Adenine Base Editor Ribonucleoproteins Delivered by Lentivirus-Like Particles Show High On-Target Base Editing and Undetectable RNA Off-Target Activities

Lyu, Pin; Lu, Zuyan; Cho, Sung-Ik; Yadav, Manish Kumar; Yoo, Kyung Whan; Atala, Anthony

doi:10.1089/crispr.2020.0095

Cited by 29 publications

(23 citation statements)

References 39 publications

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“…Virus-like particles (VLPs), assemblies of viral proteins that can infect cells but lack viral genetic material, have emerged as potentially promising vehicles for delivering gene editing agents as RNPs ( Campbell et al., 2019 ; Choi et al., 2016 ; Gee et al., 2020 ; Hamilton et al., 2021 ; Indikova and Indik, 2020 ; Lyu et al., 2019 , 2021 ; Mangeot et al., 2019 ; Yao et al., 2021 ). VLPs that deliver RNP cargoes exploit the efficiency and tissue targeting advantages of viral delivery but avoid the risks associated with viral genome integration and prolonged expression of the editing agent.…”

Section: Introductionmentioning

confidence: 99%

“…VLPs that deliver RNP cargoes exploit the efficiency and tissue targeting advantages of viral delivery but avoid the risks associated with viral genome integration and prolonged expression of the editing agent. However, existing VLP-mediated strategies for delivering gene editing agent RNPs thus far support only modest editing efficiencies with limited validation of therapeutic efficacy in vivo ( Campbell et al., 2019 ; Choi et al., 2016 ; Gee et al., 2020 ; Hamilton et al., 2021 ; Indikova and Indik, 2020 ; Lyu et al., 2019 , 2021 ; Mangeot et al., 2019 ; Yao et al., 2021 ). Indeed, to our knowledge, therapeutic levels of postnatal in vivo gene editing using RNP-packaging VLPs have not been previously reported.…”

Section: Introductionmentioning

confidence: 99%

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Engineered virus-like particles for efficient in vivo delivery of therapeutic proteins

Banskota

Raguram

Suh

et al. 2022

Cell

356

217

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Engineered virus-like particles for efficient in vivo delivery of therapeutic proteins

Banskota

Raguram

Suh

et al. 2022

Cell

356

217

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“…1 ), thus saving time and work linked to cell selection or sorting. Recent studies showed that lentivirus-like particles (LVLP) can be generated with an approach similar to the one described in the present work to obtain a CRISPR/Cas9 system using saCas9, SpCas9, or adenine-based editing systems [ 15 , 26 , 27 ]. The design of these LVLP (number and location of the MS2 coat protein, number of MS2 aptamer repeats, and 3′UTR modification) was different from ours.…”

Section: Discussionmentioning

confidence: 99%

CRISPR/Cas9-mediated gene knockout and interallelic gene conversion in human induced pluripotent stem cells using non-integrative bacteriophage-chimeric retrovirus-like particles

et al. 2022

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Background The application of CRISPR/Cas9 technology in human induced pluripotent stem cells (hiPSC) holds tremendous potential for basic research and cell-based gene therapy. However, the fulfillment of these promises relies on the capacity to efficiently deliver exogenous nucleic acids and harness the repair mechanisms induced by the nuclease activity in order to knock-out or repair targeted genes. Moreover, transient delivery should be preferred to avoid persistent nuclease activity and to decrease the risk of off-target events. We recently developed bacteriophage-chimeric retrovirus-like particles that exploit the properties of bacteriophage coat proteins to package exogenous RNA, and the benefits of lentiviral transduction to achieve highly efficient, non-integrative RNA delivery in human cells. Here, we investigated the potential of bacteriophage-chimeric retrovirus-like particles for the non-integrative delivery of RNA molecules in hiPSC for CRISPR/Cas9 applications. Results We found that these particles efficiently convey RNA molecules for transient expression in hiPSC, with minimal toxicity and without affecting the cell pluripotency and subsequent differentiation. We then used this system to transiently deliver in a single step the CRISPR-Cas9 components (Cas9 mRNA and sgRNA) to generate gene knockout with high indel rate (up to 85%) at multiple loci. Strikingly, when using an allele-specific sgRNA at a locus harboring compound heterozygous mutations, the targeted allele was not altered by NHEJ/MMEJ, but was repaired at high frequency using the homologous wild type allele, i.e., by interallelic gene conversion. Conclusions Our results highlight the potential of bacteriophage-chimeric retrovirus-like particles to efficiently and safely deliver RNA molecules in hiPSC, and describe for the first time genome engineering by gene conversion in hiPSC. Harnessing this DNA repair mechanism could facilitate the therapeutic correction of human genetic disorders in hiPSC.

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“…The cytosine deaminase activity then catalyzes deamination of a targeted cytosine (C) within the genome to uracil (U). Uracil glycosylase inhibitor (also a component in the Cas9 fusion protein) prohibits the U from being removed from the DNA, and following a round of DNA replication, a C to T mutation is placed within the targeted gene [69][70][71][72][73][74][75][76][77].…”

Section: Development Of Precision Editing Technology 41 Single Base Editormentioning

confidence: 99%

Research progress in gene editing technology

Huang

Yao

et al. 2021

Front. Biosci. (Landmark Ed)

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As a tool for modifying the genome, gene editing technology has developed rapidly in recent years, especially in the past two years. With the emergence of new gene editing technologies, such as transposon editing tools, numerous advancements have been made including precise editing of the genome, double base editing, and pilot editing. This report focuses on the development of gene editing tools in recent years, elaborates the progress made in classic editing tools, base editor and other new editing tools, and provides insights into challenges and opportunities.

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Adenine Base Editor Ribonucleoproteins Delivered by Lentivirus-Like Particles Show High On-Target Base Editing and Undetectable RNA Off-Target Activities

Cited by 29 publications

References 39 publications

Engineered virus-like particles for efficient in vivo delivery of therapeutic proteins

Engineered virus-like particles for efficient in vivo delivery of therapeutic proteins

CRISPR/Cas9-mediated gene knockout and interallelic gene conversion in human induced pluripotent stem cells using non-integrative bacteriophage-chimeric retrovirus-like particles

Research progress in gene editing technology

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