An improved method for precise genome editing in zebrafish using CRISPR-Cas9 technique

Gasanov, Eugene V.; Jędrychowska, Justyna; Pastor, Michal; Wiweger, Małgorzata; Methner, Axel; Korzh, Vladimir

doi:10.1007/s11033-020-06125-8

Cited by 4 publications

(6 citation statements)

References 15 publications

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“…In fact, the efficiency of precise genome editing can potentially be further enhanced by combining single-stranded DNA (ssDNA) with other approaches that augment precise genome editing, such as the incorporation of small molecules [12,[39][40][41]. It was reported that the repair of Cas9/sgRNA-induced DSBs via HDR was markedly increased by a combination of small molecules and single-stranded oligodeoxynucleotides (ssODNs) [42].…”

Section: Discussionmentioning

confidence: 99%

CRISPR Ribonucleoprotein-Mediated Precise Editing of Multiple Genes in Porcine Fibroblasts

Guo,

Liu,

Zhao

et al. 2024

Animals

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The multi-gene editing porcine cell model can analyze the genetic mechanisms of multiple genes, which is beneficial for accelerating genetic breeding. However, there has been a lack of an effective strategy to simultaneously perform precise multi-gene editing in porcine cells. In this study, we aimed to improve the efficiency of CRISPR RNP-mediated precise gene editing in porcine cells. CRISPR RNP, including Cas9 protein, sgRNA, and ssODN, was used to generate precise nucleotide substitutions by homology-directed repair (HDR) in porcine fetal fibroblasts (PFFs). These components were introduced into PFFs via electroporation, followed by PCR for each target site. To enhance HDR efficacy, small-molecule M3814 and phosphorothioate-modified ssODN were employed. All target DNA samples were sequenced and analyzed, and the efficiencies of different combinations of the CRISPR RNP system in target sites were compared. The results showed that when 2 μM M3814, a small molecule which inhibits NHEJ-mediated repair by blocking DNA-PKs activity, was used, there was no toxicity to PFFs. The CRISPR RNP-mediated HDR efficiency increased 3.62-fold. The combination of CRISPR RNP with 2 μM M3814 and PS-ssODNs achieved an HDR-mediated precision gene modification efficiency of approximately 42.81% in mutated cells, a 6.38-fold increase compared to the control group. Then, we used the optimized CRISPR RNP system to perform simultaneous editing of two and three loci at the INS and RLN3 genes. The results showed that the CRISPR RNP system could simultaneously edit two and three loci. The efficiency of simultaneous editing of two loci was not significantly different from that of single-gene editing compared to the efficiency of single-locus editing. The efficiency of simultaneous precise editing of INS, RLN3 exon 1, and RLN3 exon 2 was 0.29%, 0.24%, and 1.05%, respectively. This study demonstrated that a 2 μM M3814 combination with PS-ssODNs improves the efficacy of CRISPR RNP-mediated precise gene editing and allows for precise editing of up to three genes simultaneously in porcine cells.

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Section: Discussionmentioning

confidence: 99%

CRISPR Ribonucleoprotein-Mediated Precise Editing of Multiple Genes in Porcine Fibroblasts

Guo,

Liu,

Zhao

et al. 2024

Animals

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“…Transplantation of healthy NSCs to RTT patients could differentiate to generate neurons and glial cells, which could provide therapeutic benefits in RTT patients. In the in vivo study, the designed CRISPR‐Cas9 system could be texted in the zebrafish model 114–116 . Upon successful preclinical validation, therapeutic and toxicity should be evaluated in clinical trials, which, hopefully, could be translated to patients based on the consistent and positive outcomes of clinical trials.…”

Section: Disease Modelsmentioning

confidence: 99%

“…In the in vivo study, the designed CRISPR-Cas9 system could be texted in the zebrafish model. [114][115][116] Upon successful preclinical validation, therapeutic and toxicity should be evaluated in clinical trials, which, hopefully, could be translated to patients based on the consistent and positive outcomes of clinical trials.…”

Section: Gene and Stem Cell Therapymentioning

confidence: 99%

Zebrafish in understanding molecular pathophysiology, disease modeling, and developing effective treatments for Rett syndrome

Pramanik,

Bala,

Pradhan

2024

The Journal of Gene Medicine

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Rett syndrome (RTT) is a rare but dreadful X‐linked genetic disease that mainly affects young girls. It is a neurological disease that affects nerve cell development and function, resulting in severe motor and intellectual disabilities. To date, no cure is available for treating this disease. In 90% of the cases, RTT is caused by a mutation in methyl‐CpG‐binding protein 2 (MECP2), a transcription factor involved in the repression and activation of transcription. MECP2 is known to regulate several target genes and is involved in different physiological functions. Mouse models exhibit a broad range of phenotypes in recapitulating human RTT symptoms; however, understanding the disease mechanisms remains incomplete, and many potential RTT treatments developed in mouse models have not shown translational effectiveness in human trials. Recent data hint that the zebrafish model emulates similar disrupted neurological functions following mutation of the mecp2 gene. This suggests that zebrafish can be used to understand the onset and progression of RTT pathophysiology and develop a possible cure. In this review, we elaborate on the molecular basis of RTT pathophysiology in humans and model organisms, including rodents and zebrafish, focusing on the zebrafish model to understand the molecular pathophysiology and the development of therapeutic strategies for RTT. Finally, we propose a rational treatment strategy, including antisense oligonucleotides, small interfering RNA technology and induced pluripotent stem cell‐derived cell therapy.

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“…The transgenic line, Tg(Brn3c:GAP43-GFP) s356t (Xiao et al, 2005), which expresses cytosolic EGFP in inner ear and lateral line mechanosensory hair cells, was used in this study. The generation of two kcng4b mutations, C1 and C2, by CRISPR-Cas9 has been described previously (Gasanov et al, 2021).…”

Section: Animalsmentioning

confidence: 99%

“…1A, B). The C-terminal of Kcng4b polypeptide was prematurely terminated with the loss of 29 C-terminal amino-acid residues (Gasanov et al, 2021) (Fig. 1B).…”

Section: Computational Analysis Of Kcng4b Mutantsmentioning

confidence: 99%

Mutant analysis of Kcng4b reveals how the different functional states of the voltage-gated potassium channel regulate ear development

Jędrychowska

Vardanian

Wieczór

et al. 2023

Preprint

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The voltage gated (Kv) slow-inactivating delayed rectifier channel regulates the development of hollow organs of the zebrafish. The functional tetramer consists of an electrically active subunit (Kcnb1, Kv2.1) and a modulatory silent subunit (Kcng4b, Kv6.4). The two mutations in zebrafish kcng4b - kcng4b-C1 and kcng4b-C2 (Gasanov et al., 2021) - have been studied during ear development using electrophysiology, developmental biology and in silico structural modelling. kcng4b-C1 mutation causes a C-terminal truncation characterized by mild Kcng4b loss-of-function (LOF) manifested by failure of kinocilia to extend and formation of ectopic otoliths. In contrast, the kcng4b-C2-/- mutation causes the C-terminal domain to elongate and the ectopic seventh transmembrane (TM) domain to form, converting the intracellular C-terminus to an extracellular one. Kcng4b-C2 acts as a Kcng4b gain-of-function (GOF) allele. Otoliths fail to develop and kinocilia are reduced in kcng4b-C2-/-. These results show that different mutations of the silent subunit Kcng4 can affect the activity of the Kv channel and cause a wide range of developmental defects.

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An improved method for precise genome editing in zebrafish using CRISPR-Cas9 technique

Cited by 4 publications

References 15 publications

CRISPR Ribonucleoprotein-Mediated Precise Editing of Multiple Genes in Porcine Fibroblasts

CRISPR Ribonucleoprotein-Mediated Precise Editing of Multiple Genes in Porcine Fibroblasts

Zebrafish in understanding molecular pathophysiology, disease modeling, and developing effective treatments for Rett syndrome

Mutant analysis of Kcng4b reveals how the different functional states of the voltage-gated potassium channel regulate ear development

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