highly-efficient-gene-disruption-murine-human-hematopoietic

Highly Efficient Gene Disruption of Murine and Human Hematopoietic Progenitor Cells by CRISPR/Cas9

Advances in the hematopoietic stem cell (HSCs) field have been aided by methods to genetically engineer primary progenitor cells as well as animal models. ...

Baylor College of Medicine

University of Perugia

Our understanding of the mechanisms that regulate hematopoietic stem/progenitor cells (HSPCs) has been advanced by the ability to genetically manipulate mice; however, germline modification is time consuming and expensive. Here, we describe fast, efficient, and cost-effective methods to directly modify the genomes of mouse and human HSPCs using the CRISPR/Cas9 system. Using plasmid and virus-free delivery of guide RNAs alone into Cas9-expressing HSPCs or Cas9-guide RNA ribonucleoprotein (RNP) complexes into wild-type cells, we have achieved extremely efficient gene disruption in primary HSPCs from mouse (>60%) and human (∼75%). These techniques enabled rapid evaluation of the functional effects of gene loss of Eed, Suz12, and DNMT3A. We also achieved homology-directed repair in primary human HSPCs (>20%). These methods will significantly expand applications for CRISPR/Cas9 technologies for studying normal and malignant hematopoiesis.

Highly Efficient Genome Editing of Murine and Human Hematopoietic Progenitor Cells by CRISPR/Cas9.

DNA methylation is an epigenetic process involved in development, aging, and cancer. Although the advent of new molecular techniques has enhanced our knowledge of how DNA methylation alters chromatin and subsequently affects gene expression, a direct link between epigenetic marks and tumorigenesis has not been established. DNMT3A is a de novo DNA methyltransferase that has recently gained relevance because of its frequent mutation in a large variety of immature and mature hematologic neoplasms. DNMT3A mutations are early events during cancer development and seem to confer poor prognosis to acute myeloid leukemia (AML) patients making this gene an attractive target for new therapies. Here, we discuss the biology of DNMT3A and its role in controlling hematopoietic stem cell fate decisions. In addition, we review how mutant DNMT3A may contribute to leukemogenesis and the clinical relevance of DNMT3A mutations in hematologic cancers.

DNMT3A in Leukemia.

DNA methylation has widespread effects on gene expression during development. However, our ability to assign specific function to regions of DNA methylation is limited by the poor correlation between global patterns of DNA methylation and gene expression.

DNA epigenome editing using CRISPR-Cas SunTag-directed DNMT3A.

Capicua (CIC) regulates a transcriptional network downstream of the RAS/MAPK signaling cascade. In, CIC is important for many developmental processes, including embryonic patterning and specification of wing veins. In humans, CIC has been implicated in neurological diseases, including spinocerebellar ataxia type 1 (SCA1) and a neurodevelopmental syndrome. Additionally, we and others have reported mutations inin several cancers. However, whether CIC is a tumor suppressor remains to be formally tested. In this study, we found that deletion ofin adult mice causes T cell acute lymphoblastic leukemia/lymphoma (T-ALL). Using hematopoietic-specific deletion and bone marrow transplantation studies, we show that loss offrom hematopoietic cells is sufficient to drive T-ALL.-null tumors show up-regulation of the KRAS pathway as well as activation of the NOTCH1 and MYC transcriptional programs. In sum, we demonstrate that loss of CIC causes T-ALL, establishing it as a tumor suppressor for lymphoid malignancies. Moreover, we show that mouse models lacking CIC in the hematopoietic system are robust models for studying the role of RAS signaling as well as NOTCH1 and MYC transcriptional programs in T-ALL.

Article	Year
Highly Efficient Genome Editing of Murine and Human Hematopoietic Progenitor Cells by CRISPR/Cas9. Cell reports\| PubMed ID: 27783956	2016
DNMT3A in Leukemia. Cold Spring Harbor perspectives in medicine\| PubMed ID: 28003281	2017
DNA epigenome editing using CRISPR-Cas SunTag-directed DNMT3A. Genome biology\| PubMed ID: 28923089	2017
Loss of Capicua alters early T cell development and predisposes mice to T cell lymphoblastic leukemia/lymphoma. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 29382756	2018

Lorenzo Brunetti

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