A CRISPR toolbox for generating intersectional genetic mouse models for functional, molecular, and anatomical circuit mapping

In addition to single recombinase systems, the expression of two recombinases in distinct, but partially overlapping, populations allows for more defined target expression. Although the application of this method is becoming increasingly popular, its experimental implementation has been broadly restricted to manipulations of a limited set of common alleles that are often commercially produced at great expense, with costs and technical challenges associated with the production of intersectional mouse lines hindering customized approaches to many researchers Joblinks Human ADAM10 cDNA. Here, we present a simplified CRISPR toolkit for rapid, inexpensive, and facile intersectional allele production.
Results: Briefly, we produced 7 intersectional mouse lines using a dual recombinase system, one mouse line with a single recombinase system, and three embryonic stems (ES) cell lines that are designed to study the way functional, molecular, and anatomical features relate to each other in building circuits that underlie physiology and behavior.
As a proof-of-principle, we applied three of these lines to different neuronal populations for anatomical mapping and functional in vivo investigation of respiratory control.
We also generated a mouse line with a single recombinase-responsive allele that controls the expression of the calcium sensor Twitch-2B. This mouse line was applied globally to study the effects of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) on calcium release in the ovarian follicle.
Conclusions: The lines presented here are representative examples of outcomes possible with the successful application of our genetic toolkit for the facile development of diverse, modifiable animal models. This toolkit will allow labs to create single or dual recombinase effector lines easily for any cell population or subpopulation of interest when paired with the appropriate Cre and FLP recombinase mouse lines or viral vectors. We have made our tools and derivative intersectional mouse and ES cell lines openly available for non-commercial use through publicly curated repositories for plasmid DNA, ES cells, and transgenic mouse lines.

An enhanced method for nucleic acid detection with CRISPR-Cas12a using phosphorothioate modified primers and optimized gold-nanopaticle strip

CRISPR-Cas12a system has been shown promising for nucleic acid diagnostics due to its rapid, portable and accurate features. However, cleavage of the amplicons and primers by the cis– and trans-activity of Cas12a hinders the attempts to integrate the amplification and detection into a single reaction. Through phosphorothioate modification of primers, we realized onepot detection with high sensitivity using plasmids of SARS-CoV-2, HPV16 and HPV18. We also identified the activated Cas12a has a much higher affinity to C nucleotide-rich reporter than others.
By applying such reporters, the reaction time required for a lateral-flow readout was significantly reduced. Furthermore, to improve the specificity of the strip-based assay, we created a novel reporter and, when combined with a customized gold-nanopaticle strip, the readout was greatly enhanced owing to the elimination of the nonspecific signal.
This established system, termed Targeting DNA by Cas12a-based Eye Sight Testing in an One-pot Reaction (TESTOR), was validated using clinical cervical scrape samples for human papillomaviruses (HPVs) detection. Our system represents a general approach to integrating nucleic acid amplification and detection into a single reaction in CRISPR-Cas systems, highlighting its potential as a rapid, portable and accurate detection platform of nucleic acids.

Detection of plasmid contigs in draft genome assemblies using customized Kraken databases

Plasmids play an important role in bacterial evolution and mediate horizontal transfer of genes including virulence and antimicrobial resistance genes. Although short-read sequencing technologies have enabled large-scale bacterial genomics, the resulting draft genome assemblies are often fragmented into hundreds of discrete contigs. Several tools and approaches have been developed to identify plasmid sequences in such assemblies, but require trade-off between sensitivity and specificity. Here we propose using the Kraken classifier, together with a custom Kraken database comprising known chromosomal and plasmid sequences of Klebsiella pneumoniae species complex (KpSC), to identify plasmid-derived contigs in draft assemblies.
We assessed performance using Illumina-based draft genome assemblies for 82 KpSC isolates, for which complete genomes were available to supply ground truth. When benchmarked against five other classifiers (Centrifuge, RFPlasmid, mlplasmids, PlaScope and Platon), Kraken showed balanced performance in terms of overall sensitivity and specificity (90.8 and 99.4 %, respectively, for contig count; 96.5 and >99.9 %, respectively, for cumulative contig length), and the highest accuracy (96.8% vs 91.8-96.6% for contig count; 99.8% vs 99.0-99.7 % for cumulative contig length), and F1-score (94.5 % vs 84.5-94.1 %, for contig count; 98.0 % vs 88.9-96.7 % for cumulative contig length). Kraken also achieved consistent performance across our genome collection. Furthermore, we demonstrate that expanding the Kraken database with additional known chromosomal and plasmid sequences can further improve classification performance. Although we have focused here on the KpSC, this methodology could easily be applied to other species with a sufficient number of completed genomes.

Exploiting heterologous and endogenous CRISPR-Cas systems for genome editing in the probiotic Clostridium butyricum

Clostridium butyricum has been widely used as a probiotic for humans and food animals. However, the mechanisms of beneficial effects of C. butyricum on the host remain poorly understood, largely due to the lack of high-throughput genome engineering tools. Here, we report the exploitation of heterologous Type II CRISPR-Cas9 system and endogenous Type I-B CRISPR-Cas system in probiotic C. butyricum for seamless genome engineering. Although successful genome editing was achieved in C. butyricum when CRISPR-Cas9 system was employed, the expression of toxic cas9 gene result in really poor transformation, spurring us to develop an easy-applicable and high-efficient genome editing tool.
Therefore, the endogenous Type I-B CRISPR-Cas machinery located on the megaplasmid of C. butyricum was co-opted for genome editing. In vivo plasmid, interference assays identified that ACA and TAA were functional protospacer adjacent motif (PAM) sequences needed for site-specific CRISPR attacking. Using the customized endogenous CRISPR-Cas system, we successfully deleted spo0A and aldh genes in C. butyricum, yielding an efficiency of up to 100%.
Moreover, the conjugation efficiency of endogenous CRISPR-Cas system was dramatically enhanced due to the precluding expression of cas9. Altogether, the two approaches developed herein remarkably expand the existing genetic toolbox available for investigation of C. butyricum. This article is protected by copyright. All rights reserved.
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