CRISPR Journal最新文献

Safety considerations for gene therapies of inherited preleukemia syndromes, including severe congenital neutropenia (CN), are paramount. We compared several strategies for CRISPR/Cas9 gene editing of autosomal-dominant ELANE mutations in CD34⁺ cells from two CN patients head-to-head. We tested universal and allele-specific ELANE knockout, ELANE mutation correction by homology-directed repair (HDR) with AAV6, and allele-specific HDR with ssODN. All strategies were not toxic, had at least 30% editing, and rescued granulopoiesis in vitro. In contrast to published data, allele-specific indels in the last exon of ELANE also restored granulopoiesis. Moreover, by implementing patient-derived induced pluripotent stem cells for GUIDE-Seq off-target analysis, we established a clinically relevant "personalized" assessment of off-target activity of gene editing on the background of the patient's genome. We found that allele-specific approaches had the most favorable off-target profiles. Taken together, a well-defined head-to-head comparison pipeline for selecting the appropriate gene therapy is essential for diseases, with several gene editing strategies available.

Casgevy, the world's first approved CRISPR-based cell therapy, has been priced at $2.2 million per patient. Although this hefty price tag was widely anticipated, the extremely high cost of this and other cell and gene therapies poses a major ethical issue in terms of equitable access and global health. In this Perspective, we argue that lowering the prices of future CRISPR therapies is an urgent ethical imperative. Although we focus on Casgevy as a case study, much of our analysis can be extrapolated to the controversies over affordable access to other gene and cell therapies. First, we explain why this first-of-its-kind CRISPR therapy might be so expensive. We then analyze the ethical issues of equity and global health of early CRISPR treatments. Next, we discuss potential solutions to lower the prices of CRISPR gene therapies. We conclude that the approval of CRISPR transforms our obligations of justice and compels us to bring future gene therapies to the maximum possible number of patients with serious genetic diseases at affordable prices.

Gene editing in human induced pluripotent stem (iPS) cells with programmable nucleases facilitates reliable disease models, but methods using double-strand break repair often produce random on-target by-products. Prime editing (PE) combines Cas9 nickase with reverse transcriptase and PE guide RNA (pegRNA) encoding a repair template to reduce by-products. We implemented a GMP-compatible protocol for transfecting Cas9- or PE-2A-mCherry plasmids to track and fractionate human iPS cells based on PE expression level. We compared the editing outcomes of Cas9- and PE-based methods in a GFP-to-BFP conversion assay at the HEK3 benchmark locus and at the APOE Alzheimer's risk locus, revealing superior precision of PE at high expression levels. Moreover, sorting cells for PE expression level influenced allelic editing outcomes at the target loci. We expect that our findings will aid in the creation of gene-edited human iPS cells with intentional heterozygous and homozygous genotypes.

The clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) is a genome editing tool widely used in biological research and clinical therapeutics. Natural human genetic variations, through altering the sequence context of CRISPR-Cas9 target regions, can significantly affect its DNA repair outcomes and ultimately lead to different editing efficiencies. However, these effects have not been systematically studied, even as CRISPR-Cas9 is broadly applied to primary cells and patient samples that harbor such genetic diversity. Here, we present comprehensive investigations of natural genetic variations on CRISPR-Cas9 outcomes across the human genome. The utility of our analysis is illustrated in two case studies, on both preclinical discoveries of CD33 knockout in chimeric antigen receptor-T cell therapy and clinical applications of transthyretin (TTR) inactivation for treating TTR amyloidosis. We further expand our analysis to genome-scale, population-stratified common variants that may lead to gene editing disparity. Our analyses demonstrate pitfalls of failing to account for the widespread genetic variations in Cas9 target selection and how they can be effectively examined and avoided using our method. To facilitate broad access to our analysis, a web platform CROTONdb is developed, which provides predictions for all possible CRISPR-Cas9 target sites in the coding and noncoding regulatory regions, spanning over 5.38 million guide RNA targets and 90.82 million estimated variant effects. We anticipate CROTONdb having broad clinical utilities in gene and cellular therapies.

While bacteriophage applications benefit from effective phage engineering, selecting the desired genotype after subtle modifications remains challenging. Here, we describe a two-phase endogenous CRISPR-Cas-based phage engineering approach that enables selection of small defined edits in Pectobacterium carotovorum phage ZF40. We designed plasmids containing sequences homologous to ZF40 and a mini-CRISPR array. The plasmids allowed genome editing through homologous recombination and counter-selection against non-recombinant phage genomes using an endogenous type I-E CRISPR-Cas system. With this technique, we first deleted target genes and subsequently restored loci with modifications. This two-phase approach circumvented major challenges in subtle phage modifications, including inadequate sequence distinction for CRISPR-Cas counter-selection and the requirement of a protospacer-adjacent motif, limiting sequences that can be modified. Distinct 20-bp barcodes were incorporated through engineering as differential target sites for programmed CRISPR-Cas activity, which allowed quantification of phage variants in mixed populations. This method aids studies and applications that require mixtures of similar phages.

Traditional distinctions between treatment and enhancement goals for human genome editing (HGE) have animated oversight considerations, yet these categories have been complicated by the addition of prevention as a possible target for HGE applications. To assess the role these three categories might play in continued HGE governance efforts, we report on interviews with genome editing scientists and governance group members. While some accepted traditional distinctions between treatment and enhancement and rejected the latter as unacceptable, others argued that the concept of enhancement is largely irrelevant or not as morally problematic as suggested. Others described how preventive goals for HGE create gray zones where prevention and enhancement may be difficult to distinguish, which may stymie uses of HGE. We conclude by discussing the governance implications of these various understandings of treatment, prevention, and enhancement as HGE research moves beyond the treatment of serious disease to embrace longer range preventive goals.