Aug 2026· Human Gene Therapy· pp.
10430342261466427
· 0 citations· 48 references
Medicine
TL;DR
The results highlight the limitations of NHPs for the study of human CRISPR spacer specificity and contextualize human risk informed by these studies, and highlight the risks associated with off-target editing events.
Abstract
Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technology has revolutionized genetic medicine by enabling precise genome editing for therapeutic benefit. CRISPR nucleases are programmed to target genomic sites with sequence complementarity to the spacer region of an associated guide RNA. However, these nucleases may target genomic loci with sequences similar to the target site, which can lead to unintended disruption of off-target genes. The risks associated with these off-target editing events are critical to assess as CRISPR-based in vivo editing systems advance to clinical development. Nonhuman primates (NHPs) are common model species for evaluating the human safety of many therapeutic modalities, but their relevance for evaluating human CRISPR off-target activity has yet to be determined. In this study, 1,220,908 Cas12a and 6,159,066 Cas9 spacer sequences targeting human genes were designed, and off-target editing sites were computationally predicted in humans and five common NHP species. Of the 7,413 Cas12a and 570,754 Cas9 spacers meeting defined on- and off-target inclusion criteria, only 14-21% of Cas12a and 7-15% of Cas9 human off-targets per spacer are recapitulated in the genomes of NHPs commonly used in preclinical studies. These results highlight the limitations of NHPs for the study of human CRISPR spacer specificity and contextualize human risk informed by these studies.
Two edgeR-based pipelines were constructed that would allow for direct comparison between two genome-wide CRISPR screening datasets of colorectal carcinoma, the Wellcome Sanger Institute’s (Sanger) and the Broad Institute's DepMap (Achilles) datasets, and Model-based Analysis of Genome-wide CRISPR/Cas9 Knockout (MAGeCK) for comparison and validation.
Khalif Hussein, Vasileios Theocharis, A. Lindlöf· 0 citations
Five previously uncharacterized MG102-like Cas9d orthologs are identified that share the hallmark genomic, sequence, and structural features of type II-D Cas9 and establish compact MG102-like Cas9d orthologs as robust and specific genome editors and provide promising, single-AAV– compatible scaffolds for in vivo therapeutic genome editing.
Qiaochu Wang, Ahmed Saleh, G. S. Rao et al.· bioRxiv· 0 citations
A novel genome-wide CRISPR screening strategy that will facilitate the systematic engineering of novel nonviral genome editing delivery methods, where the identified novel gene hits can be further used to increase editing efficiency for other therapeutically relevant cell types.
Shivani Saxena, Meha Kabra, Amr A. Abdeen et al.· bioRxiv· 2 citations
To systematically map cellular factors constraining nonviral genome editing, influencing uptake and intracellular trafficking, we develop a genome-wide CRISPR screening platform linking perturbation of 19,114 genes to editing outcomes in human cells. We identify six negative regulators of delivery whose depletion increases editing efficiency by up to six-fold across diverse payloads, loci, and cell types. We test the top two factors, GJB2 and BET1L, in two distinct human models: correction of a pathogenic adenine base mutation in KCNJ13 and introduction of a cytosine base mutation in the GABAA receptor gene. Depletion of either improves base-editing outcomes by 6-fold, potentially through effects on delivery. In a patient-derived model of retinal channelopathy, knockdown of either gene improves lipid nanoparticle base editing efficiency by over 3.5-fold. This enables functional restoration of Kir7.1 ion channels in a subset of edited cells, highlighting cellular barriers as actionable targets to enhance the potency of genetic therapies. Low editing efficiency of nonviral delivery in post mitotic tissues presents a challenge to the field of gene therapy. Here, authors dissect the genetic regulators of nonviral delivery in post mitotic retinal epithelial cells describe strategies for improved base editor delivery and editing.
Shivani Saxena, Meha Kabra, Amr A. Abdeen et al.· Nature Communications· 0 citations
This chapter outlines a comprehensive methodology for the design, assembly, and functional assessment of CRISPR/dCas9 systems optimized for tomato to investigate pathogen-associated responses.
Ananya Mukherjee, Shrabani Basak, Raghuvir Singh et al.· Methods in molecular biology· 0 citations
A yeast selection platform is developed to engineer Cas9 with re-specified activity across multiple additional non-canonical PAMs in yeast, further demonstrating its utility as a general and programmable framework for expanding the therapeutic reach of precision genome editing.
Julia Tartaglia, Vivian Nguyen, John James Desmarais et al.· bioRxiv· 0 citations