Programmable lipid nanoparticle tropism unlocks precision RNA medicine.

Chapter 4 - Recent advances in precise plant genome editing technology

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

Predicting and Improving Insertions by Prime Editing

Targeting safety in the clinic for precise genome editing using CRISPR: a genotoxicologist's perspective.

Anzalone Prime: An Interview with Prime Editing Developer Andrew Anzalone

CRISPR Genome Editing: Into the Second Decade

The precise genome editing has not been well established in plants, largely because of the limited frequency of homology recombination and the delivery barrier of donor templates. Recently, Dr. Caixia Gao's group from the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, developed a series of plant prime editors (PPEs), which mediats the prime editing in the genomes of rice and wheat. The PPE systems are able to generate all 12 kinds of programmable base substitutions, as well desired multiplex nucleotide substitutions and small deletions or insertions without DNA double-strand breaks, thus providing versatile tools for precise plant genome editing. Herein, we introduce the structure and the editing capacity of the PPEs. The attemp on efficiency enhancements of PPEs and other PPEs are also discussed, which may provide a reference for appropriate application of PPEs in plants and also for continuous optimization of the editing tools.

Genome editing has transformed the life sciences and has exciting prospects for use in treating genetic diseases. Our laboratory developed base editing to enable precise and efficient genome editing while minimizing undesired byproducts and toxicity associated with double-stranded DNA breaks. Adenine and cytosine base editors mediate targeted A•T-to-G•C or C•G-to-T•A base pair changes, respectively, which can theoretically address most human disease-associated single-nucleotide polymorphisms. Current base editors can achieve high editing efficiencies-for example, approaching 100% in cultured mammalian cells or 70% in adult mouse neurons in vivo. Since their initial description, a large set of base editor variants have been developed with different on-target and off-target editing characteristics. Here, we describe a protocol for using base editing in cultured mammalian cells. We provide guidelines for choosing target sites, appropriate base editor variants and delivery strategies to best suit a desired application. We further describe standard base-editing experiments in HEK293T cells, along with computational analysis of base-editing outcomes using CRISPResso2. Beginning with target DNA site selection, base-editing experiments in mammalian cells can typically be completed within 1-3 weeks and require only standard molecular biology techniques and readily available plasmid constructs.

How genome editing could be used in the treatment of cardiovascular diseases.

Genome editing, a superior therapy for inherited retinal diseases

Tumor genomes often harbor a complex spectrum of single nucleotide mutations, small indels, and large chromosome rearrangements that can perturb coding and non-coding regions of the genome in ways that remain poorly understood. The mutational processes responsible for the genesis of these events are also not static; instead, they continue to operate throughout disease evolution and further diversify the genetic and phenotypic landscape of cancer cells. Mounting evidence suggests that tumor genotype can be an important determinant of disease progression and therapy response, such as by modulating the sensitivity or resistance of cancer cells to mutant-specific drugs. These types of observations have motivated efforts to treat cancers with genome-informed therapies, highlighting the need to understand how different genetic variants precisely affect gene function and overall tumor phenotypes. Variant-function maps that provide a mechanistic understanding of the biology driven by cancer-associated mutations are needed to design these types of treatment strategies. Precision genome editing technologies like base and prime editing are uniquely suited to tackle this problem. Nevertheless, deploying these methods for systematic variant-function studies and disease modeling in vivo has not been straightforward due to lack of robust and scalable platforms capable of assessing editing efficiency and precision, particularly at endogenous loci. With this goal in mind, we previously developed and applied high-throughput base editing ‘sensor’ approaches that link endogenous genome editing outcomes with synthetic DNA-based readouts and cellular fitness measurements. Using these approaches, we showed that several uncharacterized mutant p53 alleles drive cancer cell proliferation and in vivo tumor development. Building upon this work, we recently developed new prime editing guide RNA design tools and sensor-based approaches that similarly couple quantitative editing outcomes to cellular fitness, allowing us to significantly expand the breadth of cancer-associated mutations that can be interrogated using these precision genome editing technologies. In this talk, I will describe ongoing work using base and prime editing sensor libraries to probe the biological impact of thousands of functionally-distinct genetic variants in diverse types of protein-coding genes and families to learn how these influence various cancer phenotypes. I will also discuss how the implementation of these modular technologies will allow researchers to functionally disentangle the impact of endogenous and exogenous mutational processes on functional selection and tumor evolution with close to single base pair resolution. This generalizable precision genome editing framework will facilitate the functional interrogation of genetic variants across diverse biological contexts, providing much needed insight into cancer variant-function relationships that could be leveraged to develop more precise cancer treatment paradigms, including synthetic lethal therapies that exploit tumor genotype. Citation Format: Francisco J. Sánchez-Rivera, Samuel I. Gould, Alexandra N. Wuest, Kexin Dong, Grace A. Johnson, Alvin Hsu, Varun K. Narendra, Ondine Atwa, Stuart S. Levine, David R. Liu. Functional studies of genetic variation using precision genome editing [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Expanding and Translating Cancer Synthetic Vulnerabilities; 2024 Jun 10-13; Montreal, Quebec, Canada. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(6 Suppl):Abstract nr IA020.

The global climate change and unfavourable abiotic and biotic factors are limiting agricultural productivity and therefore intensifying the challenges for crop scientists to meet the rising demand for global food supply. The introduction of applied genetics to agriculture through plant breeding facilitated the development of hybrid varieties with improved crop productivity. However, the development of new varieties with the existing gene pools poses a challenge for crop breeders. Genetic engineering holds the potential to broaden genetic diversity by the introduction of new genes into crops. But the random insertion of foreign DNA into the plant’s nuclear genome often leads to transgene silencing. Recent advances in the field of plant breeding include the development of a new breeding technique called genome editing. Genome editing technologies have emerged as powerful tools to precisely modify the crop genomes at specific sites in the genome, which has been the longstanding goal of plant breeders. The precise modification of the target genome, the absence of foreign DNA in the genome-edited plants, and the faster and cheaper method of genome modification are the remarkable features of the genome-editing technology that have resulted in its widespread application in crop breeding in less than a decade. This review focuses on the advances in crop breeding through precision genome editing. This review includes: an overview of the different breeding approaches for crop improvement; genome editing tools and their mechanism of action and application of the most widely used genome editing technology, CRISPR/Cas9, for crop improvement especially for agronomic traits such as disease resistance, abiotic stress tolerance, herbicide tolerance, yield and quality improvement, reduction of anti-nutrients, and improved shelf life; and an update on the regulatory approval of the genome-edited crops. This review also throws a light on development of high-yielding climate-resilient crops through precision genome editing.

RNA-guided nucleases (RGNs) based on CRISPR systems permit installing short and large edits within eukaryotic genomes. However, precise genome editing is often hindered due to nuclease off-target activities and the multiple-copy character of the vast majority of chromosomal sequences. Dual nicking RGNs and high-specificity RGNs both exhibit low off-target activities. Here, we report that high-specificity Cas9 nucleases are convertible into nicking Cas9D10A variants whose precision is superior to that of the commonly used Cas9D10A nickase. Dual nicking RGNs based on a selected group of these Cas9D10A variants can yield gene knockouts and gene knock-ins at frequencies similar to or higher than those achieved by their conventional counterparts. Moreover, high-specificity dual nicking RGNs are capable of distinguishing highly similar sequences by ‘tiptoeing’ over pre-existing single base-pair polymorphisms. Finally, high-specificity RNA-guided nicking complexes generally preserve genomic integrity, as demonstrated by unbiased genome-wide high-throughput sequencing assays. Thus, in addition to substantially enlarging the Cas9 nickase toolkit, we demonstrate the feasibility in expanding the range and precision of DNA knockout and knock-in procedures. The herein introduced tools and multi-tier high-specificity genome editing strategies might be particularly beneficial whenever predictability and/or safety of genetic manipulations are paramount.

The rapid development of genome editing technology has brought major breakthroughs in the fields of life science and medicine. In recent years, the clustered regularly interspaced short palindromic repeats (CRISPR)-based genome editing toolbox has been greatly expanded, not only with emerging CRISPR-associated protein (Cas) nucleases, but also novel applications through combination with diverse effectors. Recently, transposon-associated programmable RNA-guided genome editing systems have been uncovered, adding myriads of potential new tools to the genome editing toolbox. CRISPR-based genome editing technology has also revolutionized cardiovascular research. Here we first summarize the advances involving newly identified Cas orthologs, engineered variants and novel genome editing systems, and then discuss the applications of the CRISPR-Cas systems in precise genome editing, such as base editing and prime editing. We also highlight recent progress in cardiovascular research using CRISPR-based genome editing technologies, including the generation of genetically modified in vitro and animal models of cardiovascular diseases (CVD) as well as the applications in treating different types of CVD. Finally, the current limitations and future prospects of genome editing technologies are discussed.

Recently developed base editing (BE), prime editing (PE), and click editing (CE) technologies enable precise and efficient genome editing with minimal risk of double-strand breaks and associated toxicity. However, their effectiveness in correcting real disease-causing mutations has not been systematically compared. Here, we aim to evaluate the potential of BE, PE, and CE technologies in rescuing the retinal degeneration-causing Pde6b (c.1976T>C, p.L659P) mutation. This site is prone to bystander effects, making it an ideal model for comparing the editing outcomes of these 3 novel technologies, particularly their editing precision. We optimized BE, PE, and CE systems in vitro using Pde6b-L659P cell models and compared their editing via deep sequencing. BE and PE had similar efficiency, but PE was the most precise, minimizing bystander edits. CE had lower efficiency and higher indel rates, needing further optimization. Using the optimal PE system for in vivo electroporation in Pde6b-L659P mice, we achieved 12.4% targeted repair with high precision, partially rescuing retinal degeneration. This study demonstrates proof of concept for the precise correction of the Pde6b-L659P mutation causing retinal degeneration using BE, PE, and CE tools. The findings offer valuable insights into the future optimization of precision gene editing techniques and their potential translational applications.