Unexpected genomic rearrangements at targeted loci associated with CRISPR/Cas9-mediated knock-in.

The CRISPR/Cas9 gene editing tool enables accessible and efficient modifications which (re)ignited molecular research in certain species. However, targeted integration of large DNA fragments using CRISPR/Cas9 can still be challenging in numerous models. To systematically compare CRISPR/Cas9's efficiency to classical homologous recombination (cHR) for insertion of large DNA fragments, we thoroughly performed and analyzed 221 experiments targeting 128 loci in mouse ES cells. Although both technologies proved efficient, CRISPR/Cas9 yielded significantly more positive clones as detected by overlapping PCRs. It also induced unexpected rearrangements around the targeted site, ultimately rendering CRISPR/Cas9 less efficient than cHR for the production of fully validated clones. These data show that CRISPR/Cas9-mediated recombination can induce complex long-range modifications at targeted loci, thus emphasizing the need for thorough characterization of any genetically modified material obtained through CRISPR-mediated gene editing before further functional studies or therapeutic use.

A new signaling cascade linking BMP4, BMPR1A, ΔNp73 and NANOG impacts on stem-like human cell properties and patient outcome.

In a significant number of cases cancer therapy is followed by a resurgence of more aggressive tumors derived from immature cells. One example is acute myeloid leukemia (AML), where an accumulation of immature cells is responsible for relapse following treatment. We previously demonstrated in chronic myeloid leukemia that the bone morphogenetic proteins (BMP) pathway is involved in stem cell fate and contributes to transformation, expansion, and persistence of leukemic stem cells. Here, we have identified intrinsic and extrinsic dysregulations of the BMP pathway in AML patients at diagnosis. BMP2 and BMP4 protein concentrations are elevated within patients' bone marrow with a BMP4-dominant availability. This overproduction likely depends on the bone marrow microenvironment, since MNCs do not overexpress BMP4 transcripts. Intrinsically, the receptor BMPR1A transcript is increased in leukemic samples with more cells presenting this receptor at the membrane. This high expression of BMPR1A is further increased upon BMP4 exposure, specifically in AML cells. Downstream analysis demonstrated that BMP4 controls the expression of the survival factor ΔNp73 through its binding to BMPR1A. At the functional level, this results in the direct induction of NANOG expression and an increase of stem-like features in leukemic cells, as shown by ALDH and functional assays. In addition, we identified for the first time a strong correlation between ΔNp73, BMPR1A and NANOG expression with patient outcome. These results highlight a new signaling cascade initiated by tumor environment alterations leading to stem-cell features and poor patients' outcome.