CRISPR-guided DNA polymerase enabling diversification of all nucleotides in a tunable window

The capacity to diversify genetic codes advances our understanding and engineering of biological systems. A method to continuously diversify user-defined regions of a genome without requiring the integration of nucleic acid libraries would enable forward genetic approaches in systems not amenable to high efficiency homologydirected integration, rapid evolution of biotechnologically useful activity through accelerated and parallelized rounds of mutagenesis and selection, and cell lineage tracking. Here we developed EvolvR, the first system that can continuously diversify all nucleotides within a tunable window length at user-defined loci. Our results demonstrate that EvolvR enables multiplexed and continuous diversification of user-defined genomic loci that will be useful for a broad range of basic and biotechnological applications

Expression of Stem Cell Markers in the Human Fetal Kidney

In the human fetal kidney (HFK) self-renewing stem cells residing in the metanephric mesenchyme (MM)/blastema are induced to form all cell types of the nephron till 34th week of gestation. Definition of useful markers is crucial for the identification of HFK stem cells. Because wilms' tumor, a pediatric renal cancer, initiates from retention of renal stem cells, we hypothesized that surface antigens previously up-regulated in microarrays of both HFK and blastema-enriched stem-like wilms' tumor xenografts (NCAM, ACVRIIB, DLK1/PREF, GPR39, FZD7, FZD2, NTRK2) are likely to be relevant markers. Comprehensive profiling of these putative and of additional stem cell markers (CD34, CD133, c-Kit, CD90, CD105, CD24) in mid-gestation HFK was performed using immunostaining and FACS in conjunction with EpCAM, an epithelial surface marker that is absent from the MM and increases along nephron differentiation and hence can be separated into negative, dim or bright fractions. No marker was specifically localized to the MM. Nevertheless, FZD7 and NTRK2 were preferentially localized to the MM and emerging tubules (<10% of HFK cells) and were mostly present within the EpCAMneg and EpCAMdim fractions, indicating putative stem/progenitor markers. In contrast, single markers such as CD24 and CD133 as well as double-positive CD24+CD133+ cells comprise >50% of HFK cells and predominantly co-express EpCAMbright, indicating they are mostly markers of differentiation. Furthermore, localization of NCAM exclusively in the MM and in its nephron progenitor derivatives but also in stroma and the expression pattern of significantly elevated renal stem/progenitor genes Six2, Wt1, Cited1, and Sall1 in NCAM+EpCAM- and to a lesser extent in NCAM+EpCAM+ fractions confirmed regional identity of cells and assisted us in pinpointing the presence of subpopulations that are putative MM-derived progenitor cells (NCAM+EpCAM+FZD7+), MM stem cells (NCAM+EpCAM-FZD7+) or both (NCAM+FZD7+). These results and concepts provide a framework for developing cell selection strategies for human renal cell-based therapies

Antibodies used in the flow cytometry assays.

<p>Antibodies used in the flow cytometry assays.</p

FACS analysis of single putative stem cell markers in HAK cells.

<p>(a) Summarizing bar graph of single marker staining in HAK cells. Data were calculated as average % of expressing cell±SD. Each marker was tested in 3 HAK. (b) Representative dot plot graphs of CD24 and CD133 co-staining demonstrate a large fraction of CD24⁺CD133⁺ cells in HAK. Quadrates were placed according to the isotype control confiding the negative staining to the lower left quadrant. Percentage of cells for each marker combination appears in the quadrant.</p

FACS analysis of single putative stem cell surface markers in HFK cells.

<p>(a) Representative flow-cytometry histograms of surface marker molecules (green) EpCAM, NCAM1, FZD7, NTRK2, CD90, CD34, CD24, CD133, and their respective isotype controls (red) in HFK (19 weeks of gestation). (b) Summarizing bar graph of single marker staining in HFK (15–19 weeks of gestation). Each marker was tested on at least 3 independent samples. Data were calculated as average % of expressing cell±SD. Each marker was tested in 10 HFK.</p

Antibodies used for IHC staining.

<p>Antibodies used for IHC staining.</p

Gene expression analysis in sorted NCAM/EpCAM subpopulations.

<p>Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis of (a) renal stem/progenitor genes (<i>Six2, Cited1, Sall1, Wt1</i> and <i>Pax2</i>), (b) vimentin and E-cadherin (c) ‘stemness’ genes (β-catenin/CTNNB1, <i>EZH2</i>, <i>BMI1</i>, <i>Nanog</i> and <i>Oct4</i>) and (d) surface marker (<i>FZD7, ACR2B, NTRK2, CD24</i> and <i>CD133</i>) gene expression in NCAM/EpCAM magnetically separated cells from HFK (15–19 weeks of gestation). Normalization was performed against control HPRT expression and RQ calculated relative to the NCAM- fraction. Data were calculated as average±SD of at least 3 independent samples. ***P<0.001, *P<0.05 versus NCAM-. <i>Sall1</i> expression in NCAM⁺ EpCAM⁺ cells was near significance (p<0.059).</p

FACS analysis of putative stem cell markers in EpCAM subpopulations.

<p>(a) Representative zebra graph of EpCAM staining and the subpopulation gating. EpCAM subpopulations were gated according to EpCAM staining intensity (negative, dim or bright) versus FSC. (b) Representative dot plot graphs of NCAM1, PSA-NCAM, FZD7, NTRK2, CD24 and CD133 expression levels in EpCAM subpopulations of HFK. Quadrates were placed according to isotype control confiding the negative staining to the lower left quadrant. Percentage of cells in each subgroup appears on the lower right quadrant. (c) Summarizing bar graphs of NCAM1, PSA- NCAM, NTRK2, FZD7, CD24 and CD133 expression levels in EpCAM subpopulations. Data are average % of cells in each subgroup±SD. Analysis of each marker was performed at least three times.</p