Browse
Submit Data
Databases
API
Help

Dataset Information

29 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Versatile and robust genome editing with Streptococcus thermophilus CRISPR1-Cas9.

ABSTRACT: Targeting definite genomic locations using CRISPR-Cas systems requires a set of enzymes with unique protospacer adjacent motif (PAM) compatibilities. To expand this repertoire, we engineered nucleases, cytosine base editors, and adenine base editors from the archetypal Streptococcus thermophilus CRISPR1-Cas9 (St1Cas9) system. We found that St1Cas9 strain variants enable targeting to five distinct A-rich PAMs and provide a structural basis for their specificities. The small size of this ortholog enables expression of the holoenzyme from a single adeno-associated viral vector for in vivo editing applications. Delivery of St1Cas9 to the neonatal liver efficiently rewired metabolic pathways, leading to phenotypic rescue in a mouse model of hereditary tyrosinemia. These robust enzymes expand and complement current editing platforms available for tailoring mammalian genomes.

SUBMITTER: Agudelo D

PROVIDER: S-EPMC6961573 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

Efficient genome editing in pathogenic mycobacteria using Streptococcus thermophilus CRISPR1-Cas9.

Project description:The ability to genetically engineer pathogenic mycobacteria has increased significantly over the last decades due to the generation of new molecular tools. Recently, the application of the Streptococcus pyogenes and the Streptococcus thermophilus CRISPR-Cas9 systems in mycobacteria has enabled gene editing and efficient CRISPR interference-mediated transcriptional regulation. Here, we converted CRISPR interference into an efficient genome editing tool for mycobacteria. We demonstrate that the Streptococcus thermophilus CRISPR1-Cas9 (Sth1Cas9) is functional in Mycobacterium marinum and Mycobacterium tuberculosis, enabling highly efficient and precise DNA breaks and indel formation, without any off-target effects. In addition, with dual sgRNAs this system can be used to generate two indels simultaneously or to create specific deletions. The ability to use the power of the CRISPR-Cas9-mediated gene editing toolbox in M. tuberculosis with a single step will accelerate research into this deadly pathogen.

| S-EPMC7612230 | biostudies-literature

Streptococcus thermophilus CRISPR-Cas9 Systems Enable Specific Editing of the Human Genome.

Project description:RNA-guided nucleases (RGNs) based on the type II CRISPR-Cas9 system of Streptococcus pyogenes (Sp) have been widely used for genome editing in experimental models. However, the nontrivial level of off-target activity reported in several human cells may hamper clinical translation. RGN specificity depends on both the guide RNA (gRNA) and the protospacer adjacent motif (PAM) recognized by the Cas9 protein. We hypothesized that more stringent PAM requirements reduce the occurrence of off-target mutagenesis. To test this postulation, we generated RGNs based on two Streptococcus thermophilus (St) Cas9 proteins, which recognize longer PAMs, and performed a side-by-side comparison of the three RGN systems targeted to matching sites in two endogenous human loci, PRKDC and CARD11. Our results demonstrate that in samples with comparable on-target cleavage activities, significantly lower off-target mutagenesis was detected using St-based RGNs as compared to the standard Sp-RGNs. Moreover, similarly to SpCas9, the StCas9 proteins accepted truncated gRNAs, suggesting that the specificities of St-based RGNs can be further improved. In conclusion, our results show that Cas9 proteins with longer or more restrictive PAM requirements provide a safe alternative to SpCas9-based RGNs and hence a valuable option for future human gene therapy applications.

| S-EPMC4786917 | biostudies-literature

Orthogonal Cas9-Cas9 chimeras provide a versatile platform for genome editing.

Project description:The development of robust, versatile and accurate toolsets is critical to facilitate therapeutic genome editing applications. Here we establish RNA-programmable Cas9-Cas9 chimeras, in single- and dual-nuclease formats, as versatile genome engineering systems. In both of these formats, Cas9-Cas9 fusions display an expanded targeting repertoire and achieve highly specific genome editing. Dual-nuclease Cas9-Cas9 chimeras have distinct advantages over monomeric Cas9s including higher target site activity and the generation of predictable precise deletion products between their target sites. At a therapeutically relevant site within the BCL11A erythroid enhancer, Cas9-Cas9 nucleases produced precise deletions that comprised up to 97% of all sequence alterations. Thus Cas9-Cas9 chimeras represent an important tool that could be particularly valuable for therapeutic genome editing applications where a precise cleavage position and defined sequence end products are desirable.

| S-EPMC6242970 | biostudies-literature

Versatile and efficient genome editing with Neisseria cinerea Cas9.

Project description:The CRISPR/Cas9 system is a versatile genome editing platform in biotechnology and therapeutics. However, the requirement of protospacer adjacent motifs (PAMs) limits the genome targeting scope. To expand this repertoire, we revisited and engineered a compact Cas9 orthologue derived from Neisseria cinerea (NcCas9) for efficient genome editing in mammal cells. We demonstrated that NcCas9 generates genome editing at target sites with N4GYAT (Y = T/C) PAM which cannot be recognized by existing Cas9s. By optimizing the NcCas9 architecture and its spacer length, editing efficacy of NcCas9 was further improved in human cells. In addition, the NcCas9-derived Base editors can efficiently generate base conversions. Six anti-CRISPR (Acr) proteins were identified as off-switches for NcCas9. Moreover, NcCas9 successfully generated efficient editing of mouse embryos by microinjection of NcCas9 mRNA and the corresponding sgRNA. Thus, the NcCas9 holds the potential to broaden the CRISPR/Cas9 toolsets for efficient gene modifications and therapeutic applications.

| S-EPMC9701194 | biostudies-literature

Targeted gene editing by transfection of in vitro reconstituted Streptococcus thermophilus Cas9 nuclease complex.

Project description: Not available

| S-EPMC4615908 | biostudies-literature

Versatile CRISPR/Cas9 Systems for Genome Editing in Ustilago maydis.

Project description:The phytopathogenic smut fungus Ustilago maydis is a versatile model organism to study plant pathology, fungal genetics, and molecular cell biology. Here, we report several strategies to manipulate the genome of U. maydis by the CRISPR/Cas9 technology. These include targeted gene deletion via homologous recombination of short double-stranded oligonucleotides, introduction of point mutations, heterologous complementation at the genomic locus, and endogenous N-terminal tagging with the fluorescent protein mCherry. All applications are independent of a permanent selectable marker and only require transient expression of the endonuclease Cas9hf and sgRNA. The techniques presented here are likely to accelerate research in the U. maydis community but can also act as a template for genome editing in other important fungi.

| S-EPMC7922307 | biostudies-literature

Harnessing CRISPR-Cas9 for Genome Editing in Streptococcus pneumoniae D39V.

Project description:CRISPR-Cas systems provide bacteria and archaea with adaptive immunity against viruses and plasmids by the detection and cleavage of invading foreign DNA. Modified versions of this system can be exploited as a biotechnological tool for precise genome editing at a targeted locus. Here, we developed a replicative plasmid that carries the CRISPR-Cas9 system for RNA-programmable genome editing by counterselection in the opportunistic human pathogen Streptococcus pneumoniae Specifically, we demonstrate an approach for making targeted markerless gene knockouts and large genome deletions. After a precise double-stranded break (DSB) is introduced, the cells' DNA repair mechanism of homology-directed repair (HDR) is exploited to select successful transformants. This is achieved through the transformation of a template DNA fragment that will recombine in the genome and eliminate recognition of the target of the Cas9 endonuclease. Next, the newly engineered strain can be easily cured from the plasmid, which is temperature sensitive for replication, by growing it at the nonpermissive temperature. This allows for consecutive rounds of genome editing. Using this system, we engineered a strain with three major virulence factors deleted. The approaches developed here could potentially be adapted for use with other Gram-positive bacteria.IMPORTANCEStreptococcus pneumoniae (the pneumococcus) is an important opportunistic human pathogen killing more than 1 million people each year. Having the availability of a system capable of easy genome editing would significantly facilitate drug discovery and efforts to identify new vaccine candidates. Here, we introduced an easy-to-use system to perform multiple rounds of genome editing in the pneumococcus by putting the CRISPR-Cas9 system on a temperature-sensitive replicative plasmid. The approaches used here will advance genome editing projects in this important human pathogen.

| S-EPMC8105017 | biostudies-literature

WT-PE: Prime editing with nuclease wild-type Cas9 enables versatile large-scale genome editing.

Project description:Large scale genomic aberrations including duplication, deletion, translocation, and other structural changes are the cause of a subtype of hereditary genetic disorders and contribute to onset or progress of cancer. The current prime editor, PE2, consisting of Cas9-nickase and reverse transcriptase enables efficient editing of genomic deletion and insertion, however, at small scale. Here, we designed a novel prime editor by fusing reverse transcriptase (RT) to nuclease wild-type Cas9 (WT-PE) to edit large genomic fragment. WT-PE system simultaneously introduced a double strand break (DSB) and a single 3' extended flap in the target site. Coupled with paired prime editing guide RNAs (pegRNAs) that have complementary sequences in their 3' terminus while target different genomic regions, WT-PE produced bi-directional prime editing, which enabled efficient and versatile large-scale genome editing, including large fragment deletion up to 16.8 megabase (Mb) pairs and chromosomal translocation. Therefore, our WT-PE system has great potential to model or treat diseases related to large-fragment aberrations.

| S-EPMC9018734 | biostudies-literature

Efficient CRISPR/Cas9 plasmids for rapid and versatile genome editing in Drosophila.

Project description:The CRISPR-associated RNA-guided nuclease Cas9 has emerged as a powerful tool for genome engineering in a variety of organisms. To achieve efficient gene targeting rates in Drosophila, current approaches require either injection of in vitro transcribed RNAs or injection into transgenic Cas9-expressing embryos. We report a simple and versatile alternative method for CRISPR-mediated genome editing in Drosophila using bicistronic Cas9/sgRNA expression vectors. Gene targeting with this single-plasmid injection approach is as efficient as in transgenic nanos-Cas9 embryos and allows the isolation of targeted knock-out and knock-in alleles by molecular screening within 2 months. Our strategy is independent of genetic background and does not require prior establishment of transgenic flies.

| S-EPMC4232553 | biostudies-literature

Efficient genome editing of an extreme thermophile, Thermus thermophilus, using a thermostable Cas9 variant.

Project description:Thermophilic organisms are extensively studied in industrial biotechnology, for exploration of the limits of life, and in other contexts. Their optimal growth at high temperatures presents a challenge for the development of genetic tools for their genome editing, since genetic markers and selection substrates are often thermolabile. We sought to develop a thermostable CRISPR-Cas9 based system for genome editing of thermophiles. We identified CaldoCas9 and designed an associated guide RNA and showed that the pair have targetable nuclease activity in vitro at temperatures up to 65 °C. We performed a detailed characterization of the protospacer adjacent motif specificity of CaldoCas9, which revealed a preference for 5'-NNNNGNMA. We constructed a plasmid vector for the delivery and use of the CaldoCas9 based genome editing system in the extreme thermophile Thermus thermophilus at 65 °C. Using the vector, we generated gene knock-out mutants of T. thermophilus, targeting genes on the bacterial chromosome and megaplasmid. Mutants were obtained at a frequency of about 90%. We demonstrated that the vector can be cured from mutants for a subsequent round of genome editing. CRISPR-Cas9 based genome editing has not been reported previously in the extreme thermophile T. thermophilus. These results may facilitate development of genome editing tools for other extreme thermophiles and to that end, the vector has been made available via the plasmid repository Addgene.

| S-EPMC8100143 | 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