Programmable deletion, replacement, integration and inversion of large DNA sequences with twin prime editing

Anzalone, Andrew V.; Gao, Xin D.; Podracky, Christopher J.; Nelson, Andrew T.; Koblan, Luke W.; Raguram, Aditya; Levy, Jay M.; Mercer, Jaron A. M.; Liu, David R.

doi:10.1038/s41587-021-01133-w

Cited by 334 publications

(246 citation statements)

References 57 publications

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“…Notably, the messager RNA (mRNA) platform has been successfully used for developing advanced vaccines and has shown potential in treating other diseases (34). CRISPR gene-editing technology corrects single-point mutations with base editors (35) and large DNA sequence deletions, replacements, integrations, and inversions with twin prime editing (36). To carry these agents, viral and non-viral platforms are required to to facilitate them into targeted cells.…”

Section: Discussionmentioning

confidence: 99%

Minimally invasive delivery of peptides to the spinal cord for behavior modulation

Gao

David

Leong

et al. 2022

Preprint

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The blood-spinal cord barrier (BSCB) tightly regulates molecular transport from the blood to the spinal cord. Herein, we present a novel approach for transient modulation of BSCB permeability and localized delivery of peptides into the spinal cord for behavior modulation with high spatial resolution. This approach utilizes optical stimulation of vasculature-targeted nanoparticles and allows delivery of BSCB-nonpermeable molecules into the spinal cord without significant glial activation or impact on animal locomotor behavior. We demonstrate minimally invasive light delivery into the spinal cord using an optical fiber and BSCB permeability modulation in the lumbar region. Our method of BSCB modulation allows delivery of bombesin, a centrally-acting and itch-inducing peptide, into the spinal cord and induces a rapid and transient increase in itching behaviors in mice. This minimally invasive approach enables behavior modulation without genetic modifications and is promising for delivering a wide range of biologics into the spinal cord for behavior modulation and potentially therapy.Significance StatementSpinal cord diseases and disorders are common and cause significant disability, including chronic pain, paralysis, cognitive impairment, and mortality. The blood-spinal cord barrier is a considerable challenge for delivery by systemic therapeutic administration. We developed an optical approach for effectively and safely delivering molecules to the spinal cord to overcome this barrier. The fiberoptic method is minimally invasive and overcomes challenges that previous technologies face, including the complicated bone structure and standing waves that complicate BSCB opening using ultrasound. Optical stimulation offers unprecedented spatial resolution for the precise delivery in intricate spinal cord structures. Significantly, our approach modulates animal behavior (i.e., itch) without genetic modifications and demonstrates the potential for delivery of biologics such as peptides into the spinal cord.

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Section: Discussionmentioning

confidence: 99%

Minimally invasive delivery of peptides to the spinal cord for behavior modulation

Gao

David

Leong

et al. 2022

Preprint

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“…Although prime editing is generally restricted to short edits of fewer than 20 bp, it is possible to introduce larger genomic deletions by targeting complementary prime editing events on both sides of the target DNA 120,121 . With a similar approach, it is possible to introduce gene-sized DNA fragments into specific loci, for example placing genes in safe harbour loci or tagging proteins with fluorescent labels 122,123 . These demonstrations of versatility, together with recent gains in efficiency, suggest that CRISPR prime editing screens are becoming broadly useful for investigating complex genetic changes.…”

Section: Insertions and Deletionsmentioning

confidence: 99%

High-content CRISPR screening

Bock

Datlinger

Chardon

et al. 2022

Nat Rev Methods Primers

334

209

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There is a need in molecular biology and biomedical research for open-ended, hypothesis-generating research, in order to discover previously unknown molecular mechanisms. Genetic screening provides a powerful approach for identifying genes, pathways and mechanisms involved in a given phenotype or biological process. This is illustrated by the many successes of forward genetics in cell lines 1 and in model organisms such as flies 2,3 , worms 4 , yeast 5 , plants 6 and fish 7 , and pioneering work in RNA interference (RNAi) screens 8,9 .CRISPR screens exploit the efficiency and flexibility of CRISPR-Cas genome editing 10 . They have become a popular and productive tool for biological discovery in a broad range of applications 11,12 . In a typical pooled CRISPR screen (FIg. 1), a CRISPR guide RNA (gRNA) library is introduced in bulk into cells, such that individual cells receive different gRNAs and are perturbed according to the gRNA received by the cell. These gRNAs are usually delivered by lentiviral transduction and are integrated into the DNA of the target cells, making it possible to efficiently determine the induced perturbations based on the gRNA sequence.

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“…These methods improved prime editing efficiency in plants. In the latest study, pairs of pegRNA were able to precisely delete 710 bp or precisely replace a sequence of 108 bp ( Anzalone et al, 2021 ). In general, the great potential of prime editing remains to be explored.…”

Section: Modified Gene-editing Systemsmentioning

confidence: 99%

Modified Gene Editing Systems: Diverse Bioengineering Tools and Crop Improvement

Zhu

2022

Front. Plant Sci.

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Gene-editing systems have emerged as bioengineering tools in recent years. Classical gene-editing systems include zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats (CRISPR) with CRISPR-associated protein 9 (Cas9) (CRISPR/Cas9), and these tools allow specific sequences to be targeted and edited. Various modified gene-editing systems have been established based on classical gene-editing systems. Base editors (BEs) can accurately carry out base substitution on target sequences, while prime editors (PEs) can replace or insert sequences. CRISPR systems targeting mitochondrial genomes and RNA have also been explored and established. Multiple gene-editing techniques based on CRISPR/Cas9 have been established and applied to genome engineering. Modified gene-editing systems also make transgene-free plants more readily available. In this review, we discuss the modifications made to gene-editing systems in recent years and summarize the capabilities, deficiencies, and applications of these modified gene-editing systems. Finally, we discuss the future developmental direction and challenges of modified gene-editing systems.

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Programmable deletion, replacement, integration and inversion of large DNA sequences with twin prime editing

Cited by 334 publications

References 57 publications

Minimally invasive delivery of peptides to the spinal cord for behavior modulation

Minimally invasive delivery of peptides to the spinal cord for behavior modulation

High-content CRISPR screening

Modified Gene Editing Systems: Diverse Bioengineering Tools and Crop Improvement

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