Assessing and advancing the safety of CRISPR-Cas tools: from DNA to RNA editing

Nature Communications, Journal Year: 2023, Volume and Issue: 14(1)

Published: Jan. 13, 2023

CRISPR-Cas gene editing has revolutionized experimental molecular biology over the past decade and holds great promise for treatment of human genetic diseases. Here we review development CRISPR-Cas9/Cas12/Cas13 nucleases, DNA base editors, prime RNA focusing on assessment improvement their precision safety, pushing limit specificity efficiency. We summarize capabilities limitations each CRISPR tool from to editing, highlight opportunities future improvements applications in basic research, as well therapeutic clinical considerations use patients.

Language: Английский

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Highly efficient prime editing by introducing same-sense mutations in pegRNA or stabilizing its structure

Nature Communications, Journal Year: 2022, Volume and Issue: 13(1)

Published: March 29, 2022

Abstract Prime editor (PE), which is developed by combining Cas9 nickase and an engineered reverse transcriptase, can mediate all twelve types of base substitutions small insertions or deletions in living cells but its efficiency remains low. Here, we develop spegRNA introducing same-sense mutations at proper positions the reverse-transcription template pegRNA to increase PE’s base-editing up-to 4,976-fold (on-average 353-fold). We also apegRNA altering secondary structure indel-editing 10.6-fold 2.77-fold). The be combined further enhance editing efficiency. When are used PE3 PE5 systems, efficiencies sPE3, aPE3, sPE5 aPE5 systems enhanced significantly. strategies this study realize highly efficient prime certain previously uneditable sites.

Language: Английский

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Precise genomic editing of pathogenic mutations in RBM20 rescues dilated cardiomyopathy

Science Translational Medicine, Journal Year: 2022, Volume and Issue: 14(672)

Published: Nov. 23, 2022

Mutations in RNA binding motif protein 20 ( RBM20 ) are a common cause of familial dilated cardiomyopathy (DCM). Many mutations cluster within an arginine/serine-rich (RS-rich) domain, which mediates nuclear localization. These induce mis-localization to form aberrant ribonucleoprotein (RNP) granules the cytoplasm cardiomyocytes and abnormal alternative splicing cardiac genes, contributing DCM. We used adenine base editing (ABE) prime (PE) correct pathogenic p.R634Q p.R636S RS-rich domain human isogenic induced pluripotent stem cell (iPSC)–derived cardiomyocytes. Using ABE R634Q iPSCs, we achieved 92% efficiency A-to-G editing, normalized restored localization RBM20, eliminated RNP granule formation. In addition, developed PE strategy R636S mutation iPSCs observed A-to-C at 40% efficiency. To evaluate potential for DCM treatment, also created Rbm20 R636Q mutant mice. Homozygous (R636Q/R636Q) mice severe dysfunction, heart failure, premature death. Systemic delivery components containing ABEmax-VRQR-SpCas9 single-guide by adeno-associated virus serotype 9 these function as assessed echocardiography extended life span. As seen sequencing analysis, correction rescued transcriptional profile treated R636Q/R636Q mice, compared gene expression untreated findings demonstrate precise genetic promising therapeutic approach

Language: Английский

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Genotoxic effects of base and prime editing in human hematopoietic stem cells

Nature Biotechnology, Journal Year: 2023, Volume and Issue: 42(6), P. 877 - 891

Published: Sept. 7, 2023

Base and prime editors (BEs PEs) may provide more precise genetic engineering than nuclease-based approaches because they bypass the dependence on DNA double-strand breaks. However, little is known about their cellular responses genotoxicity. Here, we compared state-of-the-art BEs PEs Cas9 in human hematopoietic stem progenitor cells with respect to editing efficiency, cytotoxicity, transcriptomic changes on-target genome-wide induced detrimental transcriptional that reduced efficiency repopulation xenotransplants also generated breaks genotoxic byproducts, including deletions translocations, at a lower frequency Cas9. These effects were strongest for cytidine due suboptimal inhibition of base excision repair mitigated by tailoring delivery timing editor expression through optimized mRNA design. altered mutational landscape across genome increasing load relative proportions nucleotide variants. findings raise concerns genotoxicity warrant further investigation view clinical application.

Language: Английский

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Prime editing: advances and therapeutic applications

Trends in biotechnology, Journal Year: 2023, Volume and Issue: 41(8), P. 1000 - 1012

Published: March 30, 2023

Clustered regularly interspaced short palindromic repeats-associated protein 9 (CRISPR–Cas)-mediated genome editing has revolutionized biomedical research and will likely change the therapeutic diagnostic landscape. However, CRISPR–Cas9, which edits DNA by activating double-strand break (DSB) repair pathways, is not always sufficient for gene therapy applications where precise mutation required. Prime editing, latest revolution in genome-editing technologies, can achieve any possible base substitution, insertion, or deletion without requirement DSBs. prime still its infancy, further development needed to improve efficiency delivery strategies applications. We summarize developments optimization of editor (PE) variants with improved precision. Moreover, we highlight some potential

Language: Английский

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