CRISPR/Cas9 interrogation of the mouse Pcdhg gene cluster reveals a crucial isoform-specific role for Pcdhgc4

English version

Autoři: Andrew M. Garrett ^aff001; Peter J. Bosch ^aff003; David M. Steffen ^aff003; Leah C. Fuller ^aff003; Charles G. Marcucci ^aff003; Alexis A. Koch ^aff001; Preeti Bais ^aff002; Joshua A. Weiner ^aff003; Robert W. Burgess ^aff002
Působiště autorů: Department of Pharmacology and Department of Ophthalmology, Visual, and Anatomical Sciences, Wayne State University, Detroit, Michigan, United States of America ^aff001; The Jackson Laboratory, Bar Harbor, Maine, United States of America ^aff002; Department of Biology and Iowa Neuroscience Institute, University of Iowa, Iowa City, Iowa, United States of America ^aff003
Vyšlo v časopise: CRISPR/Cas9 interrogation of the mouse Pcdhg gene cluster reveals a crucial isoform-specific role for Pcdhgc4. PLoS Genet 15(12): e32767. doi:10.1371/journal.pgen.1008554
Kategorie: Research Article
doi: https://doi.org/10.1371/journal.pgen.1008554

Souhrn

The mammalian Pcdhg gene cluster encodes a family of 22 cell adhesion molecules, the gamma-Protocadherins (γ-Pcdhs), critical for neuronal survival and neural circuit formation. The extent to which isoform diversity–a γ-Pcdh hallmark–is required for their functions remains unclear. We used a CRISPR/Cas9 approach to reduce isoform diversity, targeting each Pcdhg variable exon with pooled sgRNAs to generate an allelic series of 26 mouse lines with 1 to 21 isoforms disrupted via discrete indels at guide sites and/or larger deletions/rearrangements. Analysis of 5 mutant lines indicates that postnatal viability and neuronal survival do not require isoform diversity. Surprisingly, given reports that it might not independently engage in trans-interactions, we find that γC4, encoded by Pcdhgc4, is the only critical isoform. Because the human orthologue is the only PCDHG gene constrained in humans, our results indicate a conserved γC4 function that likely involves distinct molecular mechanisms.

Klíčová slova:

Apoptosis – Genome sequencing – Interneurons – Mammalian genomics – Polymerase chain reaction – Retina – Sequence alignment

Zdroje

1. Zipursky SL, Sanes JR. Chemoaffinity revisited: dscams, protocadherins, and neural circuit assembly. Cell. 2010;143(3):343–53. Epub 2010/10/30. doi: 10.1016/j.cell.2010.10.009 21029858.

2. Wu Q, Maniatis T. A striking organization of a large family of human neural cadherin-like cell adhesion genes. Cell. 1999;97(6):779–90. Epub 1999/06/25. doi: 10.1016/s0092-8674(00)80789-8 10380929.

3. Wu Q, Zhang T, Cheng JF, Kim Y, Grimwood J, Schmutz J, et al. Comparative DNA sequence analysis of mouse and human protocadherin gene clusters. Genome Res. 2001;11(3):389–404. Epub 2001/03/07. doi: 10.1101/gr.167301 11230163; PubMed Central PMCID: PMC311048.

4. Chen WV, Nwakeze CL, Denny CA, O'Keeffe S, Rieger MA, Mountoufaris G, et al. Pcdhalphac2 is required for axonal tiling and assembly of serotonergic circuitries in mice. Science. 2017;356(6336):406–11. Epub 2017/04/30. doi: 10.1126/science.aal3231 28450636; PubMed Central PMCID: PMC5529183.

5. Mountoufaris G, Chen WV, Hirabayashi Y, O'Keeffe S, Chevee M, Nwakeze CL, et al. Multicluster Pcdh diversity is required for mouse olfactory neural circuit assembly. Science. 2017;356(6336):411–4. Epub 2017/04/30. doi: 10.1126/science.aai8801 28450637; PubMed Central PMCID: PMC5529182.

6. Emond MR, Jontes JD. Inhibition of protocadherin-alpha function results in neuronal death in the developing zebrafish. Dev Biol. 2008;321(1):175–87. Epub 2008/07/08. doi: 10.1016/j.ydbio.2008.06.011 18602383.

7. Hasegawa S, Kobayashi H, Kumagai M, Nishimaru H, Tarusawa E, Kanda H, et al. Clustered Protocadherins Are Required for Building Functional Neural Circuits. Front Mol Neurosci. 2017;10:114. Epub 2017/05/10. doi: 10.3389/fnmol.2017.00114 28484370; PubMed Central PMCID: PMC5401904.

8. Katori S, Hamada S, Noguchi Y, Fukuda E, Yamamoto T, Yamamoto H, et al. Protocadherin-alpha family is required for serotonergic projections to appropriately innervate target brain areas. J Neurosci. 2009;29(29):9137–47. Epub 2009/07/25. doi: 10.1523/JNEUROSCI.5478-08.2009 19625505.

9. Meguro R, Hishida R, Tsukano H, Yoshitake K, Imamura R, Tohmi M, et al. Impaired clustered protocadherin-alpha leads to aggregated retinogeniculate terminals and impaired visual acuity in mice. J Neurochem. 2015;133(1):66–72. Epub 2015/02/05. doi: 10.1111/jnc.13053 25650227.

10. Ing-Esteves S, Kostadinov D, Marocha J, Sing AD, Joseph KS, Laboulaye MA, et al. Combinatorial Effects of Alpha- and Gamma-Protocadherins on Neuronal Survival and Dendritic Self-Avoidance. J Neurosci. 2018;38(11):2713–29. Epub 2018/02/14. doi: 10.1523/JNEUROSCI.3035-17.2018 29439167; PubMed Central PMCID: PMC5852656.

11. Peek SL, Mah KM, Weiner JA. Regulation of neural circuit formation by protocadherins. Cell Mol Life Sci. 2017;74(22):4133–57. Epub 2017/06/21. doi: 10.1007/s00018-017-2572-3 28631008; PubMed Central PMCID: PMC5643215.

12. Wang X, Su H, Bradley A. Molecular mechanisms governing Pcdh-gamma gene expression: evidence for a multiple promoter and cis-alternative splicing model. Genes Dev. 2002;16(15):1890–905. Epub 2002/08/03. doi: 10.1101/gad.1004802 12154121; PubMed Central PMCID: PMC186422.

13. Tasic B, Nabholz CE, Baldwin KK, Kim Y, Rueckert EH, Ribich SA, et al. Promoter choice determines splice site selection in protocadherin alpha and gamma pre-mRNA splicing. Mol Cell. 2002;10(1):21–33. Epub 2002/08/02. doi: 10.1016/s1097-2765(02)00578-6 12150904.

14. Kaneko R, Kato H, Kawamura Y, Esumi S, Hirayama T, Hirabayashi T, et al. Allelic gene regulation of Pcdh-alpha and Pcdh-gamma clusters involving both monoallelic and biallelic expression in single Purkinje cells. J Biol Chem. 2006;281(41):30551–60. Epub 2006/08/09. doi: 10.1074/jbc.M605677200 16893882.

15. Goodman KM, Rubinstein R, Thu CA, Bahna F, Mannepalli S, Ahlsen G, et al. Structural Basis of Diverse Homophilic Recognition by Clustered alpha- and beta-Protocadherins. Neuron. 2016;90(4):709–23. Epub 2016/05/11. doi: 10.1016/j.neuron.2016.04.004 27161523; PubMed Central PMCID: PMC4873334.

16. Goodman KM, Rubinstein R, Thu CA, Mannepalli S, Bahna F, Ahlsen G, et al. gamma-Protocadherin structural diversity and functional implications. Elife. 2016;5. Epub 2016/10/27. doi: 10.7554/eLife.20930 27782885; PubMed Central PMCID: PMC5106212.

17. Rubinstein R, Thu CA, Goodman KM, Wolcott HN, Bahna F, Mannepalli S, et al. Molecular logic of neuronal self-recognition through protocadherin domain interactions. Cell. 2015;163(3):629–42. Epub 2015/10/20. doi: 10.1016/j.cell.2015.09.026 26478182; PubMed Central PMCID: PMC4624033.

18. Thu CA, Chen WV, Rubinstein R, Chevee M, Wolcott HN, Felsovalyi KO, et al. Single-cell identity generated by combinatorial homophilic interactions between alpha, beta, and gamma protocadherins. Cell. 2014;158(5):1045–59. Epub 2014/08/30. doi: 10.1016/j.cell.2014.07.012 25171406; PubMed Central PMCID: PMC4183217.

19. Schreiner D, Weiner JA. Combinatorial homophilic interaction between gamma-protocadherin multimers greatly expands the molecular diversity of cell adhesion. Proc Natl Acad Sci U S A. 2010;107(33):14893–8. Epub 2010/08/04. doi: 10.1073/pnas.1004526107 20679223; PubMed Central PMCID: PMC2930437.

20. Brasch J, Goodman KM, Noble AJ, Rapp M, Mannepalli S, Bahna F, et al. Visualization of clustered protocadherin neuronal self-recognition complexes. Nature. 2019;569(7755):280–3. Epub 2019/04/12. doi: 10.1038/s41586-019-1089-3 30971825; PubMed Central PMCID: PMC6736547.

21. Rubinstein R, Goodman KM, Maniatis T, Shapiro L, Honig B. Structural origins of clustered protocadherin-mediated neuronal barcoding. Semin Cell Dev Biol. 2017;69:140–50. Epub 2017/07/27. doi: 10.1016/j.semcdb.2017.07.023 28743640; PubMed Central PMCID: PMC5582985.

22. Schmucker D, Clemens JC, Shu H, Worby CA, Xiao J, Muda M, et al. Drosophila Dscam is an axon guidance receptor exhibiting extraordinary molecular diversity. Cell. 2000;101(6):671–84. doi: 10.1016/s0092-8674(00)80878-8 WOS:000087504500013. 10892653

23. Hattori D, Chen Y, Matthews BJ, Salwinski L, Sabatti C, Grueber WB, et al. Robust discrimination between self and non-self neurites requires thousands of Dscam1 isoforms. Nature. 2009;461(7264):644–8. Epub 2009/10/02. doi: 10.1038/nature08431 19794492; PubMed Central PMCID: PMC2836808.

24. Hattori D, Demir E, Kim HW, Viragh E, Zipursky SL, Dickson BJ. Dscam diversity is essential for neuronal wiring and self-recognition. Nature. 2007;449(7159):223–7. Epub 2007/09/14. doi: 10.1038/nature06099 17851526; PubMed Central PMCID: PMC2691715.

25. Zhan XL, Clemens JC, Neves G, Hattori D, Flanagan JJ, Hummel T, et al. Analysis of Dscam diversity in regulating axon guidance in Drosophila mushroom bodies. Neuron. 2004;43(5):673–86. Epub 2004/09/02. doi: 10.1016/j.neuron.2004.07.020 15339649.

26. Hattori D, Millard SS, Wojtowicz WM, Zipursky SL. Dscam-mediated cell recognition regulates neural circuit formation. Annu Rev Cell Dev Biol. 2008;24(1):597–620. Epub 2008/10/08. doi: 10.1146/annurev.cellbio.24.110707.175250 18837673; PubMed Central PMCID: PMC2711549.

27. Fuerst PG, Bruce F, Tian M, Wei W, Elstrott J, Feller MB, et al. DSCAM and DSCAML1 function in self-avoidance in multiple cell types in the developing mouse retina. Neuron. 2009;64(4):484–97. Epub 2009/12/01. doi: 10.1016/j.neuron.2009.09.027 19945391; PubMed Central PMCID: PMC2850049.

28. Fuerst PG, Koizumi A, Masland RH, Burgess RW. Neurite arborization and mosaic spacing in the mouse retina require DSCAM. Nature. 2008;451(7177):470–4. Epub 2008/01/25. doi: 10.1038/nature06514 18216855; PubMed Central PMCID: PMC2259282.

29. Garrett AM, Jucius TJ, Sigaud LP, Tang FL, Xiong WC, Ackerman SL, et al. Analysis of Expression Pattern and Genetic Deletion of Netrin5 in the Developing Mouse. Front Mol Neurosci. 2016;9:3. Epub 2016/02/10. doi: 10.3389/fnmol.2016.00003 26858598; PubMed Central PMCID: PMC4726805.

30. Garrett AM, Khalil A, Walton DO, Burgess RW. DSCAM promotes self-avoidance in the developing mouse retina by masking the functions of cadherin superfamily members. Proc Natl Acad Sci U S A. 2018;115(43):E10216–E24. Epub 2018/10/10. doi: 10.1073/pnas.1809430115 30297418; PubMed Central PMCID: PMC6205498.

31. Prasad T, Wang X, Gray PA, Weiner JA. A differential developmental pattern of spinal interneuron apoptosis during synaptogenesis: insights from genetic analyses of the protocadherin-gamma gene cluster. Development. 2008;135(24):4153–64. Epub 2008/11/26. doi: 10.1242/dev.026807 19029045; PubMed Central PMCID: PMC2755264.

32. Su H, Marcheva B, Meng S, Liang FA, Kohsaka A, Kobayashi Y, et al. Gamma-protocadherins regulate the functional integrity of hypothalamic feeding circuitry in mice. Dev Biol. 2010;339(1):38–50. Epub 2009/12/23. doi: 10.1016/j.ydbio.2009.12.010 20025866; PubMed Central PMCID: PMC2823828.

33. Wang X, Weiner JA, Levi S, Craig AM, Bradley A, Sanes JR. Gamma protocadherins are required for survival of spinal interneurons. Neuron. 2002;36(5):843–54. Epub 2002/12/07. doi: 10.1016/s0896-6273(02)01090-5 12467588.

34. Chen WV, Alvarez FJ, Lefebvre JL, Friedman B, Nwakeze C, Geiman E, et al. Functional significance of isoform diversification in the protocadherin gamma gene cluster. Neuron. 2012;75(3):402–9. Epub 2012/08/14. doi: 10.1016/j.neuron.2012.06.039 22884324; PubMed Central PMCID: PMC3426296.

35. Garrett AM, Weiner JA. Control of CNS synapse development by {gamma}-protocadherin-mediated astrocyte-neuron contact. J Neurosci. 2009;29(38):11723–31. Epub 2009/09/25. doi: 10.1523/JNEUROSCI.2818-09.2009 19776259; PubMed Central PMCID: PMC2778296.

36. Prasad T, Weiner JA. Direct and Indirect Regulation of Spinal Cord Ia Afferent Terminal Formation by the gamma-Protocadherins. Front Mol Neurosci. 2011;4:54. Epub 2012/01/26. doi: 10.3389/fnmol.2011.00054 22275881; PubMed Central PMCID: PMC3250626.

37. Weiner JA, Wang X, Tapia JC, Sanes JR. Gamma protocadherins are required for synaptic development in the spinal cord. Proc Natl Acad Sci U S A. 2005;102(1):8–14. Epub 2004/12/03. doi: 10.1073/pnas.0407931101 15574493; PubMed Central PMCID: PMC544073.

38. Garrett AM, Schreiner D, Lobas MA, Weiner JA. gamma-protocadherins control cortical dendrite arborization by regulating the activity of a FAK/PKC/MARCKS signaling pathway. Neuron. 2012;74(2):269–76. Epub 2012/05/01. doi: 10.1016/j.neuron.2012.01.028 22542181; PubMed Central PMCID: PMC3582349.

39. Keeler AB, Schreiner D, Weiner JA. Protein Kinase C Phosphorylation of a gamma-Protocadherin C-terminal Lipid Binding Domain Regulates Focal Adhesion Kinase Inhibition and Dendrite Arborization. J Biol Chem. 2015;290(34):20674–86. Epub 2015/07/04. doi: 10.1074/jbc.M115.642306 26139604; PubMed Central PMCID: PMC4543629.

40. Molumby MJ, Keeler AB, Weiner JA. Homophilic Protocadherin Cell-Cell Interactions Promote Dendrite Complexity. Cell Rep. 2016;15(5):1037–50. Epub 2016/04/28. doi: 10.1016/j.celrep.2016.03.093 27117416; PubMed Central PMCID: PMC4856576.

41. Suo L, Lu H, Ying G, Capecchi MR, Wu Q. Protocadherin clusters and cell adhesion kinase regulate dendrite complexity through Rho GTPase. J Mol Cell Biol. 2012;4(6):362–76. Epub 2012/06/26. doi: 10.1093/jmcb/mjs034 22730554.

42. Molumby MJ, Anderson RM, Newbold DJ, Koblesky NK, Garrett AM, Schreiner D, et al. gamma-Protocadherins Interact with Neuroligin-1 and Negatively Regulate Dendritic Spine Morphogenesis. Cell Rep. 2017;18(11):2702–14. Epub 2017/03/16. doi: 10.1016/j.celrep.2017.02.060 28297673; PubMed Central PMCID: PMC5418859.

43. Lefebvre JL, Zhang Y, Meister M, Wang X, Sanes JR. gamma-Protocadherins regulate neuronal survival but are dispensable for circuit formation in retina. Development. 2008;135(24):4141–51. Epub 2008/11/26. doi: 10.1242/dev.027912 19029044; PubMed Central PMCID: PMC2644426.

44. Kostadinov D, Sanes JR. Protocadherin-dependent dendritic self-avoidance regulates neural connectivity and circuit function. Elife. 2015;4. Epub 2015/07/04. doi: 10.7554/eLife.08964 26140686; PubMed Central PMCID: PMC4548410.

45. Lefebvre JL, Kostadinov D, Chen WV, Maniatis T, Sanes JR. Protocadherins mediate dendritic self-avoidance in the mammalian nervous system. Nature. 2012;488(7412):517–21. Epub 2012/07/31. doi: 10.1038/nature11305 22842903; PubMed Central PMCID: PMC3427422.

46. Frank M, Ebert M, Shan W, Phillips GR, Arndt K, Colman DR, et al. Differential expression of individual gamma-protocadherins during mouse brain development. Mol Cell Neurosci. 2005;29(4):603–16. Epub 2005/06/21. doi: 10.1016/j.mcn.2005.05.001 15964765.

47. Fernandez-Monreal M, Kang S, Phillips GR. Gamma-protocadherin homophilic interaction and intracellular trafficking is controlled by the cytoplasmic domain in neurons. Mol Cell Neurosci. 2009;40(3):344–53. Epub 2009/01/13. doi: 10.1016/j.mcn.2008.12.002 19136062; PubMed Central PMCID: PMC2646808.

48. Hasegawa S, Kumagai M, Hagihara M, Nishimaru H, Hirano K, Kaneko R, et al. Distinct and Cooperative Functions for the Protocadherin-alpha, -beta and -gamma Clusters in Neuronal Survival and Axon Targeting. Front Mol Neurosci. 2016;9:155. Epub 2017/01/10. doi: 10.3389/fnmol.2016.00155 28066179; PubMed Central PMCID: PMC5179546.

49. Mah KM, Houston DW, Weiner JA. The gamma-Protocadherin-C3 isoform inhibits canonical Wnt signalling by binding to and stabilizing Axin1 at the membrane. Sci Rep. 2016;6:31665. Epub 2016/08/18. doi: 10.1038/srep31665 27530555; PubMed Central PMCID: PMC4987702.

50. Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, et al. Multiplex genome engineering using CRISPR/Cas systems. Science. 2013;339(6121):819–23. Epub 2013/01/05. doi: 10.1126/science.1231143 23287718; PubMed Central PMCID: PMC3795411.

51. Mali P, Yang L, Esvelt KM, Aach J, Guell M, DiCarlo JE, et al. RNA-guided human genome engineering via Cas9. Science. 2013;339(6121):823–6. Epub 2013/01/05. doi: 10.1126/science.1232033 23287722; PubMed Central PMCID: PMC3712628.

52. McKenna A, Hanna M, Banks E, Sivachenko A, Cibulskis K, Kernytsky A, et al. The Genome Analysis Toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data. Genome Res. 2010;20(9):1297–303. Epub 2010/07/21. doi: 10.1101/gr.107524.110 20644199; PubMed Central PMCID: PMC2928508.

53. Abo RP, Ducar M, Garcia EP, Thorner AR, Rojas-Rudilla V, Lin L, et al. BreaKmer: detection of structural variation in targeted massively parallel sequencing data using kmers. Nucleic Acids Res. 2015;43(3):e19. Epub 2014/11/28. doi: 10.1093/nar/gku1211 25428359; PubMed Central PMCID: PMC4330340.

54. Ono R, Ishii M, Fujihara Y, Kitazawa M, Usami T, Kaneko-Ishino T, et al. Double strand break repair by capture of retrotransposon sequences and reverse-transcribed spliced mRNA sequences in mouse zygotes. Sci Rep. 2015;5(1):12281. Epub 2015/07/29. doi: 10.1038/srep12281 26216318; PubMed Central PMCID: PMC4516963.

55. Lobas MA, Helsper L, Vernon CG, Schreiner D, Zhang Y, Holtzman MJ, et al. Molecular heterogeneity in the choroid plexus epithelium: the 22-member gamma-protocadherin family is differentially expressed, apically localized, and implicated in CSF regulation. J Neurochem. 2012;120(6):913–27. Epub 2011/11/19. doi: 10.1111/j.1471-4159.2011.07587.x 22092001; PubMed Central PMCID: PMC3296866.

56. Lewis KE. How do genes regulate simple behaviours? Understanding how different neurons in the vertebrate spinal cord are genetically specified. Philos Trans R Soc Lond B Biol Sci. 2006;361(1465):45–66. Epub 2006/03/24. doi: 10.1098/rstb.2005.1778 16553308; PubMed Central PMCID: PMC1626545.

57. Marquardt T, Ashery-Padan R, Andrejewski N, Scardigli R, Guillemot F, Gruss P. Pax6 is required for the multipotent state of retinal progenitor cells. Cell. 2001;105(1):43–55. doi: 10.1016/s0092-8674(01)00295-1 WOS:000168063300006. 11301001

58. Karczewski KJ, Francioli LC, Tiao G, Cummings BB, Alföldi J, Wang Q, et al. Variation across 141,456 human exomes and genomes reveals the spectrum of loss-of-function intolerance across human protein-coding genes. bioRxiv. 2019:531210. doi: 10.1101/531210

59. Bennett-Baker PE, Mueller JL. CRISPR-mediated isolation of specific megabase segments of genomic DNA. Nucleic Acids Res. 2017;45(19):e165. Epub 2017/10/05. doi: 10.1093/nar/gkx749 28977642; PubMed Central PMCID: PMC5737698.

60. Iyer V, Shen B, Zhang W, Hodgkins A, Keane T, Huang X, et al. Off-target mutations are rare in Cas9-modified mice. Nat Methods. 2015;12(6):479. Epub 2015/05/29. doi: 10.1038/nmeth.3408 26020497.

61. Mianne J, Chessum L, Kumar S, Aguilar C, Codner G, Hutchison M, et al. Correction of the auditory phenotype in C57BL/6N mice via CRISPR/Cas9-mediated homology directed repair. Genome Med. 2016;8(1):16. Epub 2016/02/16. doi: 10.1186/s13073-016-0273-4 26876963; PubMed Central PMCID: PMC4753642.

62. Nakajima K, Kazuno AA, Kelsoe J, Nakanishi M, Takumi T, Kato T. Exome sequencing in the knockin mice generated using the CRISPR/Cas system. Sci Rep. 2016;6(1):34703. Epub 2016/10/05. doi: 10.1038/srep34703 27698470; PubMed Central PMCID: PMC5048150.

63. Golan-Mashiach M, Grunspan M, Emmanuel R, Gibbs-Bar L, Dikstein R, Shapiro E. Identification of CTCF as a master regulator of the clustered protocadherin genes. Nucleic Acids Res. 2012;40(8):3378–91. Epub 2012/01/03. doi: 10.1093/nar/gkr1260 22210889; PubMed Central PMCID: PMC3333863.

64. Kehayova P, Monahan K, Chen W, Maniatis T. Regulatory elements required for the activation and repression of the protocadherin-alpha gene cluster. Proc Natl Acad Sci U S A. 2011;108(41):17195–200. Epub 2011/09/29. doi: 10.1073/pnas.1114357108 21949399; PubMed Central PMCID: PMC3193253.

65. Monahan K, Rudnick ND, Kehayova PD, Pauli F, Newberry KM, Myers RM, et al. Role of CCCTC binding factor (CTCF) and cohesin in the generation of single-cell diversity of protocadherin-alpha gene expression. Proc Natl Acad Sci U S A. 2012;109(23):9125–30. Epub 2012/05/03. doi: 10.1073/pnas.1205074109 22550178; PubMed Central PMCID: PMC3384188.

66. Jiang Y, Loh YE, Rajarajan P, Hirayama T, Liao W, Kassim BS, et al. The methyltransferase SETDB1 regulates a large neuron-specific topological chromatin domain. Nat Genet. 2017;49(8):1239–50. Epub 2017/07/04. doi: 10.1038/ng.3906 28671686; PubMed Central PMCID: PMC5560095.

67. Toyoda S, Kawaguchi M, Kobayashi T, Tarusawa E, Toyama T, Okano M, et al. Developmental epigenetic modification regulates stochastic expression of clustered protocadherin genes, generating single neuron diversity. Neuron. 2014;82(1):94–108. Epub 2014/04/05. doi: 10.1016/j.neuron.2014.02.005 24698270.

68. Hirayama T, Tarusawa E, Yoshimura Y, Galjart N, Yagi T. CTCF is required for neural development and stochastic expression of clustered Pcdh genes in neurons. Cell Rep. 2012;2(2):345–57. Epub 2012/08/03. doi: 10.1016/j.celrep.2012.06.014 22854024.

69. Yokota S, Hirayama T, Hirano K, Kaneko R, Toyoda S, Kawamura Y, et al. Identification of the cluster control region for the protocadherin-beta genes located beyond the protocadherin-gamma cluster. J Biol Chem. 2011;286(36):31885–95. Epub 2011/07/21. doi: 10.1074/jbc.M111.245605 21771796; PubMed Central PMCID: PMC3173131.

70. Noguchi Y, Hirabayashi T, Katori S, Kawamura Y, Sanbo M, Hirabayashi M, et al. Total expression and dual gene-regulatory mechanisms maintained in deletions and duplications of the Pcdha cluster. J Biol Chem. 2009;284(46):32002–14. Epub 2009/10/03. doi: 10.1074/jbc.M109.046938 19797050; PubMed Central PMCID: PMC2797272.

71. 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(10):1473–5. Epub 2014/01/28. doi: 10.1093/bioinformatics/btu048 24463181; PubMed Central PMCID: PMC4016707.

72. Park J, Bae S, Kim JS. Cas-Designer: a web-based tool for choice of CRISPR-Cas9 target sites. Bioinformatics. 2015;31(24):4014–6. Epub 2015/09/12. doi: 10.1093/bioinformatics/btv537 26358729.

73. Bae S, Kweon J, Kim HS, Kim JS. Microhomology-based choice of Cas9 nuclease target sites. Nat Methods. 2014;11(7):705–6. Epub 2014/06/28. doi: 10.1038/nmeth.3015 24972169.

74. Allen F, Crepaldi L, Alsinet C, Strong AJ, Kleshchevnikov V, De Angeli P, et al. Predicting the mutations generated by repair of Cas9-induced double-strand breaks. Nat Biotechnol. 2018;37(1):64. Epub 2018/11/28. doi: 10.1038/nbt.4317 30480667.

75. Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, et al. Fiji: an open-source platform for biological-image analysis. Nat Methods. 2012;9(7):676–82. Epub 2012/06/30. doi: 10.1038/nmeth.2019 22743772; PubMed Central PMCID: PMC3855844.