CrispRdesignR: A Versatile Guide RNA Design Package in R for CRISPR/Cas9 Applications

The success of CRISPR/Cas9 gene editing applications relies on the efficiency of the single guide RNA (sgRNA) used in conjunction with the Cas9 protein. Current sgRNA design software vary in the details they provide on sgRNA sequence efficiency and are almost exclusively restricted to model organisms. The crispRdesignR package aims to address these limitations by providing comprehensive sequence features of the generated sgRNAs in a single program, which allows users to predict sgRNA efficiency and design sgRNA sequences for systems that currently do not have optimized efficiency scoring methods. crispRdesignR reports extensive information on all designed sgRNA sequences with robust off-target calling and annotation and can be run in a user-friendly graphical interface. The crispRdesignR package is implemented in R and has fully editable code for specialized purposes including sgRNA design in user-provided genomes. The package is platform independent and extendable, with its source code and documentation freely available at https://github.com/dylanbeeber/crispRdesignR.

Cytosolic aggregates perturb the degradation of nontranslocated secretory and membrane proteins

A wide range of diseases are associated with the accumulation of cytosolic protein aggregates. The effects of these aggregates on various aspects of normal cellular protein homeostasis remain to be determined. Here we find that cytosolic aggregates, without necessarily disrupting proteasome function, can markedly delay the normally rapid degradation of nontranslocated secretory and membrane protein precursors. In the case of mammalian prion protein (PrP), the nontranslocated fraction is recruited into preexisting aggregates before its triage for degradation. This recruitment permits the growth and persistence of cytosolic PrP aggregates, explaining their apparent “self-conversion” seen in earlier studies of transient proteasome inhibition. For other proteins, the aggregate-mediated delay in precursor degradation led to aggregation and/or soluble residence in the cytosol, often causing aberrant cellular morphology. Remarkably, improving signal sequence efficiency mitigated these effects of aggregates. These observations identify a previously unappreciated consequence of cytosolic aggregates for nontranslocated secretory and membrane proteins, a minor but potentially disruptive population the rapid disposal of which is critical to maintaining cellular homeostasis.