The introduction of insertion-deletions (INDELs) by non-homologous end-joining (NHEJ) pathway underlies the mechanistic basis of CRISPR-Cas9-directed genome editing. Selective gene ablation using CRISPR-Cas9 is achieved by installation of a premature termination codon (PTC) from a frameshift-inducing INDEL that elicits nonsense-mediated decay (NMD) of the mutant mRNA. Here, by examining the mRNA and protein products of CRISPR targeted genes in a cell line panel with presumed gene knockouts, we detect the production of foreign mRNAs or proteins in ~50% of the cell lines. We demonstrate that these aberrant protein products stem from the introduction of INDELs that promote internal ribosomal entry, convert pseudo-mRNAs (alternatively spliced mRNAs with a PTC) into protein encoding molecules, or induce exon skipping by disruption of exon splicing enhancers (ESEs). Our results reveal challenges to manipulating gene expression outcomes using INDEL-based mutagenesis and strategies useful in mitigating their impact on intended genome-editing outcomes.

This paper presents TurboFold II, an extension of the TurboFold algorithm for predicting secondary structures for multiple RNA homologs. TurboFold II augments the structure prediction capabilities of TurboFold by additionally providing multiple sequence alignments. Probabilities for alignment of nucleotide positions between all pairs of input sequences are iteratively estimated in TurboFold II by incorporating information from both the sequence identity and secondary structures. A multiple sequence alignment is obtained from these probabilities by using a probabilistic consistency transformation and a hierarchically computed guide tree. To assess TurboFold II, its sequence alignment and structure predictions were compared with leading tools, including methods that focus on alignment alone and methods that provide both alignment and structure prediction. TurboFold II has comparable alignment accuracy with MAFFT and higher accuracy than other tools. TurboFold II also has comparable structure prediction accuracy as the original TurboFold algorithm, which is one of the most accurate methods. TurboFold II is part of the RNAstructure software package, which is freely available for download at http://rna.urmc.rochester.edu under a GPL license.

A generic sensing strategy that integrates CRISPR-Cas12a with a DNA circuit is proposed for amplified detection of microRNA.

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