Bi-FoRe: an efficient bidirectional knockin strategy to generate pairwise conditional alleles with fluorescent indicators

Abstract

Gene expression labeling and conditional manipulation of gene function are important for elaborate dissection of gene function. However, contemporary generation of pairwise dual-function knockin alleles to achieve both conditional and geno-tagging effects with a single donor has not been reported. Here we first developed a strategy based on a flipping donor named FoRe to generate conditional knockout alleles coupled with fluorescent allele-labeling through NHEJ-mediated unidirectional targeted insertion in zebrafish facilitated by the CRISPR/Cas system. We demonstrated the feasibility of this strategy at sox10 and isl1 loci, and successfully achieved Cre-induced conditional knockout of target gene function and simultaneous switch of the fluorescent reporter, allowing generation of genetic mosaics for lineage tracing. We then improved the donor design enabling efficient one-step bidirectional knockin to generate paired positive and negative conditional alleles, both tagged with two different fluorescent reporters. By introducing Cre recombinase, these alleles could be used to achieve both conditional knockout and conditional gene restoration in parallel; furthermore, differential fluorescent labeling of the positive and negative alleles enables simple, early and efficient realtime discrimination of individual live embryos bearing different genotypes prior to the emergence of morphologically visible phenotypes. We named our improved donor as Bi-FoRe and demonstrated its feasibility at the sox10 locus. Furthermore, we eliminated the undesirable bacterial backbone in the donor using minicircle DNA technology. Our system could easily be expanded for other applications or to other organisms, and coupling fluorescent labeling of gene expression and conditional manipulation of gene function will provide unique opportunities to fully reveal the power of emerging single-cell sequencing technologies.

Keywords

CRISPR/Cas / conditional knockout / allele labeling / conditional rescue / minicircle DNA

References

[1] Albert H, Dale EC, Lee E, Ow DW (1995) Site-specific integration of DNA into wild-type and mutant lox sites placed in the plant genome. Plant J 7:649–659

[2] Araki K, Araki M, Yamamura KI (2002) Site-directed integration of the cre gene mediated by Cre recombinase using a combination of mutant lox sites. Nucleic Acids Res 30:e103

[3] Araki K, Araki M, Yamamura KI (2006) Negative selection with the Diphtheria toxin A fragment gene improves frequency of Cremediated cassette exchange in ES cells. J Biochem 140:793–798

[4] Auer TO, Duroure K, De Cian A, Concordet JP, Del Bene F (2014) Highly efficient CRISPR/Cas9-mediated knock-in in zebrafish by homology-independent DNA repair. Genome Res 24:142–153

[5] Burg L, Palmer N, Kikhi K, Miroshnik ES, Rueckert H, Gaddy E, Cunningham CM, Mattonet K, Lai SL, Marin-Juez R (2018) Conditional mutagenesis by oligonucleotide-mediated integration of loxP sites in zebrafish. PLoS Genet 14:e1007754

[6] Caputo L, Witzel HR, Kolovos P, Cheedipudi S, Looso M, Mylona A, van IJcken WF, Laugwitz KL, Evans SM, Braun T (2015) The Isl1/Ldb1 complex orchestrates genome-wide chromatin organization to instruct differentiation of multipotent cardiac progenitors. Cell Stem Cell 17:287–299

[7] Carney TJ, Mosimann C (2018) Switch and trace: recombinase genetics in zebrafish. Trends Genet 34:362–378

[8] Chang NN, Sun CH, Gao L, Zhu D, Xu XF, Zhu XJ, Xiong JW, Xi JJ (2013) Genome editing with RNA-guided Cas9 nuclease in Zebrafish embryos. Cell Res 23:465–472

[9] Chen ZY, Yant SR, He CY, Meuse L, Shen S, Kay MA (2001) Linear DNAs concatemerize in vivo and result in sustained transgene expression in mouse liver. Mol Ther 3:403–410

[10] Chen ZY, He CY, Ehrhardt A, Kay MA (2003) Minicircle DNA vectors devoid of bacterial DNA result in persistent and high-level transgene expression in vivo. Mol Ther 8:495–500

[11] Dutton KA, Dutton JR, Pauliny A, Kelsh RN (2001a) A morpholino phenocopy of the colourless mutant. Genesis 30:188–189

[12] Dutton KA, Pauliny A, Lopes SS, Elworthy S, Carney TJ, Rauch J, Geisler R, Haffter P, Kelsh RN (2001b) Zebrafish colourless encodes sox10 and specifies non-ectomesenchymal neural crest fates. Development 128:4113–4125

[13] Flores DJ, Duong T, Brandenberger LO, Mitra A, Shirali A, Johnson JC, Springer D, Noguchi A, Yu ZX, Ebert SN et al (2018) Conditional ablation and conditional rescue models for Casq2 elucidate the role of development and of cell-type specific expression of Casq2 in the CPVT2 phenotype. Hum Mol Genet 27:1533–1544

[14] Geng FS, Abbas L, Baxendale S, Holdsworth CJ, Swanson AG, Slanchev K, Hammerschmidt M, Topczewski J, Whitfield TT (2013) Semicircular canal morphogenesis in the zebrafish inner ear requires the function of gpr126 (lauscher), an adhesion class G protein-coupled receptor gene. Development 140:4362–4374

[15] Guy J, Gan J, Selfridge J, Cobb S, Bird A (2007) Reversal of neurological defects in a mouse model of Rett syndrome. Science 315:1143–1147

[16] Hoshijima K, Jurynec MJ, Grunwald DJ (2016) Precise editing of the zebrafish genome made simple and efficient. Dev Cell 36:654–667

[17] Hur JK, Kim K, Been KW, Baek G, Ye S, Hur JW, Ryu SM, Lee YS, Kim JS (2016) Targeted mutagenesis in mice by electroporation of Cpf1 ribonucleoproteins. Nat Biotechnol 34:807–808

[18] Jao LE, Wente SR, Chen WB (2013) Efficient multiplex biallelic zebrafish genome editing using a CRISPR nuclease system. Proc Natl Acad Sci USA 110:13904–13909

[19] Kay MA, He CY, Chen ZY (2010) A robust system for production of minicircle DNA vectors. Nat Biotechnol 28:1287–1289

[20] Kelsh RN, Eisen JS (2000) The zebrafish colourless gene regulates development of non-ectomesenchymal neural crest derivatives. Development 127:515–525

[21] Li J, Zhang BB, Ren YG, Gu SY, Xiang YH, Huang C, Du JL (2015) Intron targeting-mediated and endogenous gene integritymaintaining knockin in zebrafish using the CRISPR/Cas9 system. Cell Res 25:634–637

[22] Li W, Zhang Y, Han B, Li L, Li M, Lu X, Chen C, Lu M, Zhang Y, Jia X (2019) One-step efficient generation of dual-function conditional knockout and geno-tagging alleles in zebrafish. Elife 8:e48081

[23] Li J, Li HY, Gu SY, Zi HX, Jiang L, Du JL (2020) One-step generation of zebrafish carrying a conditional knockout-knockin visible switch via CRISPR/Cas9-mediated intron targeting. Sci. China Life Sci. 63:59–67

[24] Lin S, Staahl BT, Alla RK, Doudna JA (2014) Enhanced homologydirected human genome engineering by controlled timing of CRISPR/Cas9 delivery. Elife 3:e04766

[25] Lister JA (2010) Transgene excision in zebrafish using the phiC31 integrase. Genesis 48:137–143

[26] Lu JJ, Maddison LA, Chen WB (2011) PhiC31 integrase induces efficient site-specific excision in zebrafish. Transgenic Res 20:183–189

[27] Luo JJ, Bian WP, Liu Y, Huang HY, Yin Q, Yang XJ, Pei DS (2018) CRISPR/Cas9-based genome engineering of zebrafish using a seamless integration strategy. FASEB J 32:5132–5142

[28] Mali P, Esvelt K, Church G (2013) Cas9 as a versatile tool for engineering biology. Nat Methods 10:957–963

[29] Mei Y, Monteiro P, Zhou Y, Kim JA, Gao X, Fu Z, Feng G (2016) Adult restoration of Shank3 expression rescues selective autistic-like phenotypes. Nature 530:481–484

[30] Moreno-Mateos MA, Fernandez JP, Rouet R, Vejnar CE, Lane MA, Mis E, Khokha MK, Doudna JA, Giraldez AJ (2017) CRISPRCpf1 mediates efficient homology-directed repair and temperature-controlled genome editing. Nat Commun 8:2024

[31] Ruehle S, Remmers F, Romo-Parra H, Massa F, Wickert M, Wortge S, Haring M, Kaiser N, Marsicano G, Pape HC (2013) Cannabinoid CB1 receptor in dorsal telencephalic glutamatergic neurons: distinctive sufficiency for hippocampus-dependent and amygdala-dependent synaptic and behavioral functions. J Neurosci 33:10264–10277

[32] Santos R, Kawauchi S, Jacobs RE, Lopez-Burks ME, Choi H, Wikenheiser J, Hallgrimsson B, Jamniczky HA, Fraser SE, Lander AD (2016) Conditional creation and rescue of nipbl-deficiency in mice reveals multiple determinants of risk for congenital heart defects. PLoS Biol 14:e2000197

[33] Shin J, Chen J, Solnica-Krezel L (2014) Efficient homologous recombination-mediated genome engineering in zebrafish using TALE nucleases. Development 141:3807–3818

[34] Sirbu IO, Zhao X, Duester G (2008) Retinoic acid controls heart anteroposterior patterning by down-regulating Isl1 through the Fgf8 pathway. Dev Dyn 237:1627–1635

[35] Sugimoto K, Hui SP, Sheng DZ, Kikuchi K (2017) Dissection of zebrafish shha function using site-specific targeting with a Credependent genetic switch. Elife 6:e24635

[36] Suzuki K, Tsunekawa Y, Hernandez-Benitez R, Wu J, Zhu J, Kim EJ, Hatanaka F, Yamamoto M, Araoka T, Li Z (2016) In vivo genome editing via CRISPR/Cas9 mediated homology-independent targeted integration. Nature 540:144–149

[37] Wilfinger A, Arkhipova V, Meyer D (2013) Cell type and tissue specific function of islet genes in zebrafish pancreas development. Dev Biol 378:25–37

[38] Witzel HR, Jungblut B, Choe CP, Crump JG, Braun T, Dobreva G (2012) The LIM protein Ajuba restricts the second heart field progenitor pool by regulating Isl1 activity. Dev Cell 23:58–70

[39] Witzel HR, Cheedipudi S, Gao R, Stainier DY, Dobreva GD (2017) Isl2b regulates anterior second heart field development in zebrafish. Sci Rep 7:41043

[40] Xiao A, Cheng ZC, Kong L, Zhu ZY, Lin S, Gao G, Zhang B (2014) CasOT: a genome-wide Cas9/gRNA off-target searching tool. Bioinformatics 30:1180–1182

[41] Zetsche B, Gootenberg JS, Abudayyeh OO, Slaymaker IM, Makarova KS, Essletzbichler P, Volz SE, Joung J, van der Oost J, Regev A (2015) Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. Cell 163:759–771

[42] Zu Y, Tong X, Wang Z, Liu D, Pan R, Li Z, Hu Y, Luo Z, Huang P, Wu Q (2013) TALEN-mediated precise genome modification by homologous recombination in zebrafish. Nat Methods 10:329–331

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