Prime editing: advances and therapeutic applications DOI Creative Commons
Zhihan Zhao, Peng Shang, Prarthana Mohanraju

et al.

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

Past, present, and future of CRISPR genome editing technologies DOI Creative Commons

Martin Pacesa,

Oana Pelea, Martin Jínek

et al.

Cell, Journal Year: 2024, Volume and Issue: 187(5), P. 1076 - 1100

Published: Feb. 1, 2024

Genome editing has been a transformative force in the life sciences and human medicine, offering unprecedented opportunities to dissect complex biological processes treat underlying causes of many genetic diseases. CRISPR-based technologies, with their remarkable efficiency easy programmability, stand at forefront this revolution. In Review, we discuss current state CRISPR gene technologies both research therapy, highlighting limitations that constrain them technological innovations have developed recent years address them. Additionally, examine summarize landscape applications context health therapeutics. Finally, outline potential future developments could shape coming years.

Language: Английский

Citations

146
Mapping the genetic landscape of DNA double-strand break repair DOI Creative Commons
Jeffrey A. Hussmann, Ling Jia, Purnima Ravisankar

et al.

Cell, Journal Year: 2021, Volume and Issue: 184(22), P. 5653 - 5669.e25

Published: Oct. 1, 2021

Highlights•Repair-seq maps the genetic dependencies of DNA repair outcomes•High-resolution signatures gene function identify unexpected relationships•DSB-induced mutations with similar sequences can result from distinct mechanisms•Repair-seq be adapted to study a broad range genome editing toolsSummaryCells double-strand breaks (DSBs) through complex set pathways critical for maintaining genomic integrity. To systematically map these pathways, we developed high-throughput screening approach called Repair-seq that measures effects thousands perturbations on introduced at targeted lesions. Using Repair-seq, profiled DSB products induced by two programmable nucleases (Cas9 and Cas12a) in presence or absence oligonucleotides homology-directed (HDR) after knockdown 476 genes involved associated processes. The resulting data enabled principled, data-driven inference end joining HDR pathways. Systematic interrogation this uncovered relationships among demonstrated outcomes superficially sequence architectures have markedly different dependencies. This work provides foundation mapping optimizing across diverse modalities.Graphical abstract

Language: Английский

Citations

141
In vivo prime editing of a metabolic liver disease in mice DOI

Desirée Böck,

Tanja Rothgangl, Lukas Villiger

et al.

Science Translational Medicine, Journal Year: 2022, Volume and Issue: 14(636)

Published: March 16, 2022

Prime editing is a highly versatile CRISPR-based genome technology that works without DNA double-strand break formation. Despite rapid technological advances, in vivo application for the treatment of genetic diseases remains challenging. Here, we developed size-reduced Sp Cas9 prime editor (PE) lacking RNaseH domain (PE2 Δ RnH ) and an intein-split construct p.1153) adeno-associated virus–mediated delivery into liver. Editing efficiencies reached 15% at Dnmt1 locus were further elevated to 58% by delivering unsplit PE2 via human adenoviral vector 5 (AdV). To provide proof concept correcting liver disease, used AdV approach repairing disease-causing Pah enu2 mutation mouse model phenylketonuria (PKU) editing. Average correction 11.1% (up 17.4%) neonates led therapeutic reduction blood phenylalanine, inducing detectable off-target mutations or prolonged inflammation. Although current PKU has limitations clinical due requirement high doses (7 × 10 14 vg/kg) induction immune responses PE, development may lead curative therapies other diseases.

Language: Английский

Citations

137
Phage-assisted evolution and protein engineering yield compact, efficient prime editors DOI Creative Commons
Jordan L. Doman, Smriti Pandey, Monica E. Neugebauer

et al.

Cell, Journal Year: 2023, Volume and Issue: 186(18), P. 3983 - 4002.e26

Published: Aug. 1, 2023

Prime editing enables a wide variety of precise genome edits in living cells. Here we use protein evolution and engineering to generate prime editors with reduced size improved efficiency. Using phage-assisted evolution, efficiencies compact reverse transcriptases by up 22-fold generated that are 516–810 base pairs smaller than the current-generation editor PEmax. We discovered different specialize types used this insight outperform PEmax PEmaxΔRNaseH, truncated dual-AAV delivery systems. Finally, Cas9 domains improve editing. These resulting (PE6a-g) enhance therapeutically relevant patient-derived fibroblasts primary human T-cells. PE6 variants also enable longer insertions be installed vivo following delivery, achieving 40% loxP insertion cortex murine brain, 24-fold improvement compared previous state-of-the-art editors.

Language: Английский

Citations

136
In vivo somatic cell base editing and prime editing DOI Creative Commons
Gregory A. Newby, David R. Liu

Molecular Therapy, Journal Year: 2021, Volume and Issue: 29(11), P. 3107 - 3124

Published: Sept. 10, 2021

Recent advances in genome editing technologies have magnified the prospect of single-dose cures for many genetic diseases. For most disorders, precise DNA correction is anticipated to best treat patients. To install desired changes with high precision, our laboratory developed base editors (BEs), which can correct four common single-base substitutions, and prime editors, any substitution, insertion, and/or deletion over a stretch dozens pairs. Compared nuclease-dependent approaches that involve double-strand breaks (DSBs) often result large percentage uncontrolled outcomes, such as mixtures insertions deletions (indels), larger deletions, chromosomal rearrangements, offer greater efficiency fewer byproducts slowly dividing or non-dividing cells, those make up cells adult animals. Both viral non-viral vivo delivery methods now been used deliver animal models, establishing serve effective agents therapeutic This review summarizes examples somatic cell (post-natal) prospects future development.

Language: Английский

Citations

133
Prime editing efficiency and fidelity are enhanced in the absence of mismatch repair DOI Creative Commons
Joana Ferreira da Silva, Gonçalo P. Oliveira, E. A. Arasa-Verge

et al.

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

Published: Feb. 9, 2022

Abstract Prime editing (PE) is a powerful genome engineering approach that enables the introduction of base substitutions, insertions and deletions into any given genomic locus. However, efficiency PE varies widely depends not only on region targeted, but also genetic background edited cell. Here, to determine which cellular factors affect efficiency, we carry out focused screen targeting 32 DNA repair factors, spanning all reported pathways. We show that, depending cell line type edit, ablation mismatch (MMR) affords 2–17 fold increase in across several human lines, types edits loci. The accumulation key MMR MLH1 MSH2 at sites argues for direct involvement control. Our results shed new light mechanism suggest how its might be optimised.

Language: Английский

Citations

128
A split prime editor with untethered reverse transcriptase and circular RNA template DOI
Bin Liu, Xiaolong Dong, Haoyang Cheng

et al.

Nature Biotechnology, Journal Year: 2022, Volume and Issue: 40(9), P. 1388 - 1393

Published: April 4, 2022

Language: Английский

Citations

119
A time-resolved, multi-symbol molecular recorder via sequential genome editing DOI Creative Commons
Junhong Choi, Wei Chen, Anna Minkina

et al.

Nature, Journal Year: 2022, Volume and Issue: 608(7921), P. 98 - 107

Published: July 6, 2022

Abstract DNA is naturally well suited to serve as a digital medium for in vivo molecular recording. However, contemporary DNA-based memory devices are constrained terms of the number distinct ‘symbols’ that can be concurrently recorded and/or by failure capture order which events occur 1 . Here we describe Typewriter, general system recording overcomes these and other limitations. For blank (‘DNA Tape’) consists tandem array partial CRISPR–Cas9 target sites, with all but first site truncated at their 5′ ends therefore inactive. Short insertional edits symbols record identity prime editing guide RNA 2 mediating edit while also shifting position ‘type guide’ one unit along Tape, is, sequential genome editing. In this proof concept demonstrate decoding thousands symbols, complex event histories short text messages; evaluate performance dozens orthogonal tapes; construct ‘long tape’ potentially capable many 20 serial events. Finally, leverage Typewriter conjunction single-cell RNA-seq reconstruct monophyletic lineage 3,257 cells find Poisson-like accumulation multicopy tape maintained across least generations 25 days vitro clonal expansion.

Language: Английский

Citations

116
Efficient targeted insertion of large DNA fragments without DNA donors DOI

Jinlin Wang,

He Zhou, Guoquan Wang

et al.

Nature Methods, Journal Year: 2022, Volume and Issue: 19(3), P. 331 - 340

Published: Feb. 28, 2022

Language: Английский

Citations

113
Efficient prime editing in mouse brain, liver and heart with dual AAVs DOI Creative Commons
Jessie R. Davis, Samagya Banskota, Jonathan M. Levy

et al.

Nature Biotechnology, Journal Year: 2023, Volume and Issue: 42(2), P. 253 - 264

Published: May 4, 2023

Abstract Realizing the promise of prime editing for study and treatment genetic disorders requires efficient methods delivering editors (PEs) in vivo. Here we describe identification bottlenecks limiting adeno-associated virus (AAV)-mediated vivo development AAV-PE vectors with increased PE expression, guide RNA stability modulation DNA repair. The resulting dual-AAV systems, v1em v3em PE-AAV, enable therapeutically relevant mouse brain (up to 42% efficiency cortex), liver 46%) heart 11%). We apply these systems install putative protective mutations Alzheimer’s disease astrocytes coronary artery hepatocytes. In PE-AAV caused no detectable off-target effects or significant changes enzymes histology. Optimized support highest unenriched levels reported date, facilitating potential diseases a component.

Language: Английский

Citations

113