Genome editing in animals with minimal PAM CRISPR-Cas9 enzymes

Vicencio, Jeremy; C, Sánchez-Bolaños; Moreno-Sánchez, Ismael; Brena, David; Vejnar, Charles E.; Kukhtar, Dmytro; Ruiz-López, Miguel; Cots-Ponjoan, Mariona; Rubio, Ana Martínez; Melero, Natalia Rodrigo; Crespo-Cuadrado, Jesús; Carolis, Carlo; Pérez-Pulido, Antonio J.; Giráldez, Antonio J.; Kleinstiver, Benjamin P.; Cerón, Julián; Moreno-Mateos, Miguel A.

doi:10.1038/s41467-022-30228-4

Cited by 36 publications

(22 citation statements)

References 67 publications

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“…Based on our results, we conclude that CBE technology is a more efficient, easier to design, and time-saving approach to introduce a specific C:G to T:A mutation into the genome and should be considered as an alternative for prime editing and HDR-based knock-in techniques. Moreover, while our manuscript was under review, the Cas9-SpRY has been shown to be efficient for classical knock-out approaches generating INDELS in zebrafish 39 . The use of the CBE4max-SpRY is expected to increase the number of potential off-targets compared to the classical base editors as it is highly flexible on the type of PAM used; however, the NGS analyses revealed a good fidelity of the CBE4max-SpRY.Indeed, the presence of only one mismatch on the off-target site decreases drastically the efficiency of base editing and for the other analyzed off-targets no mutations were detected (Fig.…”

Section: Discussionmentioning

confidence: 99%

Disease modeling by efficient genome editing using a near PAM-less base editor in vivo

et al. 2022

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Base Editors are emerging as an innovative technology to introduce point mutations in complex genomes. So far, the requirement of an NGG Protospacer Adjacent Motif (PAM) at a suitable position often limits the base editing possibility to model human pathological mutations in animals. Here we show that, using the CBE4max-SpRY variant recognizing nearly all PAM sequences, we could introduce point mutations for the first time in an animal model with high efficiency, thus drastically increasing the base editing possibilities. With this near PAM-less base editor we could simultaneously mutate several genes and we developed a co-selection method to identify the most edited embryos based on a simple visual screening. Finally, we apply our method to create a zebrafish model for melanoma predisposition based on the simultaneous base editing of multiple genes. Altogether, our results considerably expand the Base Editor application to introduce human disease-causing mutations in zebrafish.

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Section: Discussionmentioning

confidence: 99%

Disease modeling by efficient genome editing using a near PAM-less base editor in vivo

et al. 2022

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“…Of note, several optimization strategies can be considered to limit off-target effects, such as alteration of the crRNA and optimizing Cas9 concentrations [ 29 ]. While a PAM needs to be directly adjacent to the intended cleavage site to allow CRISPR-mediated editing [ 28 ], due to the variety in naturally occurring Cas-proteins and Cas-proteins with mutations in the PAM-recognition domain, a variety in PAM sequences is available to facilitate gene targeting [ 28 , 48 , 49 , 50 ]. As a result, virtually any gene or DNA region of interest can be targeted with the CRISPR system, in contrast to other gene editing tools such as ZFNs [ 30 ].…”

Section: Gene Editing Basics—mode Of Action and Different Toolsmentioning

confidence: 99%

“…adjacent to the intended cleavage site to allow CRISPR-mediated editing [28], due to the variety in naturally occurring Cas-proteins and Cas-proteins with mutations in the PAMrecognition domain, a variety in PAM sequences is available to facilitate gene targeting [28,[48][49][50]. As a result, virtually any gene or DNA region of interest can be targeted with the CRISPR system, in contrast to other gene editing tools such as ZFNs [30].…”

Section: Gene Editing Basics-mode Of Action and Different Toolsmentioning

confidence: 99%

Closing the Door with CRISPR: Genome Editing of CCR5 and CXCR4 as a Potential Curative Solution for HIV

Heeren¹

2022

BioTech

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Human immunodeficiency virus (HIV) infection can be controlled by anti-retroviral therapy. Suppressing viral replication relies on life-long medication, but anti-retroviral therapy is not without risks to the patient. Therefore, it is important that permanent cures for HIV infection are developed. Three patients have been described to be completely cured from HIV infection in recent years. In all cases, patients received a hematopoietic stem cell (HSC) transplantation due to a hematological malignancy. The HSCs were sourced from autologous donors that expressed a homozygous mutation in the CCR5 gene. This mutation results in a non-functional receptor, and confers resistance to CCR5-tropic HIV strains that rely on CCR5 to enter host cells. The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated (Cas) system is one of the methods of choice for gene editing, and the CRISPR/Cas system has been employed to target loci of interest in the context of HIV. Here, the current literature regarding CRISPR-mediated genome editing to render cells resistant to HIV (re)-infection by knocking out the co-receptors CCR5 and CXCR4 is summarized, and an outlook is provided regarding future (research) directions.

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“…VQR recognizes NGAG as efficiently as wild-type Cas9 targets NGG [17], thus broadening the range of targets. Additionally, SpG and SpRY are two modified versions of Cas9 with more relaxed PAM requirements than Cas9 [18]. Thus, they target more portions of the genome.…”

Section: Cas9 Variantsmentioning

confidence: 99%

A Decade of CRISPR-Cas Genome Editing in C. elegans

Kim¹,

Hong²,

Chen³

2022

Preprint

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CRISPR-Cas allows us to introduce desired genome editing, including mutations, epitopes, and deletions with unprecedented efficiency. The development of CRISPR-Cas has progressed to such an extent that it is now applicable in various fields with the help of model organisms. C. elegans is one of the pioneering animals in which numerous CRISPR-Cas strategies have been rapidly es-tablished over the past decade. Ironically, the emergence of numerous methods makes the right choice of method difficult. Choosing an appropriate selection or screening approach is the first step in planning a genome modification. This report summarizes the key features and applications of CRISPR-Cas methods using C. elegans and illustrates key strategies. Our overview of significant advances in CRISPR-Cas will help readers to understand current advances in genome editing and navigate various methods of CRISPR-Cas genome editing.

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Genome editing in animals with minimal PAM CRISPR-Cas9 enzymes

Cited by 36 publications

References 67 publications

Disease modeling by efficient genome editing using a near PAM-less base editor in vivo

Disease modeling by efficient genome editing using a near PAM-less base editor in vivo

Closing the Door with CRISPR: Genome Editing of CCR5 and CXCR4 as a Potential Curative Solution for HIV

A Decade of CRISPR-Cas Genome Editing in C. elegans

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