ErCas12a and T5exo-ErCas12a Mediate Simple and Efficient Genome Editing in Zebrafish

Han, Bingzhou; Zhang, Yage; Yang, Zhou; Zhang, Biao; Krueger, Christopher J.; Bi, Xuetong; Zhu, Zuoyan; Tong, Xiangjun; Zhang, Bo

doi:10.3390/biology11030411

Cited by 9 publications

(4 citation statements)

References 45 publications

(95 reference statements)

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“…Over 70% of human genes have at least one zebrafish orthologue [ 36 ], with the Xenopus genome including orthologs of ~80% of human disease genes [ 37 ]. More recently, methods to increase the genome editing efficiency of the Clustered Regularly Interspaced Short Palindromic Repeat– (CRISPR–) Cas9 system in zebrafish [ 38 , 39 , 40 , 41 , 42 , 43 ] and Xenopus [ 44 , 45 , 46 , 47 , 48 ] have led to new human disease models.…”

Section: Aquatic Freshwater Vertebrate Animal Model Advantagesmentioning

confidence: 99%

Aquatic Freshwater Vertebrate Models of Epilepsy Pathology: Past Discoveries and Future Directions for Therapeutic Discovery

Williams¹,

Mruk²

2022

IJMS

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Epilepsy is an international public health concern that greatly affects patients’ health and lifestyle. About 30% of patients do not respond to available therapies, making new research models important for further drug discovery. Aquatic vertebrates present a promising avenue for improved seizure drug screening and discovery. Zebrafish (Danio rerio) and African clawed frogs (Xenopus laevis and tropicalis) are increasing in popularity for seizure research due to their cost-effective housing and rearing, similar genome to humans, ease of genetic manipulation, and simplicity of drug dosing. These organisms have demonstrated utility in a variety of seizure-induction models including chemical and genetic methods. Past studies with these methods have produced promising data and generated questions for further applications of these models to promote discovery of drug-resistant seizure pathology and lead to effective treatments for these patients.

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Section: Aquatic Freshwater Vertebrate Animal Model Advantagesmentioning

confidence: 99%

Aquatic Freshwater Vertebrate Models of Epilepsy Pathology: Past Discoveries and Future Directions for Therapeutic Discovery

Williams¹,

Mruk²

2022

IJMS

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“…The gene editing activity and functionality of Mad7 have been confirmed in several bacteria, yeasts, filamentous fungi, HEK293 cell lines, zebrafish, rodent embryos, and stem cells (Bayarsaikhan et al, 2021;Han et al, 2022;Jarczynska et al, 2021). Similarly, Mad7 has been used to integrate donor DNA ranging in size from 1 to 14 kb into mammalian cells, making it a suitable alternative CRISPR enzyme for cell line engineering in the industrial relevant CHO production cell line (Bayarsaikhan et al, 2021).…”

mentioning

confidence: 95%

Expanding the CRISPR toolbox for Chinese hamster ovary cells with comprehensive tools for Mad7 genome editing

Rojek

Basavaraju

Nallapareddy

et al. 2023

Biotech & Bioengineering

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The production of high-value biopharmaceuticals is dominated by mammalian production cells, particularly Chinese hamster ovary (CHO) cells, which have been widely used and preferred in manufacturing processes. The discovery of CRISPR-Cas9 significantly accelerated cell line engineering advances, allowing for production yield and quality improvements. Since then, several other CRISPR systems have become appealing genome editing tools, such as the Cas12a nucleases, which provide broad editing capabilities while utilizing short guide RNAs (gRNAs) that reduce the complexity of the editing systems. One of these is the Mad7 nuclease, which has been shown to efficiently convey targeted gene disruption and insertions in several different organisms. In this study, we demonstrate that Mad7 can generate indels for gene knockout of host cell proteins in CHO cells. We found that the efficiency of Mad7 depends on the addition of protein nuclear localization signals and the gRNAs employed for genome targeting. Moreover, we provide computational tools to design Mad7 gRNAs against any genome of choice and for automated indel detection analysis from next-generation sequencing data. In summary, this paper establishes the application of Mad7 in CHO cells, thereby improving the CRISPR toolbox versatility for research and cell line engineering.

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“…Exonucleases have dominant 5′-3′ or 3′-5′ hydrolysis activity, and they participate in various DNA repair, replication, and recombination processes [26], playing crucial roles in determining the pathway choice for DSB repair [22]. Previously, several types of exonucleases including T5 exonuclease [27][28][29], RecJ exonuclease [30], MRE11 [31], as well as human exonuclease1 (hExo1) [32,33] have been applied for increasing the frequency of indels or knock-in in a variety of organisms (Additional File 2: Table. S1).…”

Section: Introductionmentioning

confidence: 99%

Exonuclease editor promotes precision of gene editing in mammalian cells

Shi,

Li,

et al. 2024

BMC Biol

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Background Many efforts have been made to improve the precision of Cas9-mediated gene editing through increasing knock-in efficiency and decreasing byproducts, which proved to be challenging. Results Here, we have developed a human exonuclease 1-based genome-editing tool, referred to as exonuclease editor. When compared to Cas9, the exonuclease editor gave rise to increased HDR efficiency, reduced NHEJ repair frequency, and significantly elevated HDR/indel ratio. Robust gene editing precision of exonuclease editor was even superior to the fusion of Cas9 with E1B or DN1S, two previously reported precision-enhancing domains. Notably, exonuclease editor inhibited NHEJ at double strand breaks locally rather than globally, reducing indel frequency without compromising genome integrity. The replacement of Cas9 with single-strand DNA break-creating Cas9 nickase further increased the HDR/indel ratio by 453-fold than the original Cas9. In addition, exonuclease editor resulted in high microhomology-mediated end joining efficiency, allowing accurate and flexible deletion of targeted sequences with extended lengths with the aid of paired sgRNAs. Exonuclease editor was further used for correction of DMD patient-derived induced pluripotent stem cells, where 30.0% of colonies were repaired by HDR versus 11.1% in the control. Conclusions Therefore, the exonuclease editor system provides a versatile and safe genome editing tool with high precision and holds promise for therapeutic gene correction.

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ErCas12a and T5exo-ErCas12a Mediate Simple and Efficient Genome Editing in Zebrafish

Cited by 9 publications

References 45 publications

Aquatic Freshwater Vertebrate Models of Epilepsy Pathology: Past Discoveries and Future Directions for Therapeutic Discovery

Aquatic Freshwater Vertebrate Models of Epilepsy Pathology: Past Discoveries and Future Directions for Therapeutic Discovery

Expanding the CRISPR toolbox for Chinese hamster ovary cells with comprehensive tools for Mad7 genome editing

Exonuclease editor promotes precision of gene editing in mammalian cells

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