A Medium-Throughput Single Cell CRISPR-Cas9 Assay to Assess Gene Essentiality.

BACKGROUND: Target selection for oncology is a crucial step in the successful development of therapeutics. Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 editing of specific loci offers an alternative method to RNA interference and small molecule inhibitors for determining whether a cell line is dependent on a specific gene product for proliferation or survival. In our initial studies using CRISPR-Cas9 to verify the dependence on EZH2 activity for proliferation of a SMARCB1/SNF5/INI1 mutant malignant rhabdoid tumor (MRT) cell line, we noted that the initial reduction in proliferation was lost over time. We hypothesized that in the few cells that retain proliferative capacity, at least one allele of EZH2 remains functional. To verify this, we developed an assay to analyze 10s-100s of clonal cell populations for target gene disruption using restriction digest and fluorescent fragment length analyses. RESULTS: Our results clearly show that in cell lines in which EZH2 is essential for proliferation, at least one potentially functional allele of EZH2 is retained in the clones that survive. CONCLUSION: This assay clearly indicates whether or not a specific gene is essential for survival and/or proliferation in a given cell line. Such data can aid the development of more robust therapeutics by increasing confidence in target selection.

Subcellular localization of alpha-synuclein in primary neuronal cultures: effect of missense mutations.

Numerous recent observations have implicated alpha-synuclein in the pathogenesis of several neurodegenerative diseases, including Parkinson's disease, Alzheimer's disease, dementia with Lewy bodies and multiple-system atrophy. Two missense mutations in the gene for alpha-synuclein have been identified in some cases of familial Parkinson's disease and it is thought that these may disrupt the normal structure of the protein and thus promote aggregation into Lewy body filaments. Here, we examine the subcellular localization of alpha-synuclein in primary cortical neurons maintained in a monolayer culture. The protein has widespread expression throughout neurons, including the nucleus, and has a discete localization in the neurites of more mature neurons, reminiscent of synaptic specializations. Interestingly, in a subpopulation of cortical neurons transfected at 13 days in vitro, we find that alpha-synuclein appears to aggregate into distinct punctate inclusions in the cytoplasm and proximal neurites. Unlike Lewy bodies, these structures are not ubiquitin positive. These regions of alpha-synuclein accumulation are observed following transfections with wild-type, Ala30Pro or Ala53Thr alpha-synuclein; neither mutation alters their frequency.

Membrane association and protein conformation of alpha-synuclein in intact neurons. Effect of Parkinson's disease-linked mutations.

Two missense mutations (Ala-30 --> Pro and Ala-53 --> Thr) in the gene encoding alpha-synuclein are associated with rare autosomal dominant forms of familial Parkinson's disease. In addition, alpha-synuclein is an abundant component of Lewy bodies in sporadic Parkinson's disease and diffuse Lewy body disease. However, the normal conformation of alpha-synuclein, its cellular localization in neurons, and the effects of the mutations remain to be determined. In the present study, we examine these questions using sensitive fluorescence resonance energy transfer techniques. Transient transfection of alpha-synuclein expression constructs into primary cortical neurons and counterstaining with the lipophilic fluorescent marker, DiI, demonstrates a close association between alpha-synuclein and cellular membranes. Both the N- and C-terminal regions of alpha-synuclein are tightly associated with membranes. A weak interaction also occurs between the N and C termini themselves. The Parkinson's disease-associated mutations have no effect on membrane interaction; however, the Ala-30 --> Pro mutation alters the three-dimensional conformation of alpha-synuclein, as measured by significantly increased fluorescence resonance energy transfer between the N and C termini.

Characterization of IS1676 from Rhodococcus erythropolis SQ1.

To develop a transposable element-based system for mutagenesis in Rhodococcus, we used the sacB gene from Bacillus subtilis to isolate a novel transposable element, IS1676, from R. erythropolis SQ1. This 1693 bp insertion sequence is bounded by imperfect (10 out of 13 bp) inverted repeats and it creates 4 bp direct repeats upon insertion. Comparison of multiple insertion sites reveals a preference for the sequence 5'-(C/T)TA(A/G)-3' in the target site. IS1676 contains a single, large (1446 bp) open reading frame with coding potential for a protein of 482 amino acids. IS1676 may be similar to an ancestral transposable element that gave rise to repetitive sequences identified in clinical isolates of Mycobacterium kansasii. Derivatives of IS1676 should be useful for analysis of Rhodococcus strains, for which few other genetic tools are currently available.