The advent of facile genome engineering using the bacterial RNA-guided CRISPR-Cas9 system in animals and plants is transforming biology. We review the history of CRISPR (clustered regularly interspaced palindromic repeat) biology from its initial discovery through the elucidation of the CRISPR-Cas9 enzyme mechanism, which has set the stage for remarkable developments using this technology to modify, regulate, or mark genomic loci in a wide variety of cells and organisms from all three domains of life. These results highlight a new era in which genomic manipulation is no longer a bottleneck to experiments, paving the way toward fundamental discoveries in biology, with applications in all branches of biotechnology, as well as strategies for human therapeutics.

The new frontier of genome engineering with CRISPR-Cas9

The efficacy and the mutation spectrum of genome editing methods can vary substantially depending on the targeted sequence. A simple, quick assay to accurately characterize and quantify the induced mutations is therefore needed. Here we present TIDE, a method for this purpose that requires only a pair of PCR reactions and two standard capillary sequencing runs. The sequence traces are then analyzed by a specially developed decomposition algorithm that identifies the major induced mutations in the projected editing site and accurately determines their frequency in a cell population. This method is cost-effective and quick, and it provides much more detailed information than current enzyme-based assays. An interactive web tool for automated decomposition of the sequence traces is available. TIDE greatly facilitates the testing and rational design of genome editing strategies.

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Easy quantitative assessment of genome editing by sequence trace decomposition

The success of the CRISPR/Cas9 genome editing technique depends on the choice of the guide RNA sequence, which is facilitated by various websites. Despite the importance and popularity of these algorithms, it is unclear to which extent their predictions are in agreement with actual measurements. We conduct the first independent evaluation of CRISPR/Cas9 predictions. To this end, we collect data from eight SpCas9 off-target studies and compare them with the sites predicted by popular algorithms. We identify problems in one implementation but found that sequence-based off-target predictions are very reliable, identifying most off-targets with mutation rates superior to 0.1 %, while the number of false positives can be largely reduced with a cutoff on the off-target score. We also evaluate on-target efficiency prediction algorithms against available datasets. The correlation between the predictions and the guide activity varied considerably, especially for zebrafish. Together with novel data from our labs, we find that the optimal on-target efficiency prediction model strongly depends on whether the guide RNA is expressed from a U6 promoter or transcribed in vitro. We further demonstrate that the best predictions can significantly reduce the time spent on guide screening. To make these guidelines easily accessible to anyone planning a CRISPR genome editing experiment, we built a new website (
 http://crispor.org
 ) that predicts off-targets and helps select and clone efficient guide sequences for more than 120 genomes using different Cas9 proteins and the eight efficiency scoring systems evaluated here.

https://hal.archives-ouvertes.fr/hal-01346049/document

Evaluation of off-target and on-target scoring algorithms and integration into the guide RNA selection tool CRISPOR.

CRISPR-Cas9 technology provides a powerful system for genome engineering. However, variable activity across different single guide RNAs (sgRNAs) remains a significant limitation. We analyzed the molecular features that influence sgRNA stability, activity and loading into Cas9 in vivo. We observed that guanine enrichment and adenine depletion increased sgRNA stability and activity, whereas differential sgRNA loading, nucleosome positioning and Cas9 off-target binding were not major determinants. We also identified sgRNAs truncated by one or two nucleotides and containing 5' mismatches as efficient alternatives to canonical sgRNAs. On the basis of these results, we created a predictive sgRNA-scoring algorithm, CRISPRscan, that effectively captures the sequence features affecting the activity of CRISPR-Cas9 in vivo. Finally, we show that targeting Cas9 to the germ line using a Cas9-nanos 3' UTR led to the generation of maternal-zygotic mutants, as well as increased viability and decreased somatic mutations. These results identify determinants that influence Cas9 activity and provide a framework for the design of highly efficient sgRNAs for genome targeting in vivo.

CRISPRscan: designing highly efficient sgRNAs for CRISPR-Cas9 targeting in vivo

CRISPOR.org is a web tool for genome editing experiments with the CRISPR-Cas9 system. It finds guide RNAs in an input sequence and ranks them according to different scores that evaluate potential off-targets in the genome of interest and predict on-target activity. The list of genomes is continuously expanded, with more 150 genomes added in the last two years. CRISPOR tries to provide a comprehensive solution from selection, cloning and expression of guide RNA as well as providing primers needed for testing guide activity and potential off-targets. Recent developments include batch design for genome-wide CRISPR and saturation screens, creating custom oligonucleotides for guide cloning and the design of next generation sequencing primers to test for off-target mutations. CRISPOR is available from http://crispor.org, including the full source code of the website and a stand-alone, command-line version.

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CRISPOR: intuitive guide selection for CRISPR/Cas9 genome editing experiments and screens.

Major advances in genome editing have recently been made possible with the development of the TALEN and CRISPR/Cas9 methods. The speed and ease of implementing these technologies has led to an explosion of mutant and transgenic organisms. A rate-limiting step in efficiently applying TALEN and CRISPR/Cas9 methods is the selection and design of targeting constructs. We have developed an online tool, CHOPCHOP (https://chopchop.rc.fas.harvard.edu), to expedite the design process. CHOPCHOP accepts a wide range of inputs (gene identifiers, genomic regions or pasted sequences) and provides an array of advanced options for target selection. It uses efficient sequence alignment algorithms to minimize search times, and rigorously predicts off-target binding of single-guide RNAs (sgRNAs) and TALENs. Each query produces an interactive visualization of the gene with candidate target sites displayed at their genomic positions and color-coded according to quality scores. In addition, for each possible target site, restriction sites and primer candidates are visualized, facilitating a streamlined pipeline of mutant generation and validation. The ease-of-use and speed of CHOPCHOP make it a valuable tool for genome engineering.

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CHOPCHOP: a CRISPR/Cas9 and TALEN web tool for genome editing

We report various modes of synchrony observed for a population of two, three and four pentanol drops in a rectangular channel at the air-water interface. Initially, the autonomous oscillations of a single 1-pentanol drop were studied in a ferroin DI water solution pre-mixed with some volume of pentanol. A pentanol drop performs continuous motion on the air-water interface due to Marangoni forces. A linear channel was prepared to study the uniaxial movement of the drop(s). Thereafter, a systematic study of the self-propelled motion of a 1-pentanol drop was reported as a function of the drop volume. Subsequently, the coupled dynamics were studied for two, three and four drops, respectively. We observed anti-phase oscillations in a pair of pentanol drops. In the case of three drops, relay synchronization was observed, wherein consecutive pairs of drops were exhibiting out-of-phase oscillations and alternate drops were performing in-phase oscillations. Four pentanol drops showed two different modes of synchrony: one was relay synchrony and the other was out-of-phase oscillations between two pairs of drops (within a pair, the drops exhibit in-phase oscillations).

Modes of synchrony in self-propelled pentanol drops.

Attrition-enhanced chiral symmetry breaking in crystals, known as Viedma deracemization, is a promising method for converting racemic solid phases into enantiomerically pure ones under non equilibrium conditions. However, many aspects of this process remain unclear. In this study, we present a new investigation into Viedma deracemization using a comprehensive kinetic rate equation continuous model based on classical primary nucleation theory, crystal growth, and Ostwald ripening. Our approach employs a fully micro-reversible kinetic scheme with a size-dependent solubility following the Gibbs-Thomson rule. To validate our model, we use data from a real NaClO3 deracemization experiment. After parametrization, the model shows spontaneous mirror symmetry breaking (SMSB) under grinding. Additionally, we identify a bifurcation scenario with a lower and upper limit of the grinding intensity that leads to deracemization, including a minimum deracemization time within this window. Furthermore, this model uncovers that SMSB is caused by multiple instances of concealed high-order autocatalysis. Our findings provide new insights into attrition-enhanced deracemization and its potential applications in chiral molecule synthesis and understanding biological homochirality.

A Complex Reaction Network Model for Spontaneous Mirror Symmetry Breaking in Viedma Deracemizations.

Dengue is a viral infection transmitted mainly by the Aedes aegypti mosquito and to a lesser extent by the Aedes albopictus. This infectious disease generally causes flu-like symptoms, but it can also lead to life-threatening symptoms. Unfortunately, the number of cases increases every year and about a third part of the world’s population is at risk of contracting this disease. To generate tools capable of containing dengue transmission, we present a novel stochastic model for the Aedes aegypti life cycle and the dengue virus transmission, taking into account all the mechanisms of transmission and parameters estimated experimentally to date. This new model describes in detail all the interactions in the stages of the life cycle of the mosquito. It also considers the environmental conditions, i.e., the breeding sites and the temperature, which are very important factors for the mosquito survival. The results show that the contagion by bite only does not provoke an epidemic outbreak when five infected, pregnant, and fed females, looking for lay eggs, arrive to a susceptible human population. However, if in addition to the bite transmission, the virus is also transmitted in vertical transmission and sexual ways, then an outbreak arises. Altogether, the transmission mechanisms and the adequate environmental conditions could explain the virus persistence in the population. Under these conditions and by considering fumigation as a way to control the mosquito population, in this new model the outbreak and the virus propagation could be avoided—but only if the control is implemented within the first two weeks of the presence of the virus.

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José M. Cruz

Papers

CHOPCHOP: a CRISPR/Cas9 and TALEN web tool for genome editing

Modes of synchrony in self-propelled pentanol drops.

A Complex Reaction Network Model for Spontaneous Mirror Symmetry Breaking in Viedma Deracemizations.

A New Stochastic Model for the Aedes aegypti Life Cycle and the Dengue Virus Transmission