Dataset Information

Generation of An Endogenous FGFR2-BICC1 Gene Fusion/58 Megabase Inversion Using Single-Plasmid CRISPR/Cas9 Editing in Biliary Cells.

ABSTRACT: Fibroblast growth factor receptor 2 (FGFR2) gene fusions are bona fide oncogenic drivers in 10-15% of intrahepatic cholangiocarcinoma (CCA), yet currently there are no cell lines publically available to study endogenous FGFR2 gene fusions. The ability of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 to generate large yet precise chromosomal rearrangements has presented the possibility of engineering endogenous gene fusions for downstream studies. In this technical report, we describe the generation of an endogenous FGFR2-Bicaudal family RNA binding protein 1 (BICC1) fusion in multiple independent cholangiocarcinoma and immortalized liver cell lines using CRISPR. BICC1 is the most common FGFR2 fusion partner in CCA, and the fusion arises as a consequence of a 58-megabase-sized inversion on chromosome 10. We replicated this inversion to generate a fusion product that is identical to that seen in many human CCA. Our results demonstrate the feasibility of generating large megabase-scale inversions that faithfully reproduce human cancer aberrations.

SUBMITTER: Reicher A

PROVIDER: S-EPMC7178239 | biostudies-literature | 2020 Apr

REPOSITORIES: biostudies-literature

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Publications

Generation of An Endogenous FGFR2-BICC1 Gene Fusion/58 Megabase Inversion Using Single-Plasmid CRISPR/Cas9 Editing in Biliary Cells.

Reicher Andreas A Harris Antoneicka L AL Prinz Felix F Kiesslich Tobias T Wei Miaoyan M Öllinger Rupert R Rad Roland R Pichler Martin M Kwong Lawrence N LN

International journal of molecular sciences 20200402 7

Fibroblast growth factor receptor 2 (FGFR2) gene fusions are bona fide oncogenic drivers in 10-15% of intrahepatic cholangiocarcinoma (CCA), yet currently there are no cell lines publically available to study endogenous FGFR2 gene fusions. The ability of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 to generate large yet precise chromosomal rearrangements has presented the possibility of engineering endogenous gene fusions for downstream studies. In ...[more]

PMID: 32252259

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Project description:Many Candida species that cause infection have diploid genomes and do not undergo classical meiosis. The application of clustered regularly interspaced short palindromic repeat-Cas9 (CRISPR-Cas9) gene editing systems has therefore greatly facilitated the generation of gene disruptions and the introduction of specific polymorphisms. However, CRISPR methods are not yet available for all Candida species. We describe here an adaption of a previously developed CRISPR system in Candida parapsilosis that uses an autonomously replicating plasmid. Guide RNAs can be introduced in a single cloning step and are released by cleavage between a tRNA and a ribozyme. The plasmid also contains CAS9 and a selectable nourseothricin SAT1 marker. It can be used for markerless editing in C. parapsilosis, C. orthopsilosis, and C. metapsilosis We also show that CRISPR can easily be used to introduce molecular barcodes and to reintroduce wild-type sequences into edited strains. Heterozygous mutations can be generated, either by careful selection of the distance between the polymorphism and the Cas9 cut site or by providing two different repair templates at the same time. In addition, we have constructed a different autonomously replicating plasmid for CRISPR-Cas9 editing in Candida tropicalis We show that editing can easily be carried out in multiple C. tropicalis isolates. Nonhomologous end joining (NHEJ) repair occurs at a high level in C. metapsilosis and C. tropicalis IMPORTANCE Candida species are a major cause of infection worldwide. The species associated with infection vary with geographical location and with patient population. Infection with Candida tropicalis is particularly common in South America and Asia, and Candida parapsilosis infections are more common in the very young. Molecular methods for manipulating the genomes of these species are still lacking. We describe a simple and efficient CRISPR-based gene editing system that can be applied in the C. parapsilosis species group, including the sister species Candida orthopsilosis and Candida metapsilosis We have also constructed a separate system for gene editing in C. tropicalis.

Project description:MotivationCRISPR/Cas9 technology has been revolutionizing the field of gene editing in recent years. Guide RNAs (gRNAs) enable Cas9 proteins to target specific genomic loci for editing. However, editing efficiency varies between gRNAs. Thus, computational methods were developed to predict editing efficiency for any gRNA of interest. High-throughput datasets of Cas9 editing efficiencies were produced to train machine-learning models to predict editing efficiency. However, these high-throughput datasets have low correlation with functional and endogenous editing. Another difficulty arises from the fact that functional and endogenous editing efficiency is more difficult to measure, and as a result, functional and endogenous datasets are too small to train accurate machine-learning models on.ResultsWe developed DeepCRISTL, a deep-learning model to predict the on-target efficiency given a gRNA sequence. DeepCRISTL takes advantage of high-throughput datasets to learn general patterns of gRNA on-target editing efficiency, and then uses transfer learning (TL) to fine-tune the model and fit it to the functional and endogenous prediction task. We pre-trained the DeepCRISTL model on more than 150 000 gRNAs, produced through the DeepHF study as a high-throughput dataset of three Cas9 enzymes. We improved the DeepHF model by multi-task and ensemble techniques and achieved state-of-the-art results over each of the three enzymes: up to 0.89 in Spearman correlation between predicted and measured on-target efficiencies. To fine-tune model weights to predict on-target efficiency of functional or endogenous datasets, we tested several TL approaches, with gradual learning being the overall best performer, both when pre-trained on DeepHF and when pre-trained on CRISPROn, another high-throughput dataset. DeepCRISTL outperformed state-of-the-art methods on all functional and endogenous datasets. Using saliency maps, we identified and compared the important features learned by the model in each dataset. We believe DeepCRISTL will improve prediction performance in many other CRISPR/Cas9 editing contexts by leveraging TL to utilize both high-throughput datasets, and smaller and more biologically relevant datasets, such as functional and endogenous datasets.Availability and implementationDeepCRISTL is available via github.com/OrensteinLab/DeepCRISTL.Supplementary informationSupplementary data are available at Bioinformatics online.

Dataset Information

Generation of An Endogenous FGFR2-BICC1 Gene Fusion/58 Megabase Inversion Using Single-Plasmid CRISPR/Cas9 Editing in Biliary Cells.

Publications

Generation of An Endogenous FGFR2-BICC1 Gene Fusion/58 Megabase Inversion Using Single-Plasmid CRISPR/Cas9 Editing in Biliary Cells.

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