Novel mosaic mice with diverse applications

Chen, Yuxin; Mao, Shaoshuai; Liu, Bo; Jing, Zhengyu; Zang, Ying; Xia, Jing; Sun, Jie; Tian, Chao

doi:10.1101/2020.03.21.001388

Cited by 3 publications

(2 citation statements)

References 40 publications

(42 reference statements)

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“…CRISPR-Cas also potentially allows novel approaches to increase the throughput of gene editing of many alleles simultaneously (246). Multi sgRNA alleles can be generated by placing a lox71 site downstream of a U6 promoter and then downstream of this a concatemers of alternating sgRNA sequences with loxKR3 lox sites.…”

Section: Rapid Generation Of Germline Mutationsmentioning

confidence: 99%

Forward and Reverse Genetics of B Cell Malignancies: From Insertional Mutagenesis to CRISPR-Cas

Dawes

Uren

2021

Front. Immunol.

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Cancer genome sequencing has identified dozens of mutations with a putative role in lymphomagenesis and leukemogenesis. Validation of driver mutations responsible for B cell neoplasms is complicated by the volume of mutations worthy of investigation and by the complex ways that multiple mutations arising from different stages of B cell development can cooperate. Forward and reverse genetic strategies in mice can provide complementary validation of human driver genes and in some cases comparative genomics of these models with human tumors has directed the identification of new drivers in human malignancies. We review a collection of forward genetic screens performed using insertional mutagenesis, chemical mutagenesis and exome sequencing and discuss how the high coverage of subclonal mutations in insertional mutagenesis screens can identify cooperating mutations at rates not possible using human tumor genomes. We also compare a set of independently conducted screens from Pax5 mutant mice that converge upon a common set of mutations observed in human acute lymphoblastic leukemia (ALL). We also discuss reverse genetic models and screens that use CRISPR-Cas, ORFs and shRNAs to provide high throughput in vivo proof of oncogenic function, with an emphasis on models using adoptive transfer of ex vivo cultured cells. Finally, we summarize mouse models that offer temporal regulation of candidate genes in an in vivo setting to demonstrate the potential of their encoded proteins as therapeutic targets.

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Section: Rapid Generation Of Germline Mutationsmentioning

confidence: 99%

Forward and Reverse Genetics of B Cell Malignancies: From Insertional Mutagenesis to CRISPR-Cas

Dawes

Uren

2021

Front. Immunol.

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“…To overcome some limitations with CRISPR library delivery to tissues, engineered mosaic mice harboring germline floxed sgRNAs linked together in tandem are in development, with one sgRNA expressed per cell following Cre-mediated recombination [179]. Double-stranded AAVs (dsAAVs), which result from a mutation on one of the viral inverted terminal repeats (ITR), have provided further improvements in viral transduction efficiency and CRISPR-mediated genome editing, although at the cost of a reduced package capacity (~2.5 kb) compared to ssAAVs [180].…”

Section: Future Directions For Transposon and Crispr Screensmentioning

confidence: 99%

CRISPR and transposon in vivo screens for cancer drivers and therapeutic targets

2020

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Human cancers harbor substantial genetic, epigenetic, and transcriptional changes, only some of which drive oncogenesis at certain times during cancer evolution. Identifying the cancer-driver alterations amongst the vast swathes of "passenger" changes still remains a major challenge. Transposon and CRISPR screens in vivo provide complementary methods for achieving this, and each platform has its own advantages. Here, we review recent major technological breakthroughs made with these two approaches and highlight future directions. We discuss how each genetic screening platform can provide unique insight into cancer evolution, including intratumoral heterogeneity, metastasis, and immune evasion, presenting transformative opportunities for targeted therapeutic intervention.

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Integrating Omics and CRISPR Technology for Identification and Verification of Genomic Safe Harbor Loci in the Chicken Genome

et al. 2023

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Background One of the most prominent questions in the field of transgenesis is ‘Where in the genome to integrate a transgene?’. Escape from epigenetic silencing and promoter shutdown of the transgene needs reliable genomic safe harbor (GSH) loci. Advances in genome engineering technologies combined with multi-omics bioinformatics data have enabled rational evaluation of GSH loci in the host genome. Currently, no validated GSH loci have been evaluated in the chicken genome. Results Here, we analyzed and experimentally examined two GSH loci in the genome of chicken cells. To this end, putative GSH loci including chicken HIPP-like (cHIPP; between DRG1 and EIF4ENIF1 genes) and chicken ROSA-like (cROSA; upstream of the THUMPD3 gene) were predicted using multi-omics bioinformatics data. Then, the durable expression of the transgene was validated by experimental characterization of continuously-cultured isogenous cell clones harboring DsRed2-ΔCMV-EGFP cassette in the predicted loci. The weakened form of the CMV promoter (ΔCMV) allowed the precise evaluation of GSH loci in a locus-dependent manner compared to the full-length CMV promoter. Conclusions cHIPP and cROSA loci introduced in this study can be reliably exploited for consistent bio-manufacturing of recombinant proteins in the genetically-engineered chickens. Also, results showed that the genomic context dictates the expression of transgene controlled by ΔCMV in GSH loci.

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Novel mosaic mice with diverse applications

Cited by 3 publications

References 40 publications

Forward and Reverse Genetics of B Cell Malignancies: From Insertional Mutagenesis to CRISPR-Cas

Forward and Reverse Genetics of B Cell Malignancies: From Insertional Mutagenesis to CRISPR-Cas

CRISPR and transposon in vivo screens for cancer drivers and therapeutic targets

Integrating Omics and CRISPR Technology for Identification and Verification of Genomic Safe Harbor Loci in the Chicken Genome

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