Good guide, bad guide:spacer sequence-dependent cleavage efficiency of Cas12a

Genome editing has recently made a revolutionary development with the introduction of the CRISPR–Cas technology. The programmable CRISPR-associated Cas9 and Cas12a nucleases generate specific dsDNA breaks in the genome, after which host DNA-repair mechanisms can be manipulated to implement the desired editing. Despite this spectacular progress, the efficiency of Cas9/Cas12a-based engineering can still be improved. Here, we address the variation in guide-dependent efficiency of Cas12a, and set out to reveal the molecular basis of this phenomenon. We established a sensitive and robust in vivo targeting assay based on loss of a target plasmid encoding the red fluorescent protein (mRFP). Our results suggest that folding of both the precursor guide (pre-crRNA) and the mature guide (crRNA) have a major influence on Cas12a activity. Especially, base pairing of the direct repeat, other than with itself, was found to be detrimental to the activity of Cas12a. Furthermore, we describe different approaches to minimize base-pairing interactions between the direct repeat and the variable part of the guide. We show that design of the 3′ end of the guide, which is not involved in target strand base pairing, may result in substantial improvement of the guide's targeting potential and hence of its genome editing efficiency

Structure-function relations of RNA molecules involved in gene expression and host defence

This PhD thesis describes the relation of the structure of RNA with its effect on protein expression and the efficacy of CRISPR-Cas12a. Direct control of protein expression by RNA is done via so-called riboswitches, which change their structure depending on the presence of a ligand or external factors like temperature. A theophylline-responsive riboswitch based on the phage T4 td group I intron is described in detail and a method is proposed to obtain riboswitches based on this intron. This method was used to generate a riboswitch library for 3-methylxanthine, a compound closely related to theophylline, and the unrelated citrulline. The second part focuses on the effect of coding and non-coding mRNA on gene translation. Coding regions that are in close proximity of other coding regions have their translation coupled to each other, and the order of the genes appears to be of surprisingly little influence. The next part investigates the effect of the 3’-UTR and the intergenic region. These non-coding elements may have a lot of influence on the translation rate, by a mechanism that is not yet understood. The last part shines a light on the influence of the RNA structure of the CRISPR-Cas12a crRNA. The pre-crRNA forms a characteristic pseudoknot structure that is required for the recognition by Cas12a, which then turns it into crRNA. The low GC content of the pseudoknot allows this structure to be outcompeted by other potential structures, which renders the pre-crRNA unrecognisable. A design strategy was proposed to improve the efficacy of CRISPR-Cas12a

Medium-throughput in vitro detection of DNA cleavage by CRISPR-Cas12a

Quantifying DNA cleavage by CRISPR-Cas nucleases is usually done by separating the cleaved products from the non-cleaved target by agarose gel electrophoresis. We devised a method that eliminates the quantification from band intensity on agarose gel, and uses a target with a fluorescent dye on the one end and a biotin on the other. Cleavage of the target will separate the dye from the biotin, and cause the dye to stay in solution when streptavidin beads are introduced. All non-cleaved target will be eliminated from solution and no longer contribute to detectable fluorescence. Cleavage will therefore increase the fluorescent signal. A control, which has no streptavidin treatment, is taken along to correct for any errors that might have been introduced by pipetting, inactivation of the fluorescent dye or release of the biotin during several steps of the procedure. With this method we were able to quantify the fraction of active Cas12a in a purification sample and assess the cleavage rate

Medium-throughput in vitro detection of DNA cleavage by CRISPR-Cas12a

Quantifying DNA cleavage by CRISPR-Cas nucleases is usually done by separating the cleaved products from the non-cleaved target by agarose gel electrophoresis. We devised a method that eliminates the quantification from band intensity on agarose gel, and uses a target with a fluorescent dye on the one end and a biotin on the other. Cleavage of the target will separate the dye from the biotin, and cause the dye to stay in solution when streptavidin beads are introduced. All non-cleaved target will be eliminated from solution and no longer contribute to detectable fluorescence. Cleavage will therefore increase the fluorescent signal. A control, which has no streptavidin treatment, is taken along to correct for any errors that might have been introduced by pipetting, inactivation of the fluorescent dye or release of the biotin during several steps of the procedure. With this method we were able to quantify the fraction of active Cas12a in a purification sample and assess the cleavage rate.</p

CRISPR type V-U1 system from Mycobacterium mucogenicum and uses thereof

The type V-U1 system from the bacterium Mycobacterium mucogenicum CCH10-A2 (Mmu) has a nuclease which binds dsDNA but it does not cleave it. Additionally, after dsDNA binding by the nuclease an RuvC-dependent interference of nascent transcript (mRNA) takes place and this mechanism has not been described before for any CRISPR system. CRISPR based gene manipulation can therefore use CRISPR endonucleases from the Type V-U1 system from Mycobacterium mucogenicum, including variant and modified endonucleases, so as to provide for methods of expression control and gene editing in cells of any living organism or of any nucleic acid in vitro.</p

Universal riboswitch for inducible gene expression

Aspects described herein relate to methods for controlling expression of RNA and polypeptides of interest using a tuneable self-splicing intron. Specifically, there is provided modified 5' and 3' exons of the T4 td intron which function as a tuneable self-splicing intron that can be introduced to any gene of interest to multiple spots in the open reading frame therefore allowing the intron to be inserted without changing the amino acid sequence of the protein of interest. Methods and a system for inducer controlled modification of a target genomic locus in a cell are also provided herein. The invention further provides kits for expressing an RNA of interest or a polypeptide of interest, and wherein the expression is in transformed host cells under the control of an inducer molecule

A growth- and bioluminescence-based bioreporter for the in vivo detection of novel biocatalysts

The use of bioreporters in high-throughput screening for small molecules is generally laborious and/or expensive. The technology can be simplified by coupling the generation of a desired compound to cell survival, causing only positive cells to stay in the pool of generated variants. Here, a dual selection/screening system was developed for the in vivo detection of novel biocatalysts. The sensor part of the system is based on the transcriptional regulator AraC, which controls expression of both a selection reporter (LeuB or KmR; enabling growth) for rapid reduction of the initially large library size and a screening reporter (LuxCDABE; causing bioluminescence) for further quantification of the positive variants. Of four developed systems, the best system was the medium copy system with KmR as selection reporter. As a proof of principle, the system was tested for the selection of cells expressing an l-arabinose isomerase derived from mesophilic Escherichia coli or thermophilic Geobacillus thermodenitrificans. A more than a millionfold enrichment of cells with l-arabinose isomerase activity was demonstrated by selection and exclusion of false positives by screening. This dual selection/screening system is an important step towards an improved detection method for small molecules, and thereby for finding novel biocatalysts

Improving heterologous membrane protein production in Escherichia coli by combining transcriptional tuning and codon usage algorithms.

High-level, recombinant production of membrane-integrated proteins in Escherichia coli is extremely relevant for many purposes, but has also been proven challenging. Here we study a combination of transcriptional fine-tuning in E. coli LEMO21(DE3) with different codon usage algorithms for heterologous production of membrane proteins. The overex- pression of 6 different membrane proteins is compared for the wild-type gene codon usage variant, a commercially codon-optimized variant, and a codon-harmonized variant. We show that transcriptional fine-tuning plays a major role in improving the production of all tested proteins. Moreover, different codon usage variants significantly improved production of some of the tested proteins. However, not a single algorithm performed consistently best for the membrane-integrated production of the 6 tested proteins. In conclusion, for improving heterologous membrane protein production in E. coli, the major effect is accomplished by transcriptional tuning. In addition, further improvements may be realized by attempting dif- ferent codon usage variants, such as codon harmonized variants, which can now be easily generated through our online Codon Harmonizer tool

Improving heterologous membrane protein production in Escherichia coli by combining transcriptional tuning and codon usage algorithms

High-level, recombinant production of membrane-integrated proteins in Escherichia coli is extremely relevant for many purposes, but has also been proven challenging. Here we study a combination of transcriptional fine-tuning in E. coli LEMO21(DE3) with different codon usage algorithms for heterologous production of membrane proteins. The overexpression of 6 different membrane proteins is compared for the wild-type gene codon usage variant, a commercially codon-optimized variant, and a codon-harmonized variant. We show that transcriptional fine-tuning plays a major role in improving the production of all tested proteins. Moreover, different codon usage variants significantly improved production of some of the tested proteins. However, not a single algorithm performed consistently best for the membrane-integrated production of the 6 tested proteins. In conclusion, for improving heterologous membrane protein production in E. coli, the major effect is accomplished by transcriptional tuning. In addition, further improvements may be realized by attempting different codon usage variants, such as codon harmonized variants, which can now be easily generated through our online Codon Harmonizer tool

Streamlined CRISPR genome engineering in wild-type bacteria using SIBR-Cas

CRISPR-Cas is a powerful tool for genome editing in bacteria. However, its efficacy is dependent on host factors (such as DNA repair pathways) and/or exogenous expression of recombinases. In this study, we mitigated these constraints by developing a simple and widely applicable genome engineering tool for bacteria which we termed SIBR-Cas (Self-splicing Intron-Based Riboswitch-Cas). SIBR-Cas was generated from a mutant library of the theophylline-dependent self-splicing T4 td intron that allows for tight and inducible control over CRISPR-Cas counter-selection. This control delays CRISPR-Cas counter-selection, granting more time for the editing event (e.g. by homologous recombination) to occur. Without the use of exogenous recombinases, SIBR-Cas was successfully applied to knock-out several genes in three wild-type bacteria species (Escherichia coli MG1655, Pseudomonas putida KT2440 and Flavobacterium IR1) with poor homologous recombination systems. Compared to other genome engineering tools, SIBR-Cas is simple, tightly regulated and widely applicable for most (non-model) bacteria. Furthermore, we propose that SIBR can have a wider application as a simple gene expression and gene regulation control mechanism for any gene or RNA of interest in bacteria