Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Unbiased assessment of genome integrity and purging of large deletions at the target locus upon CD4 T-cell CRISPR-Cas9 long range editing for CD40LG deficiency

ABSTRACT: Hyper IgM1 is an X-linked combined immunodeficiency, caused by CD40LG mutations, potentially treatable with CD4+ T-cell Cas9 gene editing with a "one-size-fits-most" corrective template. Contrary to established gene therapies, there is limited data on genomic alterations following long-range gene editing, and no consensus on the relevant assays. We developed drop-off digital PCR assays for unbiased detection of large on-target deletions and found them at high frequency upon editing human cells. Large deletions were common upon editing different loci and cell types and using alternative Cas9 and template delivery methods. In CD40LG edited T cells, on-target deletions were counter-selected in culture and further purged by enrichment for edited cells using a selector coupled to gene correction. We also validated the sensitivity of optical genome mapping for the unbiased detection of genome wide rearrangements and uncovered on-target trapping of one or more vector copies, which do not compromise functionality, upon editing using an integrase defective lentiviral donor template. No other recurring events were detected. Edited patient cells showed faithful reconstitution of CD40LG regulated expression and function with a satisfactory safety profile. Large deletions and donor template integrations should be anticipated and accounted for when designing and testing similar gene editing strategies.

SUBMITTER: Dr. Daniele Canarutto

PROVIDER: S-SCDT-10_15252-EMBJ_2023114188 | biostudies-other |

REPOSITORIES: biostudies-other

ACCESS DATA

Json Xml

Similar Datasets

Unbiased assessment of genome integrity and purging of adverse outcomes at the target locus upon editing of CD4<sup>+</sup> T-cells for the treatment of Hyper IgM1.

Project description:Hyper IgM1 is an X-linked combined immunodeficiency caused by CD40LG mutations, potentially treatable with CD4+ T-cell gene editing with Cas9 and a "one-size-fits-most" corrective template. Contrary to established gene therapies, there is limited data on the genomic alterations following long-range gene editing, and no consensus on the relevant assays. We developed drop-off digital PCR assays for unbiased detection of large on-target deletions and found them at high frequency upon editing. Large deletions were also common upon editing different loci and cell types and using alternative Cas9 and template delivery methods. In CD40LG edited T cells, on-target deletions were counter-selected in culture and further purged by enrichment for edited cells using a selector coupled to gene correction. We then validated the sensitivity of optical genome mapping for unbiased detection of genome wide rearrangements and uncovered on-target trapping of one or more vector copies, which do not compromise functionality, upon editing using an integrase defective lentiviral donor template. No other recurring events were detected. Edited patient cells showed faithful reconstitution of CD40LG regulated expression and function with a satisfactory safety profile. Large deletions and donor template integrations should be anticipated and accounted for when designing and testing similar gene editing strategies.

| S-EPMC10690452 | biostudies-literature

Expanding the range of CRISPR/Cas9-directed genome editing in soybean.

Project description:The CRISPR/Cas9 system has been widely applied for plant genome editing. The commonly used SpCas9 has been shown to rely on the protospacer adjacent motif (PAM) sequences in the canonical form NGG and non-canonical NAG. Although these PAM sequences are extensively distributed across plant genomes, a broader scope of PAM sequence is required to expand the range of genome editing. Here we report the adoption of three variant enzymes, xCas9, SpCas9-NG and XNG-Cas9, to produce targeted mutation in soybean. Sequencing results determined that xCas9 with the NGG and KGA (contains TGA and GGA) PAMs successfully induces genome editing in soybean genome. SpCas9-NG could recognize NGD (contains NGG, NGA and NGT), RGC (contains AGC and GGC), GAA and GAT PAM sites. In addition, XNG-Cas9 was observed to cleave soybean genomic regions with NGG, GAA and AGY (contains AGC and AGT) PAM. Moreover, off-target analyses on soybean editing events induced by SpCas9 and xCas9 indicated that two high-fidelity Cas9 variants including eSpCas9 (enhanced specificity SpCas9) and exCas9 (enhanced specificity xCas9) could improve the specificity of the GGA PAM sequence without reducing on-target editing efficiency. These findings significantly expand the scope of Cas9-mediated genome editing in soybean.Supplementary informationThe online version contains supplementary material available at 10.1007/s42994-021-00051-4.

| S-EPMC9590560 | biostudies-literature

Easy quantification of template-directed CRISPR/Cas9 editing.

Project description:Template-directed CRISPR/Cas9 editing is a powerful tool for introducing subtle mutations in genomes. However, the success rate of incorporation of the desired mutations at the target site is difficult to predict and therefore must be empirically determined. Here, we adapted the widely used TIDE method for quantification of templated editing events, including point mutations. The resulting TIDER method is a rapid, cheap and accessible tool for testing and optimization of template-directed genome editing strategies. A free web tool for TIDER data analysis is available at http://tide.nki.nl.

| S-EPMC6007333 | biostudies-other

CRISPR-Cas9 DNA Base-Editing and Prime-Editing.

Project description:Many genetic diseases and undesirable traits are due to base-pair alterations in genomic DNA. Base-editing, the newest evolution of clustered regularly interspaced short palindromic repeats (CRISPR)-Cas-based technologies, can directly install point-mutations in cellular DNA without inducing a double-strand DNA break (DSB). Two classes of DNA base-editors have been described thus far, cytosine base-editors (CBEs) and adenine base-editors (ABEs). Recently, prime-editing (PE) has further expanded the CRISPR-base-edit toolkit to all twelve possible transition and transversion mutations, as well as small insertion or deletion mutations. Safe and efficient delivery of editing systems to target cells is one of the most paramount and challenging components for the therapeutic success of BEs. Due to its broad tropism, well-studied serotypes, and reduced immunogenicity, adeno-associated vector (AAV) has emerged as the leading platform for viral delivery of genome editing agents, including DNA-base-editors. In this review, we describe the development of various base-editors, assess their technical advantages and limitations, and discuss their therapeutic potential to treat debilitating human diseases.

| S-EPMC7503568 | biostudies-literature

Engineering large-scale chromosomal deletions by CRISPR-Cas9.

Project description:Large-scale chromosomal deletions are a prevalent and defining feature of cancer. A high degree of tumor-type and subtype specific recurrencies suggest a selective oncogenic advantage. However, due to their large size it has been difficult to pinpoint the oncogenic drivers that confer this advantage. Suitable functional genomics approaches to study the oncogenic driving capacity of large-scale deletions are limited. Here, we present an effective technique to engineer large-scale deletions by CRISPR-Cas9 and create isogenic cell line models. We simultaneously induce double-strand breaks (DSBs) at two ends of a chromosomal arm and select the cells that have lost the intermittent region. Using this technique, we induced large-scale deletions on chromosome 11q (65 Mb) and chromosome 6q (53 Mb) in neuroblastoma cell lines. A high frequency of successful deletions (up to 30% of selected clones) and increased colony forming capacity in the 11q deleted lines suggest an oncogenic advantage of these deletions. Such isogenic models enable further research on the role of large-scale deletions in tumor development and growth, and their possible therapeutic potential.

| S-EPMC8643637 | biostudies-literature

Tissue-specific genome editing in Ciona embryos by CRISPR/Cas9.

Project description:The CRISPR/Cas9 system has ushered in a new era of targeted genetic manipulations. Here, we report the use of CRISPR/Cas9 to induce double-stranded breaks in the genome of the sea squirt Ciona intestinalis. We use electroporation to deliver CRISPR/Cas9 components for tissue-specific disruption of the Ebf (Collier/Olf/EBF) gene in hundreds of synchronized Ciona embryos. Phenotyping of transfected embryos in the 'F0' generation revealed that endogenous Ebf function is required for specification of Islet-expressing motor ganglion neurons and atrial siphon muscles. We demonstrate that CRISPR/Cas9 is sufficiently effective and specific to generate large numbers of embryos carrying mutations in a targeted gene of interest, which should allow for rapid screening of gene function in Ciona.

| S-EPMC4302896 | biostudies-other

Genome editing in maize directed by CRISPR-Cas9 ribonucleoprotein complexes.

Project description:Targeted DNA double-strand breaks have been shown to significantly increase the frequency and precision of genome editing. In the past two decades, several double-strand break technologies have been developed. CRISPR-Cas9 has quickly become the technology of choice for genome editing due to its simplicity, efficiency and versatility. Currently, genome editing in plants primarily relies on delivering double-strand break reagents in the form of DNA vectors. Here we report biolistic delivery of pre-assembled Cas9-gRNA ribonucleoproteins into maize embryo cells and regeneration of plants with both mutated and edited alleles. Using this method of delivery, we also demonstrate DNA- and selectable marker-free gene mutagenesis in maize and recovery of plants with mutated alleles at high frequencies. These results open new opportunities to accelerate breeding practices in a wide variety of crop species.

| S-EPMC5116081 | biostudies-other

Expanding CRISPR/Cas9 Genome Editing Capacity in Zebrafish Using SaCas9.

Project description:The type II CRISPR/Cas9 system has been used widely for genome editing in zebrafish. However, the requirement for the 5'-NGG-3' protospacer-adjacent motif (PAM) of Cas9 from Streptococcus pyogenes (SpCas9) limits its targeting sequences. Here, we report that a Cas9 ortholog from Staphylococcus aureus (SaCas9), and its KKH variant, successfully induced targeted mutagenesis with high frequency in zebrafish. Confirming previous findings, the SpCas9 variant, VQR, can also induce targeted mutations in zebrafish. Bioinformatics analysis of these new Cas targets suggests that the number of available target sites in the zebrafish genome can be greatly expanded. Collectively, the expanded target repertoire of Cas9 in zebrafish should further facilitate the utility of this organism for genetic studies of vertebrate biology.

| S-EPMC4978904 | biostudies-other

Synthetic CRISPR RNA-Cas9-guided genome editing in human cells.

Project description:Genome editing with the clustered, regularly interspaced, short palindromic repeats (CRISPR)-Cas9 nuclease system is a powerful technology for manipulating genomes, including introduction of gene disruptions or corrections. Here we develop a chemically modified, 29-nucleotide synthetic CRISPR RNA (scrRNA), which in combination with unmodified transactivating crRNA (tracrRNA) is shown to functionally replace the natural guide RNA in the CRISPR-Cas9 nuclease system and to mediate efficient genome editing in human cells. Incorporation of rational chemical modifications known to protect against nuclease digestion and stabilize RNA-RNA interactions in the tracrRNA hybridization region of CRISPR RNA (crRNA) yields a scrRNA with enhanced activity compared with the unmodified crRNA and comparable gene disruption activity to the previously published single guide RNA. Taken together, these findings provide a platform for therapeutic applications, especially for nervous system disease, using successive application of cell-permeable, synthetic CRISPR RNAs to activate and then silence Cas9 nuclease activity.

| S-EPMC4697396 | biostudies-other

CRISPR/Cas9 Editing for Gaucher Disease Modelling.

Project description:Gaucher disease (GD) is an autosomal recessive lysosomal storage disorder caused by mutations in the acid ?-glucosidase gene (GBA1). Besides causing GD, GBA1 mutations constitute the main genetic risk factor for developing Parkinson's disease. The molecular basis of neurological manifestations in GD remain elusive. However, neuroinflammation has been proposed as a key player in this process. We exploited CRISPR/Cas9 technology to edit GBA1 in the human monocytic THP-1 cell line to develop an isogenic GD model of monocytes and in glioblastoma U87 cell lines to generate an isogenic GD model of glial cells. Both edited (GBA1 mutant) cell lines presented low levels of mutant acid ?-glucosidase expression, less than 1% of residual activity and massive accumulation of substrate. Moreover, U87 GBA1 mutant cells showed that the mutant enzyme was retained in the ER and subjected to proteasomal degradation, triggering unfolded protein response (UPR). U87 GBA1 mutant cells displayed an increased production of interleukin-1?, both with and without inflammosome activation, ?-syn accumulation and a higher rate of cell death in comparison with wild-type cells. In conclusion, we developed reliable, isogenic, and easy-to-handle cellular models of GD obtained from commercially accessible cells to be employed in GD pathophysiology studies and high-throughput drug screenings.

| S-EPMC7246564 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data