Codon usage and expression-based features significantly improve prediction of CRISPR efficiency.

CRISPR is a precise and effective genome editing technology; but despite several advancements during the last decade, our ability to computationally design gRNAs remains limited. Most predictive models have relatively low predictive power and utilize only the sequence of the target site as input. Here we suggest a new category of features, which incorporate the target site genomic position and the presence of genes close to it. We calculate four features based on gene expression and codon usage bias indices. We show, on CRISPR datasets taken from 3 different cell types, that such features perform comparably with 425 state-of-the-art predictive features, ranking in the top 2-12% of features. We trained new predictive models, showing that adding expression features to them significantly improves their r2 by up to 0.04 (relative increase of 39%), achieving average correlations of up to 0.38 on their validation sets; and that these features are deemed important by different feature importance metrics. We believe that incorporating the target site's position, in addition to its sequence, in features such as we have generated here will improve our ability to predict, design and understand CRISPR experiments going forward.

Strong association between genomic 3D structure and CRISPR cleavage efficiency.

CRISPR is a gene editing technology which enables precise in-vivo genome editing; but its potential is hampered by its relatively low specificity and sensitivity. Improving CRISPR's on-target and off-target effects requires a better understanding of its mechanism and determinants. Here we demonstrate, for the first time, the chromosomal 3D spatial structure's association with CRISPR's cleavage efficiency, and its predictive capabilities. We used high-resolution Hi-C data to estimate the 3D distance between different regions in the human genome and utilized these spatial properties to generate 3D-based features, characterizing each region's density. We evaluated these features based on empirical, in-vivo CRISPR efficiency data and compared them to 425 features used in state-of-the-art models. The 3D features ranked in the top 13% of the features, and significantly improved the predictive power of LASSO and xgboost models trained with these features. The features indicated that sites with lower spatial density demonstrated higher efficiency. Understanding how CRISPR is affected by the 3D DNA structure provides insight into CRISPR's mechanism in general and improves our ability to correctly predict CRISPR's cleavage as well as design sgRNAs for therapeutic and scientific use.

The Causes for Genomic Instability and How to Try and Reduce Them Through Rational Design of Synthetic DNA.

Genetic engineering has revolutionized our ability to manipulate DNA and engineer organisms for various applications. However, this approach can lead to genomic instability, which can result in unwanted effects such as toxicity, mutagenesis, and reduced productivity. To overcome these challenges, smart design of synthetic DNA has emerged as a promising solution. By taking into consideration the intricate relationships between gene expression and cellular metabolism, researchers can design synthetic constructs that minimize metabolic stress on the host cell, reduce mutagenesis, and increase protein yield. In this chapter, we summarize the main challenges of genomic instability in genetic engineering and address the dangers of unknowingly incorporating genomically unstable sequences in synthetic DNA. We also demonstrate the instability of those sequences by the fact that they are selected against conserved sequences in nature. We highlight the benefits of using ESO, a tool for the rational design of DNA for avoiding genetically unstable sequences, and also summarize the main principles and working parameters of the software that allow maximizing its benefits and impact.

Fighting the battle against evolution: designing genetically modified organisms for evolutionary stability.

Synthetic biology has made significant progress in many areas, but a major challenge that has received limited attention is the evolutionary stability of synthetic constructs made of heterologous genes. The expression of these constructs in microorganisms, that is, production of proteins that are not necessary for the organism, is a metabolic burden, leading to a decrease in relative fitness and make the synthetic constructs unstable over time. This is a significant concern for the synthetic biology community, particularly when it comes to bringing this technology out of the laboratory. In this review, we discuss the issue of evolutionary stability in synthetic biology and review the available tools to address this challenge.

Evolutionary Stability Optimizer (ESO): A Novel Approach to Identify and Avoid Mutational Hotspots in DNA Sequences While Maintaining High Expression Levels.

Modern synthetic biology procedures rely on the ability to generate stable genetic constructs that keep their functionality over long periods of time. However, maintenance of these constructs requires energy from the cell and thus reduces the host's fitness. Natural selection results in loss-of-functionality mutations that negate the expression of the construct in the population. Current approaches for the prevention of this phenomenon focus on either small-scale, manual design of evolutionary stable constructs or the detection of mutational sites with unstable tendencies. We designed the Evolutionary Stability Optimizer (ESO), a software tool that enables the large-scale automatic design of evolutionarily stable constructs with respect to both mutational and epigenetic hotspots and allows users to define custom hotspots to avoid. Furthermore, our tool takes the expression of the input constructs into account by considering the guanine-cytosine (GC) content and codon usage of the host organism, balancing the trade-off between stability and gene expression, allowing to increase evolutionary stability while maintaining the high expression. In this study, we present the many features of the ESO and show that it accurately predicts the evolutionary stability of endogenous genes. The ESO was created as an easy-to-use, flexible platform based on the notion that directed genetic stability research will continue to evolve and revolutionize current applications of synthetic biology. The ESO is available at the following link: https://www.cs.tau.ac.il/~tamirtul/ESO/.

New computational model for miRNA-mediated repression reveals novel regulatory roles of miRNA bindings inside the coding region.

MOTIVATION: MicroRNAs (miRNAs) are short (∼24nt), non-coding RNAs, which downregulate gene expression in many species and physiological processes. Many details regarding the mechanism which governs miRNA-mediated repression continue to elude researchers. RESULTS: We elucidate the interplay between the coding sequence and the 3'UTR, by using elastic net regularization and incorporating translation-related features to predict miRNA-mediated repression. We find that miRNA binding sites at the end of the coding sequence contribute to repression, and that weak binding sites are linked to effective de-repression, possibly as a result of competing with stronger binding sites. Furthermore, we propose a recycling model for miRNAs dissociated from the open reading frame (ORF) by traversing ribosomes, explaining the observed link between increased ribosome density/traversal speed and increased repression. We uncover a novel layer of interaction between the coding sequence and the 3'UTR (untranslated region) and suggest the ORF has a larger role than previously thought in the mechanism of miRNA-mediated repression. AVAILABILITY AND IMPLEMENTATION: The code is freely available at https://github.com/aescrdni/miRNA_model. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Widespread non-modular overlapping codes in the coding regions.

Messenger RNAs (mRNAs) consist of a coding region (open reading frame (ORF)) and two untranslated regions (UTRs), 5'UTR and 3'UTR. Ribosomes travel along the coding region, translating nucleotide triplets (called codons) to a chain of amino acids. The coding region was long believed to mainly encode the amino acid content of proteins, whereas regulatory signals reside in the UTRs and in other genomic regions. However, in recent years we have learned that the ORF is expansively populated with various regulatory signals, or codes, which are related to all gene expression steps and additional intracellular aspects. In this paper, we review the current knowledge related to overlapping codes inside the coding regions, such as the influence of synonymous codon usage on translation speed (and, in turn, the effect of translation speed on protein folding), ribosomal frameshifting, mRNA stability, methylation, splicing, transcription and more. All these codes come together and overlap in the ORF sequence, ensuring production of the right protein at the right time.