Covalent modification by SUMO regulates a wide range of cellular processes, including transcription, cell cycle, and chromatin dynamics. To address the biological function of the SUMO pathway in mammals, we generated mice deficient for the SUMO E2-conjugating enzyme Ubc9. Ubc9-deficient embryos die at the early postimplantation stage. In culture, Ubc9 mutant blastocysts are viable, but fail to expand after 2 days and show apoptosis of the inner cell mass. Loss of Ubc9 leads to major chromosome condensation and segregation defects. Ubc9-deficient cells also show severe defects in nuclear organization, including nuclear envelope dysmorphy and disruption of nucleoli and PML nuclear bodies. Moreover, RanGAP1 fails to accumulate at the nuclear pore complex in mutant cells that show a collapse in Ran distribution. Together, these findings reveal a major role for Ubc9, and, by implication, for the SUMO pathway, in nuclear architecture and function, chromosome segregation, and embryonic viability in mammals.

We used BE-PACE to evolve new cytosine base editors (CBEs) that overcome target sequence context constraints of canonical CBEs. One evolved CBE, evoAPOBEC1-BE4max, is up to 26-fold more efficient at editing GC, a disfavored context for wild-type APOBEC1 deaminase, while maintaining efficient editing in all other sequence contexts tested. Another evolved deaminase, evoFERNY, is 29% smaller than APOBEC1 and edits efficiently in all tested sequence contexts. We also evolved a CBE based on CDA1 deaminase with much higher editing efficiency at difficult target sites. Finally, we used data from evolved CBEs to illuminate the relationship between deaminase activity, base editing efficiency, editing window width, and byproduct formation. These findings establish a system for rapid evolution of base editors and inform their use and improvement.Genome editing has revolutionized the life sciences and entered clinical trials to treat genetic diseases. 1 The use of programmable nucleases to generate double-stranded DNA breaks (DSBs) followed by homology-directed repair can introduce a wide variety of modifications but is inefficient in non-dividing cells, and is typically accompanied by an excess of unwanted insertions and deletions (indels), translocations, or other chromosomal rearrangements. 2 Base editing directly modifies target DNA bases in living cells and has become widely used to correct or install point mutations in organisms ranging from bacteria to human embryos. 3 Base editors use a catalytically impaired Cas9 to open a single-stranded DNA loop at a specified genomic site (Fig. 1a). Bases within the editing window (typically ~5 nt wide) in this region are modified by a tethered base-modification enzyme that only accepts single-stranded DNA.

Mucopolysaccharidosis type III A (MPS III A, Sanfilippo syndrome) is a rare, autosomal recessive, lysosomal storage disease characterized by accumulation of heparan sulfate secondary to defective function of the lysosomal enzyme heparan N- sulfatase (sulfamidase). Here we describe a spontaneous mouse mutant that replicates many of the features found in MPS III A in children. Brain sections revealed neurons with distended lysosomes filled with membranous and floccular materials with some having a classical zebra body morphology. Storage materials were also present in lysosomes of cells of many other tissues, and these often stained positively with periodic-acid Schiff reagent. Affected mice usually died at 7-10 months of age exhibiting a distended bladder and hepatosplenomegaly. Heparan sulfate isolated from urine and brain had nonreducing end glucosamine- N -sulfate residues that were digested with recombinant human sulfamidase. Enzyme assays of liver and brain extracts revealed a dramatic reduction in sulfamidase activity. Other lysosomal hydrolases that degrade heparan sulfate or other glycans and glycosaminoglycans were either normal, or were somewhat increased in specific activity. The MPS III A mouse provides an excellent model for evaluating pathogenic mechanisms of disease and for testing treatment strategies, including enzyme or cell replacement and gene therapy.

Acute promyelocytic leukemia (APL) is associated with chromosomal translocations that always involve the RARalpha gene, which variably fuses to one of several distinct loci, including PML or PLZF (X genes). Due to the reciprocity of the translocation, X-RARalpha and RARalpha-X fusion proteins coexist in APL blasts. PLZF-RARalpha transgenic mice (TM) develop leukemia that lacks the differentiation block at the promyelocytic stage that characterizes APL. We generated TM expressing RARalpha-PLZF and PLZF-RARalpha in their promyelocytes. RARalpha-PLZF TM do not develop leukemia. However, PLZF-RARalpha/RARalpha-PLZF double TM develop leukemia with classic APL features. We demonstrate that RARalpha-PLZF can interfere with PLZF transcriptional repression and that this is critical for APL pathogenesis, since leukemias in PLZF(-/-)/PLZF-RARalpha mutants and in PLZF-RARalpha/RARalpha-PLZF TM are indistinguishable. Thus, both products of a cancer-associated translocation are crucial in determining the distinctive features of the disease.

Background: BRCA1 and PALB2 interact with each other to promote homologous recombination and DNA double strand break repair. Results: Mice with abrogated PALB2-BRCA1 interaction show male fertility defect. Conclusion: PALB2 and BRCA1 function together to ensure normal male meiosis. Significance: This work demonstrates the importance of the PALB2-BRCA1 interaction in vivo and reveals a novel role of PALB2 in sex chromosome synapsis.

Duchenne muscular dystrophy (DMD) is an incurable neuromuscular degenerative disease, caused by a mutation in the dystrophin gene. Mdx mice recapitulate DMD features. Here we show that injection of wild-type (WT) embryonic stem cells (ESCs) into mdx blastocysts produces mice with improved pathology and function. A small fraction of WT ESCs incorporates into the mdx mouse nonuniformly to upregulate protein levels of dystrophin in the skeletal muscle. The chimeric muscle shows reduced regeneration and restores dystrobrevin, a dystrophin-related protein, in areas with high and with low dystrophin content. WT ESC injection increases the amount of fat in the chimeras to reach WT levels. ESC injection without dystrophin does not prevent the appearance of phenotypes in the skeletal muscle or in the fat. Thus, dystrophin supplied by the ESCs reverses disease in mdx mice globally in a dose-dependent manner.

Mutant mice produced by gene targeting in embryonic stem (ES) cells often have a complex or embryonic lethal phenotype. In these cases, it would be helpful to identify tissues and cell types first affected in mutant embryos by following the contribution to chimeras ofES cells homozygous for the mutant allele. Although a number of strategies for following ES cell development in vivo have been reported, each has limitations that preclude its general application. In this paper, we describe ES cell lines that can be tracked to every nucleated cell type in chimeras at all developmental stages. These lines were derived from blastocysts of mice that carry an 11-Mb ,3-globin transgene on chromosome 3. The transgene is readily detected by DNA in situ hybridization, providing an inert, nuclear-localized marker whose presence is not affected by transcriptional or translational controls. The ";WW" series of ES lines possess the essential features of previously described ES lines, including giving rise to a preponderance of male chimeras, all of which have to date exhibited germ-line transmission. In addition, clones selected for single or double targeting events form strong chimeras, demonstrating the feasibility of using WW6 cells to identify phenotypes associated with the creation of a null mutant.Embryonic stem (ES) cells are widely used for the production of mice bearing a germ-line mutation (1). The phenotypes generated are often difficult to interpret in cellular and molecular terms, especially when the gene inactivated is essential at an early developmental stage. Methods for bypassing lethality in order to identify functions for embryonic lethal genes in adult tissues have been developed (2-4). However, these strategies are tissue specific and hypothesis driven. They may miss unsuspected gene functions. An alternative approach, aimed at identifying tissues affected by a particular mutation, is to determine the contribution of homozygous mutant ES cells to the tissues of a chimera. Markers that have been used to identify the progeny of injected ES cells exploit a glycerophosphate isomerase (GPI) isozyme difference (5, 6), a difference in H2 class 1 molecules expressed at the cell surface (7) 10] was created by injection of cloned DNA containing the mouse fmaj globin gene (11). In the original founder mouse, the transgene integrated as a large, tandem repeat of -1000 copies confined to the telomeric region of chromosome 3 (9, 12-15). The transgene is not expressed and no phenotypic effects have been attributed to this insertion (16). Sensitive methods developed to detect the transgene by DNADNA in situ hybridization using a biotinylated probe have shown the hybridization signal to be precisely localized over cell nuclei (9). This transgene has been used in cell lineage studies of the original transgenic strain (12) and in studies of aggregation chimeras (17,18). In another experiment, tetraploid embryos derived from strain TgN(Hbb-bl)83Clo were aggregated with 129/Sv blastocyst-derived inner cell mass cells to...

BCCIP is a BRCA2- and CDKN1A(p21)-interacting protein that has been implicated in the maintenance of genomic integrity. To understand the in vivo functions of BCCIP, we generated a conditional BCCIP knockdown transgenic mouse model using Cre-LoxP mediated RNA interference. The BCCIP knockdown embryos displayed impaired cellular proliferation and apoptosis at day E7.5. Consistent with these results, the in vitro proliferation of blastocysts and mouse embryonic fibroblasts (MEFs) of BCCIP knockdown mice were impaired considerably. The BCCIP deficient mouse embryos die before E11.5 day. Deletion of the p53 gene could not rescue the embryonic lethality due to BCCIP deficiency, but partially rescues the growth delay of mouse embryonic fibroblasts in vitro. To further understand the cause of development and proliferation defects in BCCIP-deficient mice, MEFs were subjected to chromosome stability analysis. The BCCIP-deficient MEFs displayed significant spontaneous chromosome structural alterations associated with replication stress, including a 3.5-fold induction of chromatid breaks. Remarkably, the BCCIP-deficient MEFs had a ∼20-fold increase in sister chromatid union (SCU), yet the induction of sister chromatid exchanges (SCE) was modestly at 1.5 fold. SCU is a unique type of chromatid aberration that may give rise to chromatin bridges between daughter nuclei in anaphase. In addition, the BCCIP-deficient MEFs have reduced repair of irradiation-induced DNA damage and reductions of Rad51 protein and nuclear foci. Our data suggest a unique function of BCCIP, not only in repair of DNA damage, but also in resolving stalled replication forks and prevention of replication stress. In addition, BCCIP deficiency causes excessive spontaneous chromatin bridges via the formation of SCU, which can subsequently impair chromosome segregations in mitosis and cell division.