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Results for “off-target editing”

Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

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10 records
All sourcesNASA NTRSDOE OSTI
DOE OSTI2023

Genome editing in plants using the compact editor CasΦ

Clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins (CRISPR-Cas) systems have been developed as important tools for plant genome engineering. Here, we demonstrate that the hypercompact CasΦ nuclease is able to generate stably inherited gene edits in Arabidopsis, and that CasΦ guide RNAs can be expressed with either the Pol-III U6 promoter or a Pol-II promoter together with ribozyme mediated RNA processing. Using the Arabidopsis fwa epiallele, we show that CasΦ displays higher editing efficiency when the target locus is not DNA methylated, suggesting that CasΦ is sensitive to chromatin environment. Importantly, two CasΦ protein variants, vCasΦ and nCasΦ, both showed much higher editing efficiency relative to the wild-type CasΦ enzyme. Consistently, vCasΦ and nCasΦ yielded offspring plants with inherited edits at much higher rates compared to WTCasΦ. Extensive genomic analysis of gene edited plants showed no off-target editing, suggesting that CasΦ is highly specific. The hypercompact size, T-rich minimal protospacer adjacent motif (PAM), and wide range of working temperatures make CasΦ an excellent supplement to existing plant genome editing systems.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Packaged delivery of CRISPR–Cas9 ribonucleoproteins accelerates genome editing

Effective genome editing requires a sufficient dose of CRISPR–Cas9 ribonucleoproteins (RNPs) to enter the target cell while minimizing immune responses, off-target editing, and cytotoxicity. Clinical use of Cas9 RNPs currently entails electroporation into cells ex vivo, but no systematic comparison of this method to packaged RNP delivery has been made. Here we compared two delivery strategies, electroporation and enveloped delivery vehicles (EDVs), to investigate the Cas9 dosage requirements for genome editing. Using fluorescence correlation spectroscopy, we determined that >1300 Cas9 RNPs per nucleus are typically required for productive genome editing. EDV-mediated editing was >30-fold more efficient than electroporation, and editing occurs at least 2-fold faster for EDV delivery at comparable total Cas9 RNP doses. We hypothesize that differences in efficacy between these methods result in part from the increased duration of RNP nuclear residence resulting from EDV delivery. Our results directly compare RNP delivery strategies, showing that packaged delivery could dramatically reduce the amount of CRISPR–Cas9 RNPs required for experimental or clinical genome editing.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2022

Decorating chromatin for enhanced genome editing using CRISPR-Cas9

CRISPR-associated (Cas) enzymes have revolutionized biology by enabling RNA-guided genome editing. Homology-directed repair (HDR) in the presence of donor templates is currently the most versatile method to introduce precise edits following CRISPR-Cas-induced double-stranded DNA cuts, but HDR efficiency is generally low relative to end-joining pathways that lead to insertions and deletions (indels). We tested the hypothesis that HDR could be increased using a Cas9 construct fused to PRDM9, a chromatin remodeling factor that deposits histone methylations H3K36me3 and H3K4me3 to mediate homologous recombination in human cells. Our results show that the fusion protein contacts chromatin specifically at the Cas9 cut site in the genome to increase the observed HDR efficiency by threefold and HDR:indel ratio by fivefold compared with that induced by unmodified Cas9. HDR enhancement occurred in multiple cell lines with no increase in off-target genome editing. These findings underscore the importance of chromatin features for the balance between DNA repair mechanisms during CRISPR-Cas genome editing and provide a strategy to increase HDR efficiency.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Efficient mutagenesis and genotyping of maize inbreds using biolistics, multiplex CRISPR/Cas9 editing, and Indel-Selective PCR

CRISPR/Cas9 based genome editing has advanced our understanding of a myriad of important biological phenomena. Important challenges to multiplex genome editing in maize include assembly of large complex DNA constructs, few genotypes with efficient transformation systems, and costly/labor-intensive genotyping methods. Here we present an approach for multiplex CRISPR/Cas9 genome editing system that delivers a single compact DNA construct via biolistics to Type I embryogenic calli, followed by a novel efficient genotyping assay to identify desirable editing outcomes. We first demonstrate the creation of heritable mutations at multiple target sites within the same gene. Next, we successfully created individual and stacked mutations for multiple members of a gene family. Genome sequencing found off-target mutations are rare. Multiplex genome editing was achieved for both the highly transformable inbred line H99 and Illinois Low Protein1 (ILP1), a genotype where transformation has not previously been reported. In addition to screening transformation events for deletion alleles by PCR, we also designed PCR assays that selectively amplify deletion or insertion of a single nucleotide, the most common outcome from DNA repair of CRISPR/Cas9 breaks by non-homologous end-joining. The Indel-Selective PCR (IS-PCR) method enabled rapid tracking of multiple edited alleles in progeny populations. The ‘end to end’ pipeline presented here for multiplexed CRISPR/Cas9 mutagenesis can be applied to accelerate maize functional genomics in a broader diversity of genetic backgrounds.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Data for "Efficient Mutagenesis and Genotyping of Maize Inbreds Using Biolistics, Multiplex CRISPR/Cas9 Editing, and Indel-Selective PCR"

CRISPR/Cas9 based genome editing has advanced our understanding of a myriad of important biological phenomena. Important challenges to multiplex genome editing in maize include assembly of large complex DNA constructs, few genotypes with efficient transformation systems, and costly/labor-intensive genotyping methods. Here we present an approach for multiplex CRISPR/Cas9 genome editing system that delivers a single compact DNA construct via biolistics to Type I embryogenic calli, followed by a novel efficient genotyping assay to identify desirable editing outcomes. We first demonstrate the creation of heritable mutations at multiple target sites within the same gene. Next, we successfully created individual and stacked mutations for multiple members of a gene family. Genome sequencing found off-target mutations are rare. Multiplex genome editing was achieved for both the highly transformable inbred line H99 and Illinois Low Protein1 (ILP1), a genotype where transformation has not previously been reported. In addition to screening transformation events for deletion alleles by PCR, we also designed PCR assays that selectively amplify deletion or insertion of a single nucleotide, the most common outcome from DNA repair of CRISPR/Cas9 breaks by non-homologous end-joining. The Indel-Selective PCR (IS-PCR) method enabled rapid tracking of multiple edited alleles in progeny populations. The ‘end to end’ pipeline presented here for multiplexed CRISPR/Cas9 mutagenesis can be applied to accelerate maize functional genomics in a broader diversity of genetic backgrounds.

gene editing↗
DOE OSTI2024

Multiplexed CRISPR-Cas9 mutagenesis of rice PSBS1 noncoding sequences for transgene-free overexpression

Understanding CRISPR-Cas9’s capacity to produce native overexpression (OX) alleles would accelerate agronomic gains achievable by gene editing. To generate OX alleles with increased RNA and protein abundance, we leveraged multiplexed CRISPR-Cas9 mutagenesis of noncoding sequences upstream of the rice PSBS1 gene. We isolated 120 gene-edited alleles with varying non-photochemical quenching (NPQ) capacity in vivo—from knockout to overexpression—using a high-throughput screening pipeline. Overexpression increased OsPsbS1 protein abundance two- to threefold, matching fold changes obtained by transgenesis. Increased PsbS protein abundance enhanced NPQ capacity and water-use efficiency. Across our resolved genetic variation, we identify the role of 5'UTR indels and inversions in driving knockout/knockdown and overexpression phenotypes, respectively. Complex structural variants, such as the 252-kb duplication/inversion generated here, evidence the potential of CRISPR-Cas9 to facilitate significant genomic changes with negligible off-target transcriptomic perturbations. Our results may inform future gene-editing strategies for hypermorphic alleles and have advanced the pursuit of gene-edited, non-transgenic rice plants with accelerated relaxation of photoprotection.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2024

Engineering self-deliverable ribonucleoproteins for genome editing in the brain

The delivery of CRISPR ribonucleoproteins (RNPs) for genome editing in vitro and in vivo has important advantages over other delivery methods, including reduced off-target and immunogenic effects. However, effective delivery of RNPs remains challenging in certain cell types due to low efficiency and cell toxicity. To address these issues, we engineer self-deliverable RNPs that can promote efficient cellular uptake and carry out robust genome editing without the need for helper materials or biomolecules. Screening of cell-penetrating peptides (CPPs) fused to CRISPR-Cas9 protein identifies potent constructs capable of efficient genome editing of neural progenitor cells. Further engineering of these fusion proteins establishes a C-terminal Cas9 fusion with three copies of A22p, a peptide derived from human semaphorin-3a, that exhibits substantially improved editing efficacy compared to other constructs. We find that self-deliverable Cas9 RNPs generate robust genome edits in clinically relevant genes when injected directly into the mouse striatum. Overall, self-deliverable Cas9 proteins provide a facile and effective platform for genome editing in vitro and in vivo.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

CRISPR-COPIES: Web Tool

CRISPR/Cas system has emerged as a powerful genome-editing tool for metabolic engineering and human gene therapy. However, the conundrum of where to integrate heterologous genes on the chromosome using the CRISPR/Cas system remains an open question. Selecting a site for gene integration requires incorporation of complex criteria such as factors involved in CRISPR/Cas-mediated integration, genetic stability, and gene expression and therefore, usually requires strenuous characterization of sites on particular or different chromosomal locations. To address these issues, we developed CRISPR-COPIES, a COmputational Pipeline for the Identification of CRISPR/Cas-facilitated intEgration Sites. The tool applies ScaNN, a state-of-the-art model on the embedding-based nearest neighbor search for fast and accurate off-target search and can identify genome-wide intergenic sites for most bacterial and fungal genomes within minutes. This submission contains the code we developed to create a user-friendly web interface for CRISPR-COPIES (https://biofoundry.web.illinois.edu/copies/). We anticipate CRISPR-COPIES will serve as a useful tool for targeted DNA integration and aid in the characterization of synthetic biology toolkits, rapid strain construction to produce valuable biochemicals, and human gene and cell therapy.

Bioinformatics↗
DOE OSTI2024

CRISPR-COPIES: an in silico platform for discovery of neutral integration sites for CRISPR/Cas-facilitated gene integration

Abstract The CRISPR/Cas system has emerged as a powerful tool for genome editing in metabolic engineering and human gene therapy. However, locating the optimal site on the chromosome to integrate heterologous genes using the CRISPR/Cas system remains an open question. Selecting a suitable site for gene integration involves considering multiple complex criteria, including factors related to CRISPR/Cas-mediated integration, genetic stability, and gene expression. Consequently, identifying such sites on specific or different chromosomal locations typically requires extensive characterization efforts. To address these challenges, we have developed CRISPR-COPIES, a COmputational Pipeline for the Identification of CRISPR/Cas-facilitated intEgration Sites. This tool leverages ScaNN, a state-of-the-art model on the embedding-based nearest neighbor search for fast and accurate off-target search, and can identify genome-wide intergenic sites for most bacterial and fungal genomes within minutes. As a proof of concept, we utilized CRISPR-COPIES to characterize neutral integration sites in three diverse species: Saccharomyces cerevisiae, Cupriavidus necator, and HEK293T cells. In addition, we developed a user-friendly web interface for CRISPR-COPIES (https://biofoundry.web.illinois.edu/copies/). We anticipate that CRISPR-COPIES will serve as a valuable tool for targeted DNA integration and aid in the characterization of synthetic biology toolkits, enable rapid strain construction to produce valuable biochemicals, and support human gene and cell therapy applications.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2026

Construct design for precise DNA insertion in plants

Precise insertion of DNA sequences at targeted locations in plant genomes is pivotal for synthetic biology, genetics, and crop improvement. Construct design plays a critical role in achieving precise insertions, yet practical guidance remains limited. This review provides an in-depth overview of construct design principles and targeted DNA insertion (knock-in) strategies in plants. We assess the strengths, limitations, and construct requirements of current knock-in methods for specific applications, including short, large, and multifragment insertions. Additionally, we explore the potential of adopting advanced nonplant technologies to enhance knock-in efficiency and precision in plants. This review provides a valuable resource for facilitating the effective application of knock-in technologies to genetically improve crops with minimal off-target effects.

DNA construct↗