Microinjected transgenes, both large and small, are known to insert randomly into the mouse genome. Traditional methods of mapping a transgene are challenging, thus complicating breeding strategies and accurate interpretation of phenotypes, particularly when a transgene disrupts critical coding or noncoding sequences. As the vast majority of transgenic mouse lines remain unmapped, we developed CRISPR-Cas9 Long-Read Sequencing (CRISPR-LRS) to ascertain transgene integration loci. This novel approach mapped a wide size range of transgenes and uncovered more complex transgene-induced host genome re-arrangements than previously appreciated. CRISPR-LRS offers a facile, informative approach to establish robust breeding practices and will enable researchers to study a gene without confounding genetic issues. Finally, CRISPR-LRS will find utility in rapidly and accurately interrogating gene/genome editing fidelity in experimental and clinical settings.
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References
1.
GoodwinLO, SplinterE, DavisTL, et al.Large-scale discovery of mouse transgenic integration sites reveals frequent structural variation and insertional mutagenesis. Genome Res, 2019; 29(3):494–505; doi: 10.1101/gr.233866.117
2.
NichollsPK, BellottDW, ChoTJ, et al.Locating and characterizing a transgene integration site by nanopore sequencing. G3 (Bethesda), 2019; 9(5):1481–1486; doi: 10.1534/g3.119.300582
3.
NakanishiT, KuroiwaA, YamadaS, et al.FISH analysis of 142 EGFP transgene integration sites into the mouse genome. Genomics, 2002; 80(6):564–574; doi: 10.1006/geno.2002.7008
4.
DuboseAJ, LichtensteinST, NarisuN, et al.Use of microarray hybrid capture and next-generation sequencing to identify the anatomy of a transgene. Nucleic Acids Res, 2013; 41(6):e70; doi: 10.1093/nar/gks1463
5.
Cain-HomC, SplinterE, van MinM, et al.Efficient mapping of transgene integration sites and local structural changes in Cre transgenic mice using targeted locus amplification. Nucleic Acids Res, 2017; 45(8):e62; doi: 10.1093/nar/gkw1329
6.
SchmidtM, SchwarzwaelderK, BartholomaeC, et al.High-resolution insertion-site analysis by linear amplification-mediated PCR (LAM-PCR). Nat Methods, 2007; 4(12):1051–1057; doi: 10.1038/nmeth1103
7.
de VreePJ, de WitE, YilmazM, et al.Targeted sequencing by proximity ligation for comprehensive variant detection and local haplotyping. Nat Biotechnol, 2014; 32(10):1019–1025; doi: 10.1038/nbt.2959
8.
LiangZ, BremanAM, GrimesBR, et al.Identifying and genotyping transgene integration loci. Transgenic Res, 2008; 17(5):979–983; doi: 10.1007/s11248-008-9190-7
9.
UemuraS, NagaokaT, YokoyamaM, et al.A simple and highly efficient method to identify the integration site of a transgene in the animal genome. Neurosci Res, 2014; 80:91–94; doi: 10.1016/j.neures.2013.11.007
10.
SailerS, CoassinS, LacknerK, et al.When the genome bluffs: A tandem duplication event during generation of a novel Agmo knockout mouse model fools routine genotyping. Cell Biosci, 2021; 11(1):54; doi: 10.1186/s13578-021-00566-9
11.
LaboulayeMA, DuanX, QiaoM, et al.Mapping transgene insertion sites reveals complex interactions between mouse transgenes and neighboring endogenous genes. Front Mol Neurosci, 2018; 11:385; doi: 10.3389/fnmol.2018.00385
12.
BurgioG, TeboulL. Anticipating and identifying collateral damage in genome editing. Trends Genet, 2020; 36(12):905–914; doi: 10.1016/j.tig.2020.09.011
13.
DeamerD, AkesonM, BrantonD. Three decades of nanopore sequencing. Nat Biotechnol, 2016; 34(5):518–524; doi: 10.1038/nbt.3423
14.
KonoN, ArakawaK. Nanopore sequencing: Review of potential applications in functional genomics. Dev Growth Differ, 2019; 61(5):316–326; doi: 10.1111/dgd.12608
15.
JinekM, ChylinskiK, FonfaraI, et al.A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science, 2012; 337(6096):816–821; doi: 10.1126/science.1225829
16.
WongsurawatT, JenjaroenpunP, De LooseA, et al.A novel Cas9-targeted long-read assay for simultaneous detection of IDH1/2 mutations and clinically relevant MGMT methylation in fresh biopsies of diffuse glioma. Acta Neuropathol Commun, 2020; 8(1):87; doi: 10.1186/s40478-020-00963-0
17.
WalshT, CasadeiS, MunsonKM, et al.CRISPR-Cas9/long-read sequencing approach to identify cryptic mutations in BRCA1 and other tumour suppressor genes. J Med Genet, 2021; 58(12):850–852; doi: 10.1136/jmedgenet-2020-107320
18.
MiyamotoS, AotoK, HiraideT, et al.Nanopore sequencing reveals a structural alteration of mirror-image duplicated genes in a genome-editing mouse line. Congenit Anom (Kyoto), 2020; 60(4):120–125; doi: 10.1111/cga.12364
19.
WatsonCM, CrinnionLA, HewittS, et al.Cas9-based enrichment and single-molecule sequencing for precise characterization of genomic duplications. Lab Invest, 2020; 100(1):135–146; doi: 10.1038/s41374-019-0283-0
20.
van HaasterenJ, MunisAM, GillDR, et al.Genome-wide integration site detection using Cas9 enriched amplification-free long-range sequencing. Nucleic Acids Res, 2021; 49(3):e16; doi: 10.1093/nar/gkaa1152
21.
GabrieliT, SharimH, FridmanD, et al.Selective nanopore sequencing of human BRCA1 by Cas9-assisted targeting of chromosome segments (CATCH). Nucleic Acids Res, 2018; 46(14):e87; doi: 10.1093/nar/gky411
22.
McDonaldTL, ZhouW, CastroCP, et al.Cas9 targeted enrichment of mobile elements using nanopore sequencing. Nat Commun, 2021; 12(1):3586; doi: 10.1038/s41467-021-23918-y
23.
GilpatrickT, LeeI, GrahamJE, et al.Targeted nanopore sequencing with Cas9-guided adapter ligation. Nat Biotechnol, 2020; 38(4):433–438; doi: 10.1038/s41587-020-0407-5
24.
LyuQ, XuS, LyuY, et al.SENCR stabilizes vascular endothelial cell adherens junctions through interaction with CKAP4. Proc Natl Acad Sci U S A, 2019; 116(2):546–555; doi: 10.1073/pnas.1810729116
25.
MianoJM, RamananN, GeorgerMA, et al.Restricted inactivation of serum response factor to the cardiovascular system. Proc Natl Acad Sci U S A, 2004; 101(49):17132–17137; doi: 10.1073/pnas.0406041101
26.
LiuY, LiH, SugiuraY, et al.Ubiquitin-synaptobrevin fusion protein causes degeneration of presynaptic motor terminals in mice. J Neurosci, 2015; 35(33):11514–11531; doi: 10.1523/JNEUROSCI.5288-14.2015
27.
LabunK, MontagueTG, KrauseM, et al.CHOPCHOP v3: Expanding the CRISPR web toolbox beyond genome editing. Nucleic Acids Res, 2019; 47(W1):W171–W174; doi: 10.1093/nar/gkz365
WarthiG, FaulknerJL, DojaJ, et al.Generation and comparative analysis of an Itga8-CreER (T2) mouse with preferential activity in vascular smooth muscle cells. Nat Cardiovasc Res, 2022; 1(11):1084–1100; doi: 10.1038/s44161-022-00162-1
30.
PalmiterRD, BrinsterRL. Germ-line transformation of mice. Annu Rev Genet, 1986; 20:465–499; doi: 10.1146/annurev.ge.20.120186.002341
DymeckiSM. Flp recombinase promotes site-specific DNA recombination in embryonic stem cells and transgenic mice. Proc Natl Acad Sci U S A, 1996; 93(12):6191–6196; doi: 10.1073/pnas.93.12.6191
33.
BaurST, MaiJJ, DymeckiSM. Combinatorial signaling through BMP receptor IB and GDF5: Shaping of the distal mouse limb and the genetics of distal limb diversity. Development, 2000; 127(3):605–619; doi: 10.1242/dev.127.3.605
34.
MandelkerD, SchmidtRJ, AnkalaA, et al.Navigating highly homologous genes in a molecular diagnostic setting: a resource for clinical next-generation sequencing. Genet Med, 2016; 18(12):1282–1289; doi: 10.1038/gim.2016.58
35.
Sanford KobayashiE, BatalovS, WengerAM, et al.Approaches to long-read sequencing in a clinical setting to improve diagnostic rate. Sci Rep, 2022; 12(1):16945; doi: 10.1038/s41598-022-20113-x
36.
LogsdonGA, EichlerEE. Mining the gaps of chromosome 8. Nature, 2021. [Epub ahead of print]; doi: 10.1038/d41586-021-01095-8
37.
LogsdonGA, VollgerMR, HsiehP, et al.The structure, function and evolution of a complete human chromosome 8. Nature, 2021; 593(7857):101–107; doi: 10.1038/s41586-021-03420-7
38.
TanakaM, YokoyamaK, HayashiH, et al.CRISPR-KRISPR: A method to identify on-target and random insertion of donor DNAs and their characterization in knock-in mice. Genome Biol, 2022; 23(1):228; doi: 10.1186/s13059-022-02779-8
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