5): 440-446.
70	GIBSON D G, BENDERS G A, AXELROD K C, et al. One-step assembly in yeast of 25 overlapping DNA fragments to form a complete synthetic Mycoplasma genitalium genome[J]. Proceedings of the National Academy of Sciences of the United States of America, 2008, 105(51): 20404-20409.
71	ZHOU J T, WU R H, XUE X L, et al. CasHRA (Cas9-facilitated Homologous Recombination Assembly) method of constructing megabase-sized DNA[J]. Nucleic Acids Research, 2016, 44(14): e124.
72	MITCHELL L A, MCCULLOCH L H, PINGLAY S, et al. De novo assembly and delivery to mouse cells of a 101 kb functional human gene[J]. Genetics, 2021, 218(1). .
73	POSTMA E D, DASHKO S, BREEMEN L VAN, et al. A supernumerary designer chromosome for modular in vivo pathway assembly in Saccharomyces cerevisiae [J]. Nucleic Acids Research, 2021, 49(3): 1769-1783.
74	SHAO Y Y, LU N, WU Z F, et al. Creating a functional single-chromosome yeast[J]. Nature, 2018, 560(7718): 331-335.
75	TAGWERKER C, DUPONT C L, KARAS B J, et al. Sequence analysis of a complete 1.66 Mb Prochlorococcus marinus MED4 genome cloned in yeast[J]. Nucleic Acids Research, 2012, 40(20): 10375-10383.
76	KARAS B J, TAGWERKER C, YONEMOTO I T, et al. Cloning the Acholeplasma laidlawii PG-8A genome in Saccharomyces cerevisiae as a yeast centromeric plasmid[J]. ACS Synthetic Biology, 2012, 1(1): 22-28.
77	王培霞, 马渊, 吴毅. 大DNA体内组装技术进展[J]. 生物加工过程, 2019, 17(1): 15-22.
	WANG Peixia, MA Yuan, WU Yi. Advances in large DNA in vivo assembly[J]. Chinese Journal of Bioprocess Engineering, 2019, 17(1): 15-22.
78	NOSKOV V N, KARAS B J, YOUNG L, et al. Assembly of large, high G+C bacterial DNA fragments in yeast[J]. ACS Synthetic Biology, 2012, 1(7): 267-273.
79	KAZUKI Y, HOSHIYA H, TAKIGUCHI M, et al. Refined human artificial chromosome vectors for gene therapy and animal transgenesis[J]. Gene Therapy, 2011, 18(4): 384-393.
80	KUROIWA Y, TOMIZUKA K, SHINOHARA T, et al. Manipulation of human minichromosomes to carry greater than megabase-sized chromosome inserts[J]. Nature Biotechnology, 2000, 18(10): 1086-1090.
81	KUROIWA Y, KASINATHAN P, CHOI Y J, et al. Cloned transchromosomic calves producing human immunoglobulin[J]. Nature Biotechnology, 2002, 20(9): 889-894.
82	SANO A, MATSUSHITA H, WU H, et al. Physiological level production of antigen-specific human immunoglobulin in cloned transchromosomic cattle[J]. PLoS One, 2013, 8(10): e78119.
83	BUERSTEDDE J M, TAKEDA S. Increased ratio of targeted to random integration after transfection of chicken B cell lines[J]. Cell, 1991, 67(1): 179-188.
84	WILLBANKS A, LEARY M, GREENSHIELDS M, et al. The evolution of epigenetics: from prokaryotes to humans and its biological consequences[J]. Genetics & Epigenetics, 2016, 8: 25-36.
85	LARTIGUE C, VASHEE S, ALGIRE M A, et al. Creating bacterial strains from genomes that have been cloned and engineered in yeast[J]. Science, 2009, 325(5948): 1693-1696.
86	MEJÍA J E, WILLMOTT A, LEVY E, et al. Functional complementation of a genetic deficiency with human artificial chromosomes[J]. The American Journal of Human Genetics, 2001, 69(2): 315-326.
87	SHI J F, MA Y F, ZHU J, et al. A review on electroporation-based intracellular delivery[J]. Molecules, 2018, 23(11): 3044.
88	STEWART M P, LANGER R, JENSEN K F. Intracellular delivery by membrane disruption: Mechanisms, strategies, and concepts[J]. Chemical Reviews, 2018, 118(16): 7409-7531.
89	GNIRKE A, HUXLEY C, PETERSON K, et al. Microinjection of intact 200- to 500-kb fragments of YAC DNA into mammalian cells[J]. Genomics, 1993, 15(3): 659-667.
90	WADE-MARTINS R, SMITH E R, TYMINSKI E, et al. An infectious transfer and expression system for genomic DNA loci in human and mouse cells[J]. Nature Biotechnology, 2001, 19(11): 1067-1070.
91	MORALLI D, MONACO Z L. Developing de novo human artificial chromosomes in embryonic stem cells using HSV-1 amplicon technology[J]. Chromosome Research, 2015, 23(1): 105-110.
92	SUN T Q, FENSTERMACHER D A, VOS J M H. Human artificial episomal chromosomes for cloning large DNA fragments in human cells[J]. Nature Genetics, 1994, 8(1): 33-41.
93	SUZUKI T, KAZUKI Y, HARA T, et al. Current advances in microcell-mediated chromosome transfer technology and its applications[J]. Experimental Cell Research, 2020, 390(1): 111915.
94	BROWN D M, CHAN Y A, DESAI P J, et al. Efficient size-independent chromosome delivery from yeast to cultured cell lines[J]. Nucleic Acids Research, 2016, 45(7): e50.
95	LI L P, BLANKENSTEIN T. Generation of transgenic mice with megabase-sized human yeast artificial chromosomes by yeast spheroplast-embryonic stem cell fusion[J]. Nature Protocols, 2013, 8(8): 1567-1582.
96	MARSCHALL P, MALIK N, LARIN Z. Transfer of YACs up to 2.3 Mb intact into human cells with polyethylenimine[J]. Gene Therapy, 1999, 6(9): 1634-1637.
97	MACDONALD L E, KAROW M, STEVENS S, et al. Precise and in situ genetic humanization of 6 Mb of mouse immunoglobulin genes[J]. Proceedings of the National Academy of Sciences of the United States of America, 2014, 111(14): 5147-5152.
98	MURPHY A J, MACDONALD L E, STEVENS S, et al. Mice with megabase humanization of their immunoglobulin genes generate antibodies as efficiently as normal mice[J]. Proceedings of the National Academy of Sciences of the United States of America, 2014, 111(14): 5153-5158.
99	LARTIGUE C, GLASS J I, ALPEROVICH N, et al. Genome transplantation in bacteria: changing one species to another[J]. Science, 2007, 317(5838): 632-638.
100	MONTOLIU L. Purification of yeast artificial chromosome DNA for microinjection using pulsed-field gel electrophoresis and ultrafiltration[J]. Cold Spring Harbor Protocols, 2018. doi:10.1101/pdb.prot093948
101	MONTOLIU L. Large-scale preparation of yeast agarose plugs to isolate yeast artificial chromosome DNA[J]. Cold Spring Harbor Protocols, 2018. DOI: 10.1101/pdb.prot093955
102	LEE J T, JAENISCH R. A method for high efficiency YAC lipofection into murine embryonic stem cells[J]. Nucleic Acids Research, 1996, 24(24): 5054-5055.
103	DOHERTY A M, FISHER E M. Microcell-mediated chromosome transfer (MMCT): small cells with huge potential[J]. Mammalian Genome, 2003, 14(9): 583-592.
104	SINENKO S A, PONOMARTSEV S V, TOMILIN A N. Human artificial chromosomes for pluripotent stem cell-based tissue replacement therapy[J]. Experimental Cell Research, 2020, 389(1): 111882.
105	LISKOVYKH M, LEE N C, LARIONOV V, et al. Moving toward a higher efficiency of microcell-mediated chromosome transfer[J]. Molecular Therapy-Methods & Clinical Development, 2016, 3: 16043.
106	SUZUKI T, KAZUKI Y, OSHIMURA M, et al. Highly efficient transfer of chromosomes to a broad range of target cells using Chinese hamster ovary cells expressing murine leukemia virus-derived envelope proteins[J]. PLoS One, 2016, 11(6): e0157187.
107	JAKOBOVITS A, MOORE A L, GREEN L L, et al. Germ-line transmission and expression of a human-derived yeast artificial chromosome[J]. Nature, 1993, 362(6417): 255-258.
108	LI L P, LAMPERT J C, CHEN X J, et al. Transgenic mice with a diverse human T cell antigen receptor repertoire[J]. Nature Medicine, 2010, 16(9): 1029-1034.
109	SUZUKI N, ITOU T, HASEGAWA Y, et al. Cell to cell transfer of the chromatin-packaged human β-globin gene cluster[J]. Nucleic Acids Research, 2009, 38(5): e33.
110	O'DOHERTY A, RUF S, MULLIGAN C, et al. An aneuploid mouse strain carrying human chromosome 21 with Down syndrome phenotypes[J]. Science, 2005, 309(5743): 2033-2037.
111	KAZUKI Y, HIRATSUKA M, TAKIGUCHI M, et al. Complete genetic correction of iPS cells from Duchenne muscular dystrophy[J]. Molecular Therapy, 2010, 18(2): 386-393.
112	TAYLOR L D, CARMACK C E, HUSZAR D, et al. Human immunoglobulin transgenes undergo rearrangement, somatic mutation and class switching in mice that lack endogenous IgM[J]. International Immunology, 1994, 6(4): 579-591.
113	GREEN L L, HARDY M C, MAYNARD-CURRIE C E, et al. Antigen-specific human monoclonal antibodies from mice engineered with human Ig heavy and light chain YACs[J]. Nature Genetics, 1994, 7(1): 13-21.
114	LONBERG N, TAYLOR L D, HARDING F A, et al. Antigen-specific human antibodies from mice comprising four distinct genetic modifications[J]. Nature, 1994, 368(6474): 856-859.
115	SCHWARTZBERG P L, GOFF S P, ROBERTSON E J. Germ-line transmission of a c-abl mutation produced by targeted gene disruption in ES cells[J]. Science, 1989, 246(4931): 799-803.
116	ZIJLSTRA M, LI E, SAJJADI F, et al. Germ-line transmission of a disrupted beta 2-microglobulin gene produced by homologous recombination in embryonic stem cells[J]. Nature, 1989, 342(6248): 435-438.
117	MANSOUR S L, THOMAS K R, CAPECCHI M R. Disruption of the proto-oncogene int-2 in mouse embryo-derived stem cells: a general strategy for targeting mutations to non-selectable genes[J]. Nature, 1988, 336(6197): 348-352.
118	BRÜGGEMANN M, CASKEY H M, TEALE C, et al. A repertoire of monoclonal antibodies with human heavy chains from transgenic mice[J]. Proceedings of the National Academy of Sciences of the United States of America, 1989, 86(17): 6709-6713.
119	XU J L, XU K, JUNG S, et al. Nanobodies from camelid mice and llamas neutralize SARS-CoV-2 variants[J]. Nature, 2021, 595(7866): 278-282.
120	MATSUSHITA H, SANO A, WU H, et al. Species-specific chromosome engineering greatly improves fully human polyclonal antibody production profile in cattle[J]. PLoS One, 2015, 10(6): e0130699.
121	GARDNER C L, SUN C Q, LUKE T, et al. Antibody preparations from human transchromosomic cows exhibit prophylactic and therapeutic efficacy against venezuelan equine encephalitis virus[J]. Journal of Virology, 2017, 91(14): e00226.
122	LUKE T, WU H, ZHAO J C, et al. Human polyclonal immunoglobulin G from transchromosomic bovines inhibits MERS-CoV in vivo [J]. Science Translational Medicine, 2016, 8(326): 326ra21.
123	BEIGEL J H, VOELL J, KUMAR P, et al. Safety and tolerability of a novel, polyclonal human anti-MERS coronavirus antibody produced from transchromosomic cattle: a phase 1 randomised, double-blind, single-dose-escalation study[J]. The Lancet Infectious Diseases, 2018, 18(4): 410-418.
124	NIU D, WEI H J, LIN L, et al. Inactivation of porcine endogenous retrovirus in pigs using CRISPR-Cas9[J]. Science, 2017, 357(6357): 1303-1307.
125	YUE Y N, XU W H, KAN Y N, et al. Extensive germline genome engineering in pigs[J]. Nature Biomedical Engineering, 2021, 5(2): 134-143.
126	KAZUKI Y, KOBAYASHI K, AUEVIRIYAVIT S, et al. Trans-chromosomic mice containing a human CYP3A cluster for prediction of xenobiotic metabolism in humans[J]. Human Molecular Genetics, 2012, 22(3): 578-592.
127	UNO N, ABE S, OSHIMURA M, et al. Combinations of chromosome transfer and genome editing for the development of cell/animal models of human disease and humanized animal models[J]. Journal of Human Genetics, 2018, 63(2): 145-156.
128	KAZUKI Y, KOBAYASHI K, HIRABAYASHI M, et al. Humanized UGT2 and CYP3A transchromosomic rats for improved prediction of human drug metabolism[J]. Proceedings of the National Academy of Sciences of the United States of America, 2019, 116(8): 3072-3081.

[1]	Junyi WANG, Xiaole WU, Yueyang CAO, Bingzhi LI. Genome design and synthesis: from replication to rational design [J]. Synthetic Biology Journal, 2021, 2(2): 247-255.
[2]	Yi LI, Zhenquan LIN, Zihe LIU. Advances in yeast based adaptive laboratory evolution [J]. Synthetic Biology Journal, 2021, 2(2): 287-301.
[3]	Hui WANG, Junbiao DAI, Zhouqing LUO. Reading, editing, and writing techniques for genome research [J]. Synthetic Biology Journal, 2020, 1(5): 503-515.