Anzalone Prime: An Interview with Prime Editing Developer Andrew Anzalone

PASTE, Don't Cut: Genome Editing Tool Looks Beyond CRISPR and Prime

Multiplex precision gene editing by a surrogate prime editor in rice

Predicting and Improving Insertions by Prime Editing

PASTE: The Way Forward for Large DNA Insertions.

CRISPR Genome Editing: Into the Second Decade

Optimized prime editing efficiently generates glyphosate-resistant rice plants carrying homozygous TAP-IVS mutation in EPSPS

Toward CRISPR Therapies for Cardiomyopathies.

Precise Modifications of Both Exogenous and Endogenous Genes in Rice by Prime Editing

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.

The potential to therapeutically alter the genome is one of the remarkable scientific developments in recent years. Genome editing technologies have provided an opportunity to precisely alter genomic sequence(s) in eukaryotic cells as a treatment option for various genetic disorders. These technologies allow the correction of harmful mutations in patients by precise nucleotide editing. Genome editing technologies such as CRISPR (clustered regularly interspaced short palindromic repeat) and base editors have greatly contributed to the practical applications of gene editing. However, these technologies have certain limitations, including imperfect editing, undesirable mutations, off-target effects, and lack of potential to simultaneously edit multiple loci. Recently, prime editing (PE) has emerged as a new gene editing technology with the potential to overcome the above-mentioned limitations. Interestingly, PE not only has higher specificity but also does not require double-strand breaks. In addition, a minimum possibility of potential off-target mutant sites makes PE a preferred choice for therapeutic gene editing. Furthermore, PE has the potential to introduce insertion and deletions of all 12 single-base mutations at target sequences. Considering its potential, PE has been applied as a treatment option for genetic diseases including hemoglobinopathies. β-Thalassemia, for example, one of the most significant blood disorders characterized by reduced levels of functional hemoglobin, could potentially be treated using PE. Therapeutic reactivation of the γ-globin gene in adult β-thalassemia patients through PE technology is considered a promising therapeutic strategy. The current review aims to briefly discuss the genome editing strategies and potential applications of PE for the treatment of β-thalassemia. In addition, the review will also focus on challenges associated with the use of PE.

The acceptance of new crop varieties by consumers is contingent on the presence of consumer-preferred traits, which include sensory attributes, nutritional value, industrial products and bioactive compounds production. Recent developments in genome editing technologies provide novel insight to identify gene functions and improve the various qualitative and quantitative traits of commercial importance in plants. Various conventional as well as advanced gene-mutagenesis techniques such as physical and chemical mutagenesis, CRISPR-Cas9, Cas12 and base editors are used for the trait improvement in crops. To meet consumer demand, breakthrough biotechnologies, especially CRISPR-Cas have received a fair share of scientific and industrial interest, particularly in plant genome editing. CRISPR-Cas is a versatile tool that can be used to knock out, replace and knock-in the desired gene fragments at targeted locations in the genome, resulting in heritable mutations of interest. This review highlights the existing literature and recent developments in CRISPR-Cas technologies (base editing, prime editing, multiplex gene editing, epigenome editing, gene delivery methods) for reliable and precise gene editing in plants. This review also discusses the potential of gene editing exhibited in crops for the improvement of consumer-demanded traits such as higher nutritional value, colour, texture, aroma/flavour, and production of industrial products such as biofuel, fibre, rubber and pharmaceuticals. In addition, the bottlenecks and challenges associated with gene editing system, such as off targeting, ploidy level and the ability to edit organelle genome have also been discussed.

Systematic Mapping of Gene-Editable Mutations in GATA2 and SAMD9/SAMD9L Syndromes

AAV-mediated gene editing lights up the lung

Implementation of therapeutic in vivo gene editing using CRISPR/Cas relies on potent delivery of gene editing tools. Administration of ribonucleoprotein (RNP) complexes consisting of Cas protein and single guide RNA (sgRNA) offers short-lived editing activity and safety advantages over conventional viral and non-viral gene and RNA delivery approaches. By engineering lentivirus-derived nanoparticles (LVNPs) to facilitate RNP delivery, we demonstrate effective administration of SpCas9 as well as SpCas9-derived base and prime editors (BE/PE) leading to gene editing in recipient cells. Unique Gag/GagPol protein fusion strategies facilitate RNP packaging in LVNPs, and refinement of LVNP stoichiometry supports optimized LVNP yield and incorporation of therapeutic payload. We demonstrate near instantaneous target DNA cleavage and complete RNP turnover within 4 days. As a result, LVNPs provide high on-target DNA cleavage and lower levels of off-target cleavage activity compared to standard RNP nucleofection in cultured cells. LVNPs accommodate BE/sgRNA and PE/epegRNA RNPs leading to base editing with reduced bystander editing and prime editing without detectable indel formation. Notably, in the mouse eye, we provide the first proof-of-concept for LVNP-directed in vivo gene disruption. Our findings establish LVNPs as promising vehicles for delivery of RNPs facilitating donor-free base and prime editing without formation of double-stranded DNA breaks.

Over 6500 Mendelian disorders have been documented, with approximately 4500 genes linked to these conditions. The majority of inherited diseases present in childhood and, currently, lack effective treatments, which imposes significant economic and psychological burdens on families and society. Gene editing, particularly base editing, offers an effective and safe strategy for repairing pathogenic point mutations. It has the potential to become a treatment, even a cure, for rare diseases. Currently, multiple gene editing-related drugs have entered clinical trials. In this chapter, we summarize the various gene editing systems, including CRISPR/Cas, base editing, and prime editing. We then focus on the current research progress of base editing in correcting pathogenic mutations. This includes applications such as building animal models, correcting mutations in various diseases, germline cell editing, delivery methods, and approved clinical trials. Finally, we discuss current challenges related to delivery methods, efficiency, precision, and cost.