An overview of currently available molecular Cas-tools for precise genome modification

Kondrateva, Ekaterina; Demchenko, Anna; Лавров, А. В.; Smirnikhina, Svetlana

doi:10.1016/j.gene.2020.145225

Cited by 5 publications

(4 citation statements)

References 93 publications

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“…). Mosaicism occurs at various frequencies [ 85 ], and the type of gene editor and modification [ 90 , 91 ], introduction method, animal species, and introduced component of gene editors (e.g., expression plasmid, mRNA, and protein/nuclease) seem to be potential factors.…”

Section: Current Status and Future Prospects Of Gene-edited Pigsmentioning

confidence: 99%

Current status of the application of gene editing in pigs

2021

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Genetically modified animals, especially rodents, are widely used in biomedical research. However, non-rodent models are required for efficient translational medicine and preclinical studies. Owing to the similarity in the physiological traits of pigs and humans, genetically modified pigs may be a valuable resource for biomedical research. Somatic cell nuclear transfer (SCNT) using genetically modified somatic cells has been the primary method for the generation of genetically modified pigs. However, site-specific gene modification in porcine cells is inefficient and requires laborious and time-consuming processes. Recent improvements in gene-editing systems, such as zinc finger nucleases, transcription activator-like effector nucleases, and the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (CRISPR/Cas) system, represent major advances. The efficient introduction of site-specific modifications into cells via gene editors dramatically reduces the effort and time required to generate genetically modified pigs. Furthermore, gene editors enable direct gene modification during embryogenesis, bypassing the SCNT procedure. The application of gene editors has progressively expanded, and a range of strategies is now available for porcine gene engineering. This review provides an overview of approaches for the generation of genetically modified pigs using gene editors, and highlights the current trends, as well as the limitations, of gene editing in pigs.

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Section: Current Status and Future Prospects Of Gene-edited Pigsmentioning

confidence: 99%

Current status of the application of gene editing in pigs

2021

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“…Further improvements to editing efficiency were achieved by designing the Cas nuclease in such a manner that it generates a nick in the non-edited DNA strand, thereby stimulating cellular repair of the non-edited strand using the edited strand as a template [18]. Additional advancements were made with the replacement of wild-type SpCas with its other variants, linker optimization, and the injection of a second fragment of UGI [20].…”

Section: A New Generation Of Genome Editing Tools: Base Editing and Prime Editingmentioning

confidence: 99%

Recent Advances in CRISPR/Cas9-Based Genome Editing Tools for Cardiac Diseases

Schreurs

Sacchetto

Colpaert

et al. 2021

IJMS

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In the past two decades, genome editing has proven its value as a powerful tool for modeling or even treating numerous diseases. After the development of protein-guided systems such as zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), which for the first time made DNA editing an actual possibility, the advent of RNA-guided techniques has brought about an epochal change. Based on a bacterial anti-phage system, the CRISPR/Cas9 approach has provided a flexible and adaptable DNA-editing system that has been able to overcome several limitations associated with earlier methods, rapidly becoming the most common tool for both disease modeling and therapeutic studies. More recently, two novel CRISPR/Cas9-derived tools, namely base editing and prime editing, have further widened the range and accuracy of achievable genomic modifications. This review aims to provide an overview of the most recent developments in the genome-editing field and their applications in biomedical research, with a particular focus on models for the study and treatment of cardiac diseases.

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“…More than 90 Cas nucleases, including Cas9 and Cas12a (Cpf1), have been identified thus far 11 , 12 and used to develop genome editing tools. 13 Recently, Cas nucleases with a relatively small size, such as Cas12e (CasX) 14 and Cas12j (CasΦ), 15 were reported. Cas12f1, which was discovered in metagenomic data sets, 16 has attracted much attention due to its small size.…”

Section: Introductionmentioning

confidence: 99%

Miniature CRISPR-Cas12f1-Mediated Single-Nucleotide Microbial Genome Editing Using 3′-Truncated sgRNA

2023

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The CRISPR-Cas system has been used as a convenient tool for genome editing because the nuclease that cuts the target DNA and the guide RNA that recognizes the target are separated into modules. Cas12f1, which has a smaller size than that of other Cas nucleases, is easily loaded into vectors and is emerging as a new genome editing tool. In this study, AsCas12f1 was used to negatively select only Escherichia coli cells obtained by oligonucleotide-directed genome editing. Although double-, triple-, and quadruple-base substitutions were accurately and efficiently performed in the genome, the performance of single-base editing was poor. To resolve this limitation, we serially truncated the 3′-end of sgRNAs and determined the maximal truncation required to maintain the target DNA cleavage activity of Cas12f1. Negative selection of single-nucleotide-edited cells was efficiently performed with the maximally 3′-truncated sgRNA–Cas12f1 complex in vivo . Moreover, Sanger sequencing showed that the accuracy of single-nucleotide substitution, insertion, and deletion in the microbial genome was improved. These results demonstrated that a truncated sgRNA approach could be widely used for accurate CRISPR-mediated genome editing.

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An overview of currently available molecular Cas-tools for precise genome modification

Cited by 5 publications

References 93 publications

Current status of the application of gene editing in pigs

Current status of the application of gene editing in pigs

Recent Advances in CRISPR/Cas9-Based Genome Editing Tools for Cardiac Diseases

Miniature CRISPR-Cas12f1-Mediated Single-Nucleotide Microbial Genome Editing Using 3′-Truncated sgRNA

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