PCR is a common method to produce desired DNA fragments from templates. The oligonucleotide primers used for PCR must contain annealing sequences that are usually 20. CRISPR RNA- guided Fok. I nucleases repair a PAH variant in a phenylketonuria model. Abstract. The CRISPR/Cas. In this study, we used a modified CRISPR system, which employs the fusion of inactive Cas. Cas. 9) and the Fok. I endonuclease (Fok. I- d. Cas. 9) to correct the most common variant (allele frequency 2. PAH) gene - c. 1. C> T (p. Arg. 40. Trp) - as an approach toward curing phenylketonuria (PKU). PKU is the most common inherited diseases in amino acid metabolism. It leads to severe neurological and neuropsychological symptoms if untreated or late diagnosed. Correction of the disease- causing variants could rescue residual PAH activity and restore normal function. Co- expression of a single guide RNA plasmid, a Fok. · CRISPR RNA-guided FokI nucleases repair a PAH variant in a phenylketonuria model. · Inhibitors of Differentiation (ID1, ID2, ID3 and ID4) genes are neuronal targets of MeCP2 that are elevated in Rett syndrome. 爱问共享资料是爱问自主研发的资料共享平台,包含了千万网友上传的多种格式的文档,同时也提供海量资料的免费下载. I- d. Cas. 9- zs. Green. 1 plasmid, and the presence of a single- stranded oligodeoxynucleotide in PAH_c. C> T COS- 7 cells – an in vitro model for PKU – corrected the PAH variant and restored PAH activity. Also in this system, the HDR enhancer RS- 1 improved correction efficiency. This proof- of- concept indicates the potential of the Fok. I- d. Cas. 9 system for precision medicine, in particular for targeting PKU and other monogenic metabolic diseases. ![]() ![]() Phenylketonuria (PKU, OMIM 2. PAH, EC 1. 1. 4. 1. PAH converts phenylalanine (Phe) to tyrosine (Tyr). The cofactor tetrahydrobiopterin (BH4), iron and molecular oxygen are required for this process. PAH deficiency results in the accumulation of Phe in the body including the brain. If untreated or diagnosed late, its high concentration leads to severe neurological and neuropsychological symptoms, including intellectual disability, seizures, ataxia, motor deficits and behavioral problems. ![]() Because of the severity of the disease, newborn screening for PKU has been instituted in many countries. Lifelong restriction of dietary Phe is the established treatment for preventing neuronal damage. However, poor compliance by PKU patients and the heavy economic burden challenge the dietary management. Although the restriction of dietary Phe improves cognitive performance, other neuropsychological parameters such as choice reaction time, attention, and executive functions are impaired compared to healthy controls even in the best treated individuals. An additional supplement of tetrahydrobiopterin (BH4; sapropterin dihydrochloride) may be beneficial for some PKU patients with a mild pheotype. However, the ideal approach for curing PKU would be to correct variants in the PAH gene directly through gene therapy. Since PKU is an autosomal recessive inherited disorder, only individuals with PAH variants located in both alleles develop symptoms. Even if only one allele is corrected, the residual PAH activity would be sufficiently increased, abolishing or reducing PKU symptoms. In patients with severe classic PKU, the c. C> T (p. Arg. 40. Trp) single nucleotide variant is the most frequent one in PAH (allele frequency 2. Arg. 40. 8Trp]; [Arg. Trp] the most common genotype (genotype frequency 1. Thus, PAH_c. 1. 22. C> T was chosen as the target in this study. The most promising genetic repair of variants is achievable through homologous recombination repair (homology directed repair; HDR) after the introduction of DNA double- stranded breaks (DSBs) close to the variant. DSBs can be generated by different genome editing technologies, such as zinc- finger nucleases (ZFNs), transcription activator- like effector nucleases (TALENs) and the recently developed technique of clustered regularly interspaced short palindromic repeats (CRISPR)4. The CRISPR/Cas. 9 system was first identified as a prokaryotic defense mechanism against bacteriophages, and is currently being used for site- specific genome editing. It is composed of a single- guide RNA (sg. RNA) containing a 2. Cas. 9 nuclease that enables the cleavage of genomic DNA. The DSBs then stimulate the DNA repair pathway through non- homologous end joining (NHEJ) and HDR. Of these mechanisms, HDR can generate the desired sequence replacement at the DSBs through the usage of a donor DNA template, which corrects the specific variant within the genome. Several studies have recently reported encouraging results using CRISPR/Cas. Duchenne muscular dystrophy and retinitis pigmentosa in vitro and in vivo. However, there are still two major challenges in the CRISPR/Cas. HDR efficiency. 5,1. To overcome these problems, different strategies were adopted in this study. First, we chose the CRISPR RNA- guided Fok. I nuclease system (Fok. I- d. Cas. 9 system)1. CRISPR/Cas. 9 system as the specific genome editing tool. The Fok. I- d. Cas. RNAs and Fok. I- d. Cas. 9 nuclease, which is a fusion of inactive Cas. Cas. 9) and the Fok. I endonuclease. The Fok. I- d. Cas. 9 system differs from CRISPR/Cas. Fok. I- d. Cas. 9: sg. RNAs is required. Moreover, we employed 3- (N- benzylsulfamoyl)- 4- 9. N- (4- bromophenyl)benzamide (RS- 1), an HDR enhancer. RAD5. 1- stimulatory compound. By this means, we successfully performed a genetic repair of the c. C> T PAH variant, which for the first time corrected the point variant and rescued both PAH protein expression and activity. Results. Establishment of the PAH_c. C> T COS- 7 cell line as an in vitro PKU model. The PAH_c. 1. 22. C> T variant was introduced into COS- 7 cells by transfection with an appropriately modified vector DNA. Single clones were sub- cultured for at least 1. Sanger sequencing analysis proved that the “T” point variant was appropriately located in PAH (Fig. Reverse transcription PCR (RT- PCR) confirmed that PAH_c. C> T was expressed in the transfected COS- 7 cells at the RNA level at a similar level as the wild- type protein used in control; untransfected COS- 7 cells exhibited no PAH RNA (Fig. Concurrently, Western blot analysis with an antibody against the wild- type protein (PAH_WT) revealed a basically complete loss of the PAH protein in the cells transfected with the mutated gene version (Fig. PAH_c. 1. 22. 2C> T COS- 7 cell line was established as a PKU in vitro model. The PAH_c. 1. 22. C> T variant was corrected using the Fok. I- d. Cas. 9 system. The Fok. I- d. Cas. CRISPR/Cas. 9 system insofar as only binding of a Fok. I- d. Cas. 9 dimer can cleave DNA (Fig. After electroporation of COS- 7 cells with expression vectors of the Fok. I- d. Cas. 9 gene and the sg. RNA, flow cytometry analysis showed that approximately 1. Fig. 2b). In presence of RS- 1, PAH expression in PAH_c. C> T COS- 7 cells treated with 0. M of the single- stranded oligodeoxynucleotide (ss. ODN) that represents the replacement sequence was significantly higher than the PAH expression of PAH_c. C> T COS- 7 cells that were not transfected with the vectors. A lower ss. ODN concentration had no apparent effect on PAH expression (Fig. Furthermore, liquid chromatography- electrospray ionization tandem mass spectrometry was employed to evaluate the PAH activity after repair (Fig. Western blot analysis. Sanger sequencing analysis was performed to confirm the correction in all groups. The 1 n. M ss. ODN and 0. M ss. ODN groups showed double- peaks at the corrected position regardless of RS- 1 treatment, demonstrating the presence of both a “T” and a “C” at the PAH_c. C> T variant in the cell population (Supplementary Fig. To examine the correction rate, we amplified by PCR target DNA from the 1 n. M ss. ODN with RS- 1 treatment group and verified the sequence by TA- cloning. Sequence analysis of the DNA of 3. C” (Fig. 2e). To assess the possible off- targeting introduced by Fok. I- d. Cas. 9- directed cleavage, two potential off- target sites were predicted by Cas. OT (Supplementary Table 1). Both potential sites were analyzed together with the PAH gene on- target site by Sanger sequencing. None of the analyzed regions showed evidence of off- target cleavage (data not shown). Correction of the PAH_c. C> T variant in COS- 7 cells. Discussion. Although the restriction of dietary Phe and/or supplementation with BH4 can substantially alleviate clinical symptoms of PKU, patients do not manage to maintain dietary treatment for life. They suffer from impaired neuropsychological and partly from intellectual function and PKU women are at risk for the maternal PKU syndrome. The ideal approach to treating this disease would be a correction of the PAH variant. PKU gene therapy has been developed over the past two decades using an animal model. Researchers delivered the wild- type PAH gene to tissues with an adeno- associated virus (AAV) vector.
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