Rapid clonal identification of biallelic CRISPR/Cas9 knock-ins using SNEAK PEEC

Singh, Sameer; Banerjee, Anoosha; Broeck, Arnaud Vanden; Klinge, Sebastian

doi:10.1038/s41598-023-28732-8

Cited by 3 publications

(3 citation statements)

References 24 publications

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“…A biallelically tagged MK67I-GFP cell line was generated using an in-house genome editing platform called SNEAK PEEC ( 74 ) ( fig. S1A ).…”

Section: Methodsmentioning

confidence: 99%

Principles of human pre-60Sbiogenesis

Broeck

Klinge

2023

Preprint

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During early stages of human large ribosomal subunit (60S) biogenesis, an ensemble of assembly factors establishes and fine-tunes the essential RNA functional centers of pre-60Sparticles by an unknown mechanism. Here, we report a series of cryo-electron microscopy structures of human nucleolar and nuclear pre-60Sassembly intermediates at resolutions of 2.5-3.2 angstroms. These structures show how protein interaction hubs tether assembly factor complexes to nucleolar particles and how GTPases and ATPases couple irreversible nucleotide hydrolysis steps to the installation of functional centers. Nuclear stages highlight how a conserved RNA processing complex, the rixosome, couples large-scale RNA conformational changes to pre-rRNA processing by the RNA degradation machinery. Our ensemble of human pre-60Sparticles provides a rich foundation to elucidate the molecular principles of ribosome formation.

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“…A biallelically tagged MK67I-GFP cell line was generated using an in-house genome editing platform called SNEAK PEEC ( 74 ) ( fig. S1A ).…”

Section: Methodsmentioning

confidence: 99%

Principles of human pre-60Sbiogenesis

Broeck

Klinge

2023

Preprint

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“…Even though increasing evidence supports the suitability of the CRISPR/Cas9 system for both modeling [ 2 , 14 ] and drug development [ 15 , 16 ], several challenges, such as finding alternatives for increasing the limited efficiency of the CRISPR/Cas9-mediated knock-in, are still to be overcome. For instance, genome editing in human cells mainly results in monoallelic modifications, where only a single gene copy is edited while the second copy remains unaltered or even undergoes undesired modifications due to the Cas9-mediated DSB [ 17 ]. Currently, antibiotics, or fluorescence-activated cell-sorting (FACS), are commonly used to enrich edited cells, followed by clone screening to detect biallelic modifications [ 17 , 18 ].…”

Section: Introductionmentioning

confidence: 99%

“…For instance, genome editing in human cells mainly results in monoallelic modifications, where only a single gene copy is edited while the second copy remains unaltered or even undergoes undesired modifications due to the Cas9-mediated DSB [ 17 ]. Currently, antibiotics, or fluorescence-activated cell-sorting (FACS), are commonly used to enrich edited cells, followed by clone screening to detect biallelic modifications [ 17 , 18 ]. Even though these strategies work for ex vivo approaches, they are unsuitable for in vivo ones.…”

Section: Introductionmentioning

confidence: 99%

Current Strategies for Increasing Knock-In Efficiency in CRISPR/Cas9-Based Approaches

Leal,

Herreno-Pachón,

Benincore-Flórez

et al. 2024

IJMS

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Since its discovery in 2012, the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) system has supposed a promising panorama for developing novel and highly precise genome editing-based gene therapy (GT) alternatives, leading to overcoming the challenges associated with classical GT. Classical GT aims to deliver transgenes to the cells via their random integration in the genome or episomal persistence into the nucleus through lentivirus (LV) or adeno-associated virus (AAV), respectively. Although high transgene expression efficiency is achieved by using either LV or AAV, their nature can result in severe side effects in humans. For instance, an LV (NCT03852498)- and AAV9 (NCT05514249)-based GT clinical trials for treating X-linked adrenoleukodystrophy and Duchenne Muscular Dystrophy showed the development of myelodysplastic syndrome and patient’s death, respectively. In contrast with classical GT, the CRISPR/Cas9-based genome editing requires the homologous direct repair (HDR) machinery of the cells for inserting the transgene in specific regions of the genome. This sophisticated and well-regulated process is limited in the cell cycle of mammalian cells, and in turn, the nonhomologous end-joining (NHEJ) predominates. Consequently, seeking approaches to increase HDR efficiency over NHEJ is crucial. This manuscript comprehensively reviews the current alternatives for improving the HDR for CRISPR/Cas9-based GTs.

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Principles of human pre-60 S biogenesis

Broeck

Klinge

2023

Science

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During the early stages of human large ribosomal subunit (60 S ) biogenesis, an ensemble of assembly factors establishes and fine-tunes the essential RNA functional centers of pre-60 S particles by an unknown mechanism. Here, we report a series of cryo–electron microscopy structures of human nucleolar and nuclear pre-60 S assembly intermediates at resolutions of 2.5 to 3.2 angstroms. These structures show how protein interaction hubs tether assembly factor complexes to nucleolar particles and how guanosine triphosphatases and adenosine triphosphatase couple irreversible nucleotide hydrolysis steps to the installation of functional centers. Nuclear stages highlight how a conserved RNA-processing complex, the rixosome, couples large-scale RNA conformational changes with pre–ribosomal RNA processing by the RNA degradation machinery. Our ensemble of human pre-60 S particles provides a rich foundation with which to elucidate the molecular principles of ribosome formation.

show abstract

Rapid clonal identification of biallelic CRISPR/Cas9 knock-ins using SNEAK PEEC

Cited by 3 publications

References 24 publications

Principles of human pre-60Sbiogenesis

Principles of human pre-60Sbiogenesis

Current Strategies for Increasing Knock-In Efficiency in CRISPR/Cas9-Based Approaches

Principles of human pre-60 S biogenesis

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