The specificity of hpCasMINI for gene editing [Tag-seq]
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE270588 | GEO | 2024/06/29
REPOSITORIES: GEO
Similar Datasets
Project description:High specificity of e\ngineered nucleases ensures precise genome editing. Couple methods were developed to identify off-target sites of CRISPR/Cas9, but hardly any high-throughput sequencing method can unequivocally determine their targeting efficiencies. Here we describe a comprehensive method, primer-extension-mediated sequencing (PEM-seq), which could sensitively detect CRISPR/Cas9 off-target sites as well as assess their editing efficiency by quantifying DNA interference events at on-target sites. Demonstrated by PEM-seq, we generated a high-fidelity Cas9 variant FeCas9 that possesses similar targeting ability as the wild-type while with extremely low off-target activities. Moreover, we provided further evidences for the broader range of xCas9 protospacer adjacent sequence. We also found the AcrIIA4 inhibitor could inhibit both on- and off-target activities of SpCas9, but it suppressed SpCas9 cleavage at the off-target loci not so efficiently as at the on-target sites. Finally, we believe PEM-seq is applicable to optimizing genome editing strategy for clinical purpose or creating animal model.
2019-04-03 | GSE116231 | GEO
Project description:The type V-I CRISPR-Cas system is becoming increasingly attractive for its potential utility in gene editing. However, natural nucleases often exhibit low efficiency, limiting their application. Here, we utilized structure-guided rational design and combinatorial protein engineering to optimize an uncharacterized Cas12i nuclease, Cas12i3. Accordingly, we developed Cas-SF01, a Cas12i3 variant that exhibits significantly improved gene-editing activity in mammalian cells and plants. Cas-SF01 displays comparable or superior editing performance compared to SpCas9 or recently engineered Cas12 nucleases. Further analysis of PAM recognition showed that Cas-SF01 has an expanded PAM range and effectively recognizes NTTN and noncanonical NATN and TTVN PAMs. Additionally, we identified an amino acid substitution, D876R, that markedly reduced the off-target effect while maintaining high on-target activity, leading to the development of Cas-SF01HiFi (high-fidelity Cas-SF01). Finally, we demonstrated that Cas-SF01 has robust gene-editing activity in both the monocot plant rice and dicot plant pepper. Our results suggest that Cas-SF01 can serve as a robust gene-editing platform with high efficiency and specificity for future genome editing applications across different organisms.
2023-11-05 | GSE236755 | GEO
Project description: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 genome editing 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.
2020-12-08 | GSE153471 | GEO
Project description:Prime editing is a highly versatile CRISPR-based genome editing technology with the potential to correct the vast majority of genetic defects1. However, correction of a disease phenotype in vivo in somatic tissues has not been achieved yet. Here, we establish proof-of-concept for in vivo prime editing, that resulted in rescue of a metabolic liver disease. We first develop a size-reduced prime editor (PE) lacking the RNaseH domain of the reverse transcriptase (SpCas9-PERnH), and a linker- and NLS-optimized intein-split PE construct (SpCas9-PE p.1153) for delivery by adeno-associated viruses (AAV). Systemic dual AAV-mediated delivery of this variant in neonatal mice enables installation of a transversion mutation at the Dnmt1 locus with 15% efficiency on average. Next, we targeted the disease-causing mutation in the phenylalanine hydroxylase (Pah)enu2 mouse model for phenylketonuria (PKU). Correction rates of 1.5% using the dual AAV approach could be increased to up to 14% by delivery of full-length SpCas9-PE via adenoviral vector 5 (AdV5), leading to full restoration of physiological blood phenylalanine (L-Phe) levels below 120 µmol/L. Our study demonstrates in vivo prime editing in the liver at two independent loci, emphasizing the potential of PEs for future therapeutic applications.
2021-12-29 | GSE174757 | GEO
Project description:We use RNA-seq to study the transcriptome wide specificity profiles of A-to-I RNA editing by split-ADAR2 constructs.
2022-01-19 | GSE158656 | GEO
Project description:This study aims to predict the activity and specificity of CRISPR/Cas9 by deep learning at genome-scale among different cell lines. Here, we have focused on embracing and modifying a system for evaluating SpCas9 activity of on-target and off-target using >1,000,000 guide RNAs (gRNAs) covering ~20,000 protein-coding genes and ~10,000 non-coding genes in synthetic constructs with a high-throughput manner. With the help of deep learning algorithms in the field of artificial intelligence, three prediction models with the best generalization performance now are constructed: Aidit_Cas9-ON, Aidit_Cas9-OFF, and Aidit_Cas9-DSB. Moreover, through systematically investigating the influence of diverse cellular environment on gRNA activity and specificity, we noticed that distinct features are favored from H1 cell line compared with the other 2 cell lines for on-target activity and the overall distribution of repair outcomes is markedly different across 3 cell lines, especially in Jurkat. Finally, we identify a key effect protein DNTT strongly influences editing outcomes induced by CRISPR/Cas9. We confirm that this study will greatly facilitate CRISPR-based genome editing.
2023-05-24 | GSE181774 | GEO
Project description:Proteome-derived peptide libraries are a powerful tool for the investigation of protease specificity, conventionally involving the biotinylation of cleavage products to enable for their enrichment. Here we present a modified strategy for protease specificity profiling using proteome-derived peptide libraries in a tag-free manner. In comparison to the original method the new protocol is faster, avoids cost- and time-intensive enrichment steps, and enables probing for lysine selectivity. In the new workflow, peptide libraries are generated by endoproteolytic digestion of proteomes without chemical modification of primary amines prior to exposure to a protease under investigation. After incubation with the test protease, treated and control library are differentially labeled using stable isotopes by means of reductive dimethylation. Upon analysis by liquid chromatography mass spectrometry, cleavage products of the test protease appear as peptides enriched for the respective isotopic label. With “PICS 2.0” we identified more than 2800 cleavage sites in five specificity experiments for four proteases, including trypsin, caspase-3, and chlamydial protease-like activity factor (CPAF). Hence, this method is a valuable addition to the different strategies currently employed for specificity profiling of proteases
2016-05-02 | PXD002785 | Pride
Project description:We identified robust cleavage by non-canonically-targeted SpCas9 that target protospacer with a few bases from the canonical NGG PAM in biochemistry assay. To determine whether this spaced SpCas9 cleavage exists in human cells, we employed high-throughput genome-wide translocation sequencing (LAM-HTGTS) using SpCas9 with three independent canonical sgRNAs served as bait (bait-A, bait-L and bait-D) to compared the cleavage activities of canonically-targeted and non-canonically-targeted SpCas9 in eight locus (RAG1B-D, RAG1K-O) in chromosome 11. We found that almost all 1bp-target shifted SpCas9 generated substantial DSBs as the canonical controls. While detected less frequently, 2bp-targeted shifted SpCas9 generated significant amounts of DSBs in RAG1D and RAG1L locus. These findings underscore the spaced PAM-mediated DSBs occurred in living cells.
2022-02-28 | GSE192459 | GEO
Project description:CRISPRs and TALENs are efficient systems for gene editing in many organisms including plants. In many cases the CRISPR-Cas or TALEN modules are expressed in the plant cell only transiently. Theoretically, transient expression of the editing modules should limit unexpected effects compared to stable transformation. However, very few studies have measured the off-target and unpredicted effects of editing strategies on the plant genome, and none of them have compared these two major editing systems. We conducted a comprehensive genome-wide investigation of off-target mutations using either a CRISPR-Cas9 or a TALEN strategy. We observed a similar number of SNVs and InDels for the two editing strategies compared to control non-transfected plants, with an average of 8.25 SNVs and 19.5 InDels for the CRISPR-edited plants, and an average of 17.5 SNVs and 32 InDels for the TALEN-edited plants. Interestingly, a comparable number of SNVs and InDels could be detected in the PEG-treated control plants. This shows that except for the on-target modifications, the gene editing tools used in this study did not show a significant off-target activity nor unpredicted effects on the genome, and that the PEG treatment in itself was probably the main source of mutations found in the edited plants.
2022-03-03 | E-MTAB-11497 | biostudies-arrayexpress
Project description:Precise and broad scope genome editing based on high-specificity Cas9 nickases
| PRJNA642789 | ENA