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Effects of faf1 gene knockout by CRISPR/Cas9 on zebrafish cartilage and sarcomere development



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Effects of faf1 gene knockout by CRISPR/Cas9 on zebrafish cartilage and sarcomere development



Department of Biochemistry and Molecular Biology, Department of Developmental Biology, College of Basic Medical Sciences, Chongqing Medical University, Chongqing, 400016, China


faf1 zebrafish CRISPR/Cas9 cartilage development delayed pigmentation sarcoidosis

Q75; Q954.48; Q959.468

Objective     To determine the effect of knocking down zebrafish faf1 gene by CRISPR/Cas9 editing technique. Methods     gRNA was designed and prepared for the faf1 gene of zebrafish, and gRNA was mixed with Cas9 mRNA by microinjection into zebrafish single cell embryos. The mutant F0 generation zebrafish was screened out by enzyme digestion and gene sequencing. The mutant F0 was genetically outcrossed with the wild-type zebrafish to get the F1 heterozygous zebrafish, and the genotype of zebrafish was detected by microscopic observation. Results     The faf1 gRNA and Cas9 mRNA were successfully prepared. The gRNA (gRNA6) located in the exon 6 of faf1 could shift the faf1 gene into frameshift mutations. The mutation type MU1 was screened out and the somatic cytochrome deposition delay was observed in this heterozygous zebrafish. At 4 d post fertilization (dpf), there were sarcomeric dysplasia and head shrinkage, increased hyoid angle and other craniofacial cartilage deformities. And the zebrafish died at 8~9 dpf. Conclusion     CRISPR/Cas9 knocking out the faf1 gene produces a new phenotype for zebrafish, with delayed pigment deposition and nodule-like change in tail muscle section.


[1]MENGES C W, ALTOMARE D A, TESTA J R. FASassociated factor 1 (FAF1): diverse functions and implications for oncogenesis[J]. Cell Cycle, 2009, 8(16): 2528-2534. DOI:10.4161/cc.8.16.9280.
[2]CHEN J, GE L, LIU A, et al. Identification of pathways related to FAF1/H. pyloriassociated gastric carcinogenesis through an integrated approach based on iTRAQ quantification and literature review[J]. J Proteomics, 2016, 131: 163-176. DOI:10.1016/j.jprot.2015.10.026.
[3]KANG H J, MOON H S, CHUNG H W. The expression of FASassociated factor 1 and heat shock protein 70 in ovarian cancer[J]. Obstet Gynecol Sci, 2014, 57(4): 281-290. DOI:10.5468/ogs.2014.57.4.281.
[4]BEA S, SALAVERRIA I, ARMENGOL L, et al. Uniparental disomies, homozygous deletions, amplifications, and target genes in mantle cell lymphoma revealed by integrative highresolution whole-genome profiling[J]. Blood, 2009, 113(13): 3059-3069. DOI: 10.1182/blood2008-07-170183.
[5]ADHAM I M, KHULAN J, HELD T, et al. Fasassociated factor(FAF1) is required for the early cleavage-stages of mouse embryo[J]. Mol Hum Reprod, 2008, 14(4): 207-213. DOI:10.1093/molehr/gan009.
[6]GHASSIBE-SABBAGH M, DESMYTER L, LANGENBERG T, et al. FAF1, a gene that is disrupted in cleft palate and has conserved function in zebrafish[J]. Am J Hum Genet, 2011, 88(2): 150-161. DOI: 10.1016/j.ajhg.2011.01.003.
[7]ZHANG L, ZHOU F, LI Y, et al. Fas-associated factor 1 is a scaffold protein that promotes beta-transducin repeat-containing protein (beta-TrCP)-mediated beta-catenin ubiquitination and degradation[J]. J Biol Chem, 2012, 287(36): 30701-30710. DOI: 10.1074/jbc.M112.353524.
[8]ZHANG L, ZHOU F, VAN LAAR T, et al. Fas-associated factor 1 antagonizes Wnt signaling by promoting beta-catenin degradation[J]. Mol Biol Cell, 2011, 22(9): 1617-1624. DOI: 10.1091/mbc.E10-12-0985.
[9]CHANG N, SUN C, GAO L, et al. Genome editing with RNA-guided Cas9 nuclease in zebrafish embryos[J]. Cell Res, 2013, 23(4): 465-472. DOI: 10.1038/cr.2013.45.
[10]MARESCA M, LIN V G, GUO N, et al. Obligate ligation-gated recombination (ObLiGaRe): custom-designed nuclease-mediated targeted integration through nonhomologous end joining[J]. Genome Res, 2013, 23(3): 539-546. DOI: 10.1101/gr.145441.112.
[11]O’CONNELL M R, OAKES B L, STERNBERG S H, et al. Programmable RNA recognition and cleavage by CRISPR/Cas9[J]. Nature, 2014, 516(7530): 263-266. DOI: 10.1038/nature 13769.
[12]RAN F A, HSU P D, WRIGHT J, et al. Genome engineering using the CRISPRCas9 system[J]. Nat Protoc, 2013, 8(11): 2281-2308. DOI: 10.1038/nprot.2013.143.
[13]KIMMEL C B, BALLARD W W, KIMMEL S R, et al. Stages of embryonic development of the zebrafish[J]. Dev Dyn, 1995, 203(3): 253-310. DOI: 10.1002/aja.1002030302.
[14]WALKER M B, KIMMEL C B. A two-color acidfree cartilage and bone stain for zebrafish larvae[J]. Biotech Histochem, 2007, 82(1): 23-28. DOI:10.1080/1052029070 1333558.
[15]BROCAL I, WHITE R J, DOOLEY C M, et al. Efficient identification of CRISPR/Cas9-induced insertions/deletions by direct germline screening in zebrafish[J]. BMC Genomics, 2016, 17: 259. DOI: 10.1186/s12864-016-2563-z.
[16]SCHILLING T F, KIMMEL C B. Segment and cell type lineage restrictions during pharyngeal arch development in the zebrafish embryo[J].Development, 1994, 120(3): 483-494.


Last Update: 2017-09-04