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Clonally select lines after CRISPR/cas editing are not isogenic

ABSTRACT: Clonally select lines after CRISPR/cas editing are not isogenic

PROVIDER: PRJNA899295 | ENA |

REPOSITORIES: ENA

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Clonally select lines after CRISPR/cas editing are not isogenic

Project description:Clonally select lines after CRISPR/cas editing are not isogenic

| PRJNA899296 | ENA

Clonally Selected Lines After CRISPR-Cas Editing Are Not Isogenic.

Project description:The CRISPR-Cas9 system has enabled researchers to precisely modify/edit the sequence of a genome. A typical editing experiment consists of two steps: (1) editing cultured cells; (2) cell cloning and selection of clones with and without intended edit, presumed to be isogenic. The application of CRISPR-Cas9 system may result in off-target edits, whereas cloning will reveal culture-acquired mutations. We analyzed the extent of the former and the latter by whole genome sequencing in three experiments involving separate genomic loci and conducted by three independent laboratories. In all experiments we hardly found any off-target edits, whereas detecting hundreds to thousands of single nucleotide mutations unique to each clone after relatively short culture of 10-20 passages. Notably, clones also differed in copy number alterations (CNAs) that were several kb to several mb in size and represented the largest source of genomic divergence among clones. We suggest that screening of clones for mutations and CNAs acquired in culture is a necessary step to allow correct interpretation of DNA editing experiments. Furthermore, since culture associated mutations are inevitable, we propose that experiments involving derivation of clonal lines should compare a mix of multiple unedited lines and a mix of multiple edited lines.

| S-EPMC10123805 | biostudies-literature

Cas-SF01, an engineered Cas12i nuclease is an efficient genome editing tool in mammalian cells and plants

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

Engineered miniature CRISPR-Cas system for mammalian genome regulation and editing

Project description:Compact and versatile CRISPR-Cas systems will enable genome engineering applications through high-efficiency delivery in a wide variety of contexts. Here we create an efficient miniature Cas system (CasMINI) engineered from the type V-F Cas12f (Cas14) system by guide RNA and protein engineering, which is less than half the size of currently used CRISPR systems (Cas9 or Cas12a). We demonstrate that CasMINI can drive high levels of gene activation (up to thousands-fold increases), while the natural Cas12f system fails to function in mammalian cells. We show that the CasMINI system has comparable activities to Cas12a for gene activation, is highly specific, and allows for robust base editing and gene editing. We expect that CasMINI can be broadly useful for cell engineering and gene therapy applications ex vivo and in vivo.

2021-09-10 | GSE180773 | GEO

Discovery and evaluation of CRISPR/Cas-mediated systems for site-specific RNA editing

Project description:In this study we report a comparative, transcriptome-wide analysis of CRISPR/Cas-based RNA-editing technologies in HEK293T cells. Targeted RNA editing technologies hold great process in the area of post-transcriptional genomic engineering and impermanent therapeutic correction of disease-causing variants, though off-target editing events still remain a concern. Here, we perform paired-end poly(A)+ purified RNA-seq to characterize transcriptome-wide in HEK293T cells transfected with catalytically-dead Cas9-, Cas13b, or Cas13d fused to human ADAR2 deaminase domain containing a hyperactive editing mutation (E488Q) along with either CYFIP2¬-targeting or non-targeting gRNA, in duplicate. We find that all technologies tested introduced widespread but distinct off-target mutations in the transcriptome, with the majority of edits occurring in 3’UTR and CDS regions of genes. Many of these edits, however, were in transcripts with low coverage, with the majority of editing rates being below 20%. This study is the first to characterize Cas-based RNA editing technologies in a direct manner, and reveals the relative similarity of currently available methods both in terms of on-target and off-target efficacy.

2020-11-01 | GSE152684 | GEO

DNA epigenome editing using CRISPR-Cas SunTag-directed DNMT3A

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Expression Data from YUGEN8 parental cell line and its clonally related isogenic variants Clone 1A, Clone 2A, and Clone 2B hereby labeled as GEN, Cl.1A, Cl.2A, and Cl.2B respectively

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Engineered miniature CRISPR-Cas system for mammalian genome regulation and editing

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WGS of KCNQ2 R201C patient derived iPSC and its isogenic line

Project description:Using prime editing, we generated an isogenic line of an iPSC line of an individual with a pathogenic KCNQ2 R201C mutation. WGS on the mutant line and two isogenic clones was performed to identify potential off target effects

| EGAS00001007231 | EGA

Efficient engineering of human and mouse primary cells using peptide-assisted genome editing

Project description:Simple and efficient delivery of CRISPR genome editing systems in primary cells remains a major challenge. Here, we describe an engineered Peptide-Assisted Genome Editing (PAGE) CRISPR-Cas system for rapid and robust editing of primary cells. PAGE couples a cell-penetrating Cas protein with a cell-penetrating endosomal escape peptide in a 30-minute incubation that yields up to ~98% editing efficiency in primary human and mouse T cells. PAGE provides a broadly generalizable platform for next generation genome engineering in primary cells. CITATION INFORMATION: Zhang Zhen, Baxter Amy E, Ren Diqiu, Qin Kunhua, Chen Zeyu, Collins Sierra M., Huang Hua, Komar Chad A., Bailer Peter F., Parker Jared B., Blobel Gerd A., Kohli Rahul M., Wherry E. John*, Berger Shelley,*, and Shi Junwei*. Peptide-assisted genome editing permits efficient CRISPR engineering of primary T cells.

2023-03-20 | GSE223805 | GEO

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