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Cas13 is a unique family of CRISPR endonucleases exhibiting programmable binding and cleavage of RNAs and is a strong candidate for eukaryotic RNA knockdown in the laboratory and the clinic. However, sequence-specific binding of Cas13 to the target RNA unleashes non-specific bystander RNA cleavage, or collateral activity, which may confound knockdown experiments and raises concerns for therapeutic applications. Although conserved across orthologs and robust in cell-free and bacterial environments, the extent of collateral activity in mammalian cells remains disputed. Here, we investigate Cas13d collateral activity in the context of an RNA-targeting therapy for myotonic dystrophy type 1, a disease caused by a transcribed long CTG repeat expansion. We find that when targeting CUGn RNA in HeLa and other cell lines, Cas13d depletes endogenous and transgenic RNAs, interferes with critical cellular processes, and activates stress response and apoptosis pathways. We also observe collateral effects when targeting other repetitive and unique transgenic sequences, and we provide evidence for collateral activity when targeting highly expressed endogenous transcripts. To minimize collateral activity for repeat-targeting Cas13d therapeutics, we introduce gRNA excision for negative-autoregulatory optimization (GENO), a simple strategy that leverages crRNA processing to control Cas13d expression and is easily integrated into an AAV gene therapy. We argue that thorough assessment of collateral activity is necessary when applying Cas13d in mammalian cells and that implementation of GENO illustrates the advantages of compact and universally robust regulatory systems for Cas-based gene therapies.

While the importance of RNA localization in highly differentiated cells is well appreciated, basic principles of RNA localization in skeletal muscle remain poorly characterized. Here, we develop a method to detect and quantify single molecule RNA localization patterns in skeletal myofibers, and uncover a critical role for directed transport of RNPs in muscle. We find that RNAs localize and are translated along sarcomere Z-disks, dispersing tens of microns from progenitor nuclei, regardless of encoded protein function. We find that directed transport along the lattice-like microtubule network of myofibers becomes essential to achieve this localization pattern as muscle development progresses; disruption of this network leads to extreme accumulation of RNPs and nascent protein around myonuclei. Our observations suggest that global active RNP transport may be required to distribute RNAs in highly differentiated cells and reveal fundamental mechanisms of gene regulation, with consequences for myopathies caused by perturbations to RNPs or microtubules., It is increasingly recognised that the spatial localisation of RNA is important for proper cellular function. Here, the authors investigate RNA localisation in skeletal muscle and develop methods to show that global active transport of RNA is required to maintain dispersion of gene products in the large muscle syncytium.

RNA-guided endonucleases such as Cas9 provide efficient on-target genome editing in cells but may also cleave at off-target loci throughout the genome. Engineered variants of Streptococcus pyogenes Cas9 (SpCas9) have been developed to globally reduce off-target activity, but individual off-targets may remain, or on-target activity may be compromised. In order to evolve against activity at specific off-targets while maintaining strong on-target editing, we developed a novel M13 bacteriophage-mediated selection method. Using this method, sequential rounds of positive and negative selection are used to identify mutations to Cas9 that enhance or diminish editing activity at particular genomic sequences. We also introduce scanning mutagenesis of oligo-directed targets (SMOOT), a comprehensive mutagenesis method to create highly diverse libraries of Cas9 variants that can be challenged with phage-based selection. Our platform identifies novel SpCas9 mutants which mitigate cleavage against off-targets both in biochemical assays and in T-cells while maintaining higher on-target activity than previously described variants. We describe an evolved variant, S. pyogenes Adapted to Reduce Target Ambiguity Cas9 (SpartaCas), composed of the most enriched mutations, each of unknown function. This evolved Cas9 mutant reduces off-target cleavage while preserving efficient editing at multiple therapeutically relevant targets. Directed evolution of Cas9 using our system demonstrates an improved structure-independent methodology to effectively engineer nuclease activity.

Encapsulating bacteria within constrained microenvironments can promote the manifestation of specialized behaviors. Using double-emulsion droplet-generating microfluidic synthesis, live Bacillus subtilis bacteria were encapsulated in a semi-permeable membrane composed of poly(ethylene glycol)-b-poly(D,L-lactic acid) (mPEG-PDLLA). This polymer membrane was sufficiently permeable to permit exponential bacterial growth, metabolite-induced gene expression, and rapid biofilm growth. The biodegradable microparticles retained structural integrity for several days and could be successfully degraded with time or sustained bacterial activity. Microencapsulated B. subtilis successfully captured and contained sodium selenite added outside the polymersomes, converting the selenite into elemental selenium nanoparticles that were selectively retained inside the polymer membrane. This remediation of selenium using polymersomes has high potential for reducing the toxicity of environmental selenium contamination, as well as allowing selenium to be harvested from areas not amenable to conventional waste or water treatment.