Focal segmental glomerulosclerosis (FSGS) is defined by focal (involving few glomeruli) and segmental sclerosis of the glomerular tuft that manifests with nephrotic syndrome. Mutations in genes involved in the maintenance of structure and function of podocytes have been found in a minority of these patients. A family with adult-onset autosomal dominant FSGS was recently found to carry a new germline missense heterozygous mutation (p.G189R) in the octapeptide domain of the transcription factor PAX2. Here, we efficiently corrected this point mutation in patient-derived induced pluripotent stem cells (iPSCs) by means of CRISPR-Cas9-based homology-directed repair. The iPSC lines were differentiated into podocytes, which were tested for their motility. Editing the PAX2 p.G189R mutation restored podocyte motility, which was altered in podocytes derived from patient iPSCs.
BaruaM, StellacciE, StellaL, et al.Mutations in PAX2 associate with adult-onset FSGS. J Am Soc Nephrol, 2014; 25:1942–1953. DOI: 10.1681/ASN.2013070686.
4.
DresslerGR, DouglassEC. Pax-2 is a DNA-binding protein expressed in embryonic kidney and Wilms tumor. Proc Natl Acad Sci U S A, 1992; 89:1179–1183.
5.
SharmaR, Sanchez-FerrasO, BouchardM. Pax genes in renal development, disease and regeneration. Semin Cell Dev Biol, 2015; 44:97–106. DOI: 10.1016/j.semcdb.2015.09.016.
6.
AbrahamS, PaknikarR, BhumbraS, et al.The Groucho-associated phosphatase PPM1B displaces Pax transactivation domain interacting protein (PTIP) to switch the transcription factor Pax2 from a transcriptional activator to a repressor. J Biol Chem, 2015; 290:7185–7194. DOI: 10.1074/jbc.M114.607424.
7.
HarshmanLA, BrophyPD. PAX2 in human kidney malformations and disease. Pediatr Nephrol, 2012; 27:1265–1275. DOI: 10.1007/s00467-011-2053-0.
8.
IatropoulosP, DainaE, MeleC, et al.Discordant phenotype in monozygotic twins with renal coloboma syndrome and a PAX2 mutation. Pediatr Nephrol, 2012; 27:1989–1993. DOI: 10.1007/s00467-012-2205-x.
9.
CaiY, BrophyPD, LevitanI, et al.Groucho suppresses Pax2 transactivation by inhibition of JNK-mediated phosphorylation. EMBO J, 2003; 22:5522–5529. DOI: 10.1093/emboj/cdg536.
10.
WagnerK-D, WagnerN, GuoJ-K, et al.An inducible mouse model for PAX2-dependent glomerular disease: insights into a complex pathogenesis. Curr Biol CB, 2006; 16:793–800. DOI: 10.1016/j.cub.2006.02.072.
11.
YeL, SwingenC, ZhangJ. Induced pluripotent stem cells and their potential for basic and clinical sciences. Curr Cardiol Rev, 2013; 9:63–72.
12.
CiampiO, IaconeR, LongarettiL, et al.Generation of functional podocytes from human induced pluripotent stem cells. Stem Cell Res, 2016; 17:130–139. DOI: 10.1016/j.scr.2016.06.001.
13.
BenedettiV, BriziV, GuidaP, et al.Engineered kidney tubules for modeling patient-specific diseases and drug discovery. EBioMedicine, 2018; 33:253–268. DOI: 10.1016/j.ebiom.2018.06.005.
14.
JinekM, ChylinskiK, FonfaraI, et al.A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science, 2012; 337:816–821. DOI: 10.1126/science.1225829.
15.
MaliP, YangL, EsveltKM, et al.RNA-guided human genome engineering via Cas9. Science, 2013; 339:823–826. DOI: 10.1126/science.1232033.
16.
CongL, RanFA, CoxD, et al.Multiplex genome engineering using CRISPR/Cas systems. Science, 2013; 339:819–823. DOI: 10.1126/science.1231143.
17.
GasiunasG, BarrangouR, HorvathP, et al.Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria. Proc Natl Acad Sci U S A, 2012; 109:E2579–2586. DOI: 10.1073/pnas.1208507109.
18.
SymingtonLS, GautierJ. Double-strand break end resection and repair pathway choice. Annu Rev Genet, 2011; 45:247–271. DOI: 10.1146/annurev-genet-110410-132435.
19.
KwartD, PaquetD, TeoS, et al.Precise and efficient scarless genome editing in stem cells using CORRECT. Nat Protoc, 2017; 12:329–354. DOI: 10.1038/nprot.2016.171.
20.
MaX, ChenX, JinY, et al.Small molecules promote CRISPR-Cpf1-mediated genome editing in human pluripotent stem cells. Nat Commun, 2018; 9:1303. DOI: 10.1038/s41467-018-03760-5.
21.
RichardsonCD, RayGJ, DeWittMA, et al.Enhancing homology-directed genome editing by catalytically active and inactive CRISPR-Cas9 using asymmetric donor DNA. Nat Biotechnol, 2016; 34:339–344. DOI: 10.1038/nbt.3481.
RanFA, HsuPD, WrightJ, et al.Genome engineering using the CRISPR-Cas9 system. Nat Protoc, 2013; 8:2281–2308. DOI: 10.1038/nprot.2013.143.
24.
DoenchJG, FusiN, SullenderM, et al.Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9. Nat Biotechnol, 2016; 34:184–191. DOI: 10.1038/nbt.3437.
25.
GuschinDY, WaiteAJ, KatibahGE, et al.A rapid and general assay for monitoring endogenous gene modification. Methods Mol Biol, 2010; 649:247–256. DOI: 10.1007/978-1-60761-753-2_15.
26.
YeJ, CoulourisG, ZaretskayaI, et al.Primer-BLAST: a tool to design target-specific primers for polymerase chain reaction. BMC Bioinformatics, 2012; 13:134. DOI: 10.1186/1471-2105-13-134.
27.
HaeusslerM, SchönigK, EckertH, et al.Evaluation of off-target and on-target scoring algorithms and integration into the guide RNA selection tool CRISPOR. Genome Biol, 2016; 17:148. DOI: 10.1186/s13059-016-1012-2.
28.
BrinkmanEK, ChenT, AmendolaM, et al.Easy quantitative assessment of genome editing by sequence trace decomposition. Nucleic Acids Res, 2014; 42:e168. DOI: 10.1093/nar/gku936.
29.
SchwarzJM, CooperDN, SchuelkeM, et al.MutationTaster2: mutation prediction for the deep-sequencing age. Nat Methods, 2014; 11:361–362. DOI: 10.1038/nmeth.2890.
30.
LessardS, FrancioliL, AlfoldiJ, et al.Human genetic variation alters CRISPR-Cas9 on- and off-targeting specificity at therapeutically implicated loci. Proc Natl Acad Sci U S A, 2017; 114:E11257–E11266. DOI: 10.1073/pnas.1714640114.
31.
LiHL, GeeP, IshidaK, et al.Efficient genomic correction methods in human iPS cells using CRISPR-Cas9 system. Methods, 2016; 101:27–35. DOI: 10.1016/j.ymeth.2015.10.015.
32.
PaquetD, KwartD, ChenA, et al.Efficient introduction of specific homozygous and heterozygous mutations using CRISPR/Cas9. Nature, 2016; 533:125–129. DOI: 10.1038/nature17664.
33.
YangL, GuellM, ByrneS, et al.Optimization of scarless human stem cell genome editing. Nucleic Acids Res, 2013; 41:9049–9061. DOI: 10.1093/nar/gkt555.
34.
TakayamaK, IgaiK, HagiharaY, et al.Highly efficient biallelic genome editing of human ES/iPS cells using a CRISPR/Cas9 or TALEN system. Nucleic Acids Res, 2017; 45:5198–5207. DOI: 10.1093/nar/gkx130.
35.
RiesenbergS, MaricicT. Targeting repair pathways with small molecules increases precise genome editing in pluripotent stem cells. Nat Commun, 2018; 9:2164. DOI: 10.1038/s41467-018-04609-7.
36.
MaruyamaT, DouganSK, TruttmannMC, et al.Increasing the efficiency of precise genome editing with CRISPR-Cas9 by inhibition of nonhomologous end joining. Nat Biotechnol, 2015; 33:538–542. DOI: 10.1038/nbt.3190.
37.
KondrashovA, Duc HoangM, SmithJGW, et al.Simplified footprint-free Cas9/CRISPR editing of cardiac-associated genes in human pluripotent stem cells. Stem Cells Dev, 2018; 27:391–404. DOI: 10.1089/scd.2017.0268.
38.
HockemeyerD, SoldnerF, BeardC, et al.Efficient targeting of expressed and silent genes in human ESCs and iPSCs using zinc-finger nucleases. Nat Biotechnol, 2009; 27:851–857. DOI: 10.1038/nbt.1562.
39.
LiLB, MaC, AwongG, et al.Silent IL2RG gene editing in human pluripotent stem cells. Mol Ther, 2016; 24:582–591. DOI: 10.1038/mt.2015.190.
40.
WangK, TangX, LiuY, et al.Efficient generation of orthologous point mutations in pigs via CRISPR-assisted ssODN-mediated homology-directed repair. Mol Ther Nucleic Acids, 2016; 5:e396. DOI: 10.1038/mtna.2016.101.
41.
InuiM, MiyadoM, IgarashiM, et al.Rapid generation of mouse models with defined point mutations by the CRISPR/Cas9 system. Sci Rep, 2014; 4:5396. DOI: 10.1038/srep05396.
42.
MénoretS, De CianA, TessonL, et al.Homology-directed repair in rodent zygotes using Cas9 and TALEN engineered proteins. Sci Rep, 2015; 5:14410. DOI: 10.1038/srep14410.
43.
HallG, GbadegesinRA. Translating genetic findings in hereditary nephrotic syndrome: the missing loops. Am J Physiol Renal Physiol, 2015; 309:F24–28. DOI: 10.1152/ajprenal.00683.2014.
44.
YangJW, DettmarAK, KronbichlerA, et al.Recent advances of animal model of focal segmental glomerulosclerosis. Clin Exp Nephrol, 2018; 22:752–763. DOI: 10.1007/s10157-018-1552-8.
DingWY, SaleemMA. Current concepts of the podocyte in nephrotic syndrome. Kidney Res Clin Pract, 2012; 31:87–93. DOI: 10.1016/j.krcp.2012.04.323.
47.
WangL, EllisMJ, GomezJA, et al.Mechanisms of the proteinuria induced by Rho GTPases. Kidney Int, 2012; 81:1075–1085. DOI: 10.1038/ki.2011.472.
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