Browse
Submit Data
Databases
API
Help

Dataset Information

10 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Enhanced Cas12a multi-gene regulation using a CRISPR array separator.

ABSTRACT: The type V-A Cas12a protein can process its CRISPR array, a feature useful for multiplexed gene editing and regulation. However, CRISPR arrays often exhibit unpredictable performance due to interference between multiple guide RNA (gRNAs). Here, we report that Cas12a array performance is hypersensitive to the GC content of gRNA spacers, as high-GC spacers can impair activity of the downstream gRNA. We analyze naturally occurring CRISPR arrays and observe that natural repeats always contain an AT-rich fragment that separates gRNAs, which we term a CRISPR separator. Inspired by this observation, we design short, AT-rich synthetic separators (synSeparators) that successfully remove the disruptive effects between gRNAs. We further demonstrate enhanced simultaneous activation of seven endogenous genes in human cells using an array containing the synSeparator. These results elucidate a previously underexplored feature of natural CRISPR arrays and demonstrate how nature-inspired engineering solutions can improve multi-gene control in mammalian cells.

SUBMITTER: Magnusson JP

PROVIDER: S-EPMC8478413 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

Next-generation CRISPR gene-drive systems using Cas12a nuclease.

Project description:One method for reducing the impact of vector-borne diseases is through the use of CRISPR-based gene drives, which manipulate insect populations due to their ability to rapidly propagate desired genetic traits into a target population. However, all current gene drives employ a Cas9 nuclease that is constitutively active, impeding our control over their propagation abilities and limiting the generation of alternative gene drive arrangements. Yet, other nucleases such as the temperature sensitive Cas12a have not been explored for gene drive designs in insects. To address this, we herein present a proof-of-concept gene-drive system driven by Cas12a that can be regulated via temperature modulation. Furthermore, we combined Cas9 and Cas12a to build double gene drives capable of simultaneously spreading two independent engineered alleles. The development of Cas12a-mediated gene drives provides an innovative option for designing next-generation vector control strategies to combat disease vectors and agricultural pests.

| S-EPMC10567717 | biostudies-literature

Development of Cas12a-Based Cell-Free Small-Molecule Biosensors via Allosteric Regulation of CRISPR Array Expression.

Project description:Cell-free biosensors can detect various molecules, thus promising to transform the landscape of diagnostics. Here, we developed a simple, rapid, sensitive, and field-deployable small-molecule detection platform based on allosteric transcription factor (aTF)-regulated expression of a clustered regularly interspaced short palindromic repeats (CRISPR) array coupled to Cas12a activity. To this end, we engineered an expression cassette harboring a T7 promoter, an aTF binding sequence, a Cas12a CRISPR array, and protospacer adjacent motif-flanked Cas12a target sequences. In the presence of the ligand, dissociation of the aTF allows transcription of the CRISPR array; this leads to activation of Cas12a collateral activity, which cleaves a single-stranded DNA linker to free a quenched fluorophore, resulting in a rapid, significant increase of fluorescence. As a proof of concept, we used TetR as the aTF to detect different tetracycline antibiotics with high sensitivity and specificity and a simple, hand-held visualizer to develop a fluorescence-based visual readout. We also adapted a mobile phone application to further simplify the interpretation of the results. Finally, we showed that the reagents could be lyophilized to facilitate storage and distribution. This detection platform represents a valuable addition to the toolbox of cell-free, CRISPR-based biosensors, with great potential for in-field deployment to detect non-nucleic acid small molecules.

| S-EPMC8943526 | biostudies-literature

Modeling pediatric AML FLT3 mutations using CRISPR/Cas12a- mediated gene editing.

Project description:Clustered regularly interspaced palindromic repeats (CRISPR) with the associated (Cas) nuclease complexes have democratized genetic engineering through their precision and ease-of-use. We have applied a variation of this technology, known as CRISPR-directed mutagenesis (CDM), to reconstruct genetic profiles within the FLT3 gene of AML patients. We took advantage of the versatility of CDM and built expression vectors that, in combination with a specifically designed donor DNA fragment, recapitulate simple and complex mutations within the FLT3 gene. We generate insertions and point mutations including combinations of these mutations originating from individual patient samples. We then analyze how these complex genetic profiles modulate transformation of Ba/F3 cells. Our results show that FLT3 expression plasmids bearing patient-specific single or multiple mutations recapitulate cellular transformation properties induced by FLT3 ITDs and modify their sensitivity or resistance in response to established AML drugs as a function of these complex mutations.

| S-EPMC8822598 | biostudies-literature

Gene targeting in adult organs using in vivo cleavable donor plasmids for CRISPR-Cas9 and CRISPR-Cas12a.

Project description:The CRISPR-Cas system for in vivo genome editing is a powerful tool for gene therapy against several diseases. We have previously developed the pCriMGET_9-12a system, an in vivo cleavable donor plasmid for precise targeted knock-in of exogenous DNA by both Cas9 and Cas12a. Here, we show that the pCriMGET_9-12a system can be applied for in vivo in-frame knock-in of exogenous DNA in adult mouse liver by hydrodynamic delivery of the targeting plasmids. The in vivo cleavable pCriMGET_9-12a donor plasmids significantly increased the knock-in efficiency of both CRISPR-Cas9 and CRISPR-Cas12a in the adult mouse liver compared to uncleavable donor plasmids. This strategy also achieved in-frame reporter gene knock-in without indel mutations. Therefore, in vivo gene targeting using the pCriMGET_9-12a system may contribute to the establishment of safer, more precise, versatile and efficient gene therapy methods in adult organs.

| S-EPMC10982285 | biostudies-literature

An enhanced CRISPR repressor for targeted mammalian gene regulation.

Project description:The RNA-guided endonuclease Cas9 can be converted into a programmable transcriptional repressor, but inefficiencies in target-gene silencing have limited its utility. Here we describe an improved Cas9 repressor based on the C-terminal fusion of a rationally designed bipartite repressor domain, KRAB-MeCP2, to nuclease-dead Cas9. We demonstrate the system's superiority in silencing coding and noncoding genes, simultaneously repressing a series of target genes, improving the results of single and dual guide RNA library screens, and enabling new architectures of synthetic genetic circuits.

| S-EPMC6129399 | biostudies-literature

CRISPR-Cas12a delivery by DNA-mediated bioresponsive editing for cholesterol regulation.

Project description:CRISPR-Cas12a represents an efficient tool for genome editing in addition to the extensively investigated CRISPR-Cas9. However, development of efficient nonviral delivery system for CRISPR-Cas12a remains challenging. Here, we demonstrate a DNA nanoclew (NC)-based carrier for delivery of Cas12a/CRISPR RNA (crRNA) ribonucleoprotein (RNP) toward regulating serum cholesterol levels. The DNA NC could efficiently load the Cas12a/crRNA RNP through complementation between the DNA NC and the crRNA. Addition of a cationic polymer layer condensed the DNA-templated core and allowed further coating of a charge reversal polymer layer, which makes the assembly negatively charged under a physiological pH but reverts to positive charge under an acidic environment. When Pcsk9 was selected as the target gene because of its important role in regulating the level of serum cholesterol, efficient Pcsk9 disruption was observed in vivo (~48%), significantly reducing the expression of PCSK9 and gaining the therapeutic benefit of cholesterol control (~45% of cholesterol reduction).

| S-EPMC7239642 | biostudies-literature

A Cas12a-based CRISPR interference system for multigene regulation in mycobacteria.

Project description:Mycobacteria are responsible for a heavy global disease burden, but their relative genetic intractability has long frustrated research efforts. The introduction of clustered regularly interspaced short palindromic repeats (CRISPR) interference (CRISPRi) has made gene repression in mycobacteria much more efficient, but limitations of the prototypical Cas9-based platform, for example, in multigene regulation, remain. Here, we introduce an alternative CRISPRi platform for mycobacteria that is based on the minimal type V Cas12a enzyme in combination with synthetic CRISPR arrays. This system is simple, tunable, reversible, can efficiently regulate essential genes and multiple genes simultaneously, and works as efficiently in infected macrophages as it does in vitro. Together, Cas12a-based CRISPRi provides a facile tool to probe higher-order genetic interactions in mycobacteria including Mycobacterium tuberculosis (Mtb), which will enable the development of synthetically lethal drug targets and the study of genes conditionally essential during infection.

| S-EPMC8363830 | biostudies-literature

Multiplex gene editing by CRISPR-Cpf1 using a single crRNA array.

Project description:Targeting of multiple genomic loci with Cas9 is limited by the need for multiple or large expression constructs. Here we show that the ability of Cpf1 to process its own CRISPR RNA (crRNA) can be used to simplify multiplexed genome editing. Using a single customized CRISPR array, we edit up to four genes in mammalian cells and three in the mouse brain, simultaneously.

| S-EPMC5225075 | biostudies-literature

Efficient Genome Editing in <i>Populus</i> Using CRISPR/Cas12a.

Project description:The ability to create targeted mutations using clustered regularly inter-spaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 in support of forest tree biotechnology is currently limited. CRISPR/Cas12a is a novel CRISPR effector protein that not only broadens the CRISPR/Cas targeting range but also enables the generation of large-fragment deletions. In this study, a CRISPR/Cas12a system was evaluated for the induction of targeted mutations in the woody tree poplar (Populus alba × Populus glandulosa). Three Cas12a nucleases, namely, AsCas12a (Acidaminococcus sp. BV3L6), LbCas12a (Lachnospiraceae bacterium ND2006), and FnCas12a (Francisella tularensis subsp. novicidain U112), were used. We knocked out multiple targets of the phytoene desaturase gene 8 (PDS) using the CRISPR/Cas12a genome-targeting system, and the results indicated that the AsCas12a system is the most efficient. We further optimized the co-cultivation temperature after Agrobacterium-mediated transformation from 22 to 28°C to increase the Cas12a nuclease editing efficiency in poplar. AsCas12a showed the highest mutation efficiency, at 70%, and the majority of editing sites were composed of large-fragment deletions. By using this simple and high-efficiency CRISPR/Cas12a system, multiple targets can be modified to obtain multigene simultaneous knockout mutants in tree species, which will provide powerful tools with which to facilitate genetic studies of forest trees.

| S-EPMC7720674 | biostudies-literature

Predicting CRISPR-Cas12a guide efficiency for targeting using machine learning.

Project description:Genome editing through the development of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat)-Cas technology has revolutionized many fields in biology. Beyond Cas9 nucleases, Cas12a (formerly Cpf1) has emerged as a promising alternative to Cas9 for editing AT-rich genomes. Despite the promises, guide RNA efficiency prediction through computational tools search still lacks accuracy. Through a computational meta-analysis, here we report that Cas12a target and off-target cleavage behavior are a factor of nucleotide bias combined with nucleotide mismatches relative to the protospacer adjacent motif (PAM) site. These features helped to train a Random Forest machine learning model to improve the accuracy by at least 15% over existing algorithms to predict guide RNA efficiency for the Cas12a enzyme. Despite the progresses, our report underscores the need for more representative datasets and further benchmarking to reliably and accurately predict guide RNA efficiency and off-target effects for Cas12a enzymes.

| S-EPMC10581463 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data