CRISPR-Switch regulates sgRNA activity by Cre recombination for sequential editing of two loci.
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ABSTRACT:
SUBMITTER: Chylinski K
PROVIDER: S-EPMC6884486 | biostudies-literature | 2019 Nov
REPOSITORIES: biostudies-literature
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Project description:Cell behavior is controlled by complex gene regulatory networks. Although studies have uncovered diverse roles of individual genes, it has been challenging to record or control sequential genetic events in living cells. In this study, we designed two cellular chain reaction systems that enable sequential sgRNA activation in mammalian cells using a nickase Cas9 tethering of a cytosine nucleotide deaminase (nCas9-CDA). In these systems, thymidine (T)-to-cytosine (C) substitutions in the scaffold region of the sgRNA or the TATA box-containing loxP sequence (TATAloxP) are corrected by the nCas9-CDA, leading to activation of the next sgRNA. These reactions can occur multiple times, resulting in cellular chain reactions. As a proof of concept, we established a chain reaction by repairing sgRNA scaffold mutations in 293 T cells. Importantly, the results obtained in yeast or in vitro did not match those obtained in mammalian cells, suggesting that in vivo chain reactions need to be optimized in appropriate cellular contexts. Our system may lay the foundation for building cellular chain reaction systems that have a broad utility in the future biomedical research.
| S-EPMC8668876 | biostudies-literature
Project description:MotivationThe development of clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) technology has provided a simple yet powerful system for targeted genome editing. In recent years, this system has been widely used for various gene editing applications. The CRISPR editing efficacy is mainly dependent on the single guide RNA (sgRNA), which guides Cas9 for genome cleavage. While there have been multiple attempts at improving sgRNA design, there is a pressing need for greater sgRNA potency and generalizability across various experimental conditions.ResultsWe employed a unique plasmid library expressed in human cells to quantify the potency of thousands of CRISPR/Cas9 sgRNAs. Differential sequence and structural features among the most and least potent sgRNAs were then used to train a machine learning algorithm for assay design. Comparative analysis indicates that our new algorithm outperforms existing CRISPR/Cas9 sgRNA design tools.Availability and implementationThe new sgRNA design tool is freely accessible as a web application, http://crispr.wustl.edu.Supplementary informationSupplementary data are available at Bioinformatics online.
| S-EPMC7203743 | biostudies-literature
Project description:CRISPR/Cas9 is a valuable tool for both basic and applied research that has been widely applied to different plant species. Nonetheless, a systematical assessment of the efficiency of this method is not available for the allotetraploid Brassica napus-an important oilseed crop. In this study, we examined the mutation efficiency of the CRISPR/Cas9 method for 12 genes and also determined the pattern, specificity and heritability of these gene modifications in B. napus. The average mutation frequency for a single-gene targeted sgRNA in the T0 generation is 65.3%. For paralogous genes located in conserved regions that were targeted by sgRNAs, we observed mutation frequencies that ranged from 27.6% to 96.6%. Homozygotes were readily found in T0 plants. A total of 48.2% of the gene mutations, including homozygotes, bi-alleles, and heterozygotes were stably inherited as classic Mendelian alleles in the next generation (T1) without any new mutations or reversions. Moreover, no mutation was found in the putative off-target sites among the examined T0 plants. Collectively, our results demonstrate that CRISPR/Cas9 is an efficient tool for creating targeted genome modifications at multiple loci that are stable and inheritable in B. napus. These findings open many doors for biotechnological applications in oilseed crops.
| S-EPMC5548805 | biostudies-literature
Project description:BACKGROUND:The CRISPR/Cas (clustered regularly interspaced short palindromic repeat and CRISPR-associated nucleases) based technologies have revolutionized genome engineering. While their use for prokaryotic genome editing is expanding, some limitations remain such as possible off-target effects and design constraints. These are compounded when performing systematic genome editing at distinct loci or when targeting repeated sequences (e.g. multicopy genes or mobile genetic elements). To overcome these limitations, we designed an approach using the same sgRNA and CRISPR-Cas9 system to independently perform gene editing at different loci. RESULTS:We developed a two-step procedure based on the introduction by homologous recombination of 'bait' DNA at the vicinity of a gene copy of interest before inducing CRISPR-Cas9 activity. The introduction of a genetic tool encoding a CRISPR-Cas9 complex targeting this 'bait' DNA induces a double strand break near the copy of interest. Its repair by homologous recombination can lead either to reversion or gene copy-specific editing. The relative frequencies of these events are linked to the impact of gene editing on cell fitness. In our study, we used this technology to successfully delete the native copies of two xenogeneic silencers lsr2 paralogs in Streptomyces ambofaciens. We observed that one of these paralogs is a candidate-essential gene since its native locus can be deleted only in the presence of an extra copy. CONCLUSION:By targeting 'bait' DNA, we designed a 'generic' CRISPR-Cas9 toolkit that can be used to edit different loci. The differential action of this CRISPR-Cas9 system is exclusively based on the specific recombination between regions surrounding the gene copy of interest. This approach is suitable to edit multicopy genes. One such particular example corresponds to the mutagenesis of candidate-essential genes that requires the presence of an extra copy of the gene before gene disruption. This opens new insights to explore gene essentiality in bacteria and to limit off-target effects during systematic CRISPR-Cas9 based approaches.
| S-EPMC6425556 | biostudies-literature
Project description:Cas9 nucleases can be programmed with single guide RNAs (sgRNAs) to mediate gene editing. High CRISPR/Cas9-mediated gene knockout efficiencies are essential for genetic screens and critically depend on the properties of the sgRNAs used. The specificity of an sgRNA is defined by its targeting sequence. Here, we discovered that two short sequence motifs at the 3' end of the targeting sequence are almost exclusively present in inefficient sgRNAs of published sgRNA-activity datasets. By specific knock-in of sgRNA target sequences with or without these motifs and quantitative measurement of knockout efficiency, we show that the presence of these motifs in sgRNAs per se results in a 10-fold reduction of gene knockout frequencies. Mechanistically, the cause of the low efficiency differs between the two motifs. These sequence motifs are relevant for future sgRNA design approaches and studies of Cas9-DNA interactions.
| S-EPMC6352712 | biostudies-literature
Project description:CCCTC-binding factor (CTCF)-mediated stable topologically associating domains (TADs) play a critical role in constraining interactions of DNA elements that are located in neighboring TADs. CTCF plays an important role in regulating the spatial and temporal expression of HOX genes that control embryonic development, body patterning, hematopoiesis, and leukemogenesis. However, it remains largely unknown whether and how HOX loci associated CTCF boundaries regulate chromatin organization and HOX gene expression. In the current protocol, a specific sgRNA pooled library targeting all CTCF binding sites in the HOXA/B/C/D loci has been generated to examine the effects of disrupting CTCF-associated chromatin boundaries on TAD formation and HOX gene expression. Through CRISPR-Cas9 genetic screening, the CTCF binding site located between HOXA7/HOXA9 genes (CBS7/9) has been identified as a critical regulator of oncogenic chromatin domain, as well as being important for maintaining ectopic HOX gene expression patterns in MLL-rearranged acute myeloid leukemia (AML). Thus, this sgRNA library screening approach provides novel insights into CTCF mediated genome organization in specific gene loci and also provides a basis for the functional characterization of the annotated genetic regulatory elements, both coding and noncoding, during normal biological processes in the post-human genome project era.
| S-EPMC7607627 | biostudies-literature
Project description:Background:When developing CRISPR/Cas9 systems for crops, it is crucial to invest time characterizing the genome editing efficiency of the CRISPR/Cas9 cassettes, especially if the transformation system is difficult or time-consuming. Cotton is an important crop for the production of fiber, oil, and biofuel. However, the cotton stable transformation is usually performed using Agrobacterium tumefaciens taking between 8 and 12 months to generate T0 plants. Furthermore, cotton is a heterotetraploid and targeted mutagenesis is considered to be difficult as many genes are duplicated in this complex genome. The application of CRISPR/Cas9 in cotton is severely hampered by the long and technically challenging genetic transformation process, making it imperative to maximize its efficiency. Results:In this study, we provide a new system to evaluate and validate the efficiency of CRISPR/Cas9 cassettes in cotton using a transient expression system. By using this system, we could select the most effective CRISPR/Cas9 cassettes before the stable transformation. We have also optimized the existing cotton CRISPR/Cas9 system to achieve vastly improved mutagenesis efficiency by incorporating an endogenous GhU6 promoter that increases sgRNA expression levels over the Arabidopsis AtU6-29 promoter. The 300 bp GhU6.3 promoter was cloned and validated using the transient expression system. When sgRNAs were expressed under the control of the GhU6.3 promoter in CRISPR/Cas9 cassettes, expression levels were 6-7 times higher than those provided by the AtU6-29 promoter and CRISPR/Cas9-mediated mutation efficiency was improved 4-6 times. Conclusions:This study provides essential improvements to maximize CRISPR/Cas9-mediated mutation efficiency by reducing risk and workload for the application of CRISPR/Cas9 approaches in the targeted mutagenesis of cotton.
| S-EPMC6169012 | biostudies-literature
Project description:The past decade has been a golden age for microbiology, marked by the discovery of an unprecedented increase in the number of novel bacterial species. Yet gaining biological knowledge of those organisms has not kept pace with sequencing efforts. To unlock this genetic potential there is an urgent need for generic (i.e. non-species specific) genetic toolboxes. Recently, we developed a method, termed chassis-independent recombinase-assisted genome engineering (CRAGE), enabling the integration and expression of large complex gene clusters directly into the chromosomes of diverse bacteria. Here we expand upon this technology by incorporating CRISPR-Cas9 allowing precise genome editing across multiple bacterial species. To do that we have developed a landing pad that carries one wild-type and two mutant lox sites to allow integration of foreign DNA at two locations through Cre-lox recombinase-mediated cassette exchange (RMCE). The first RMCE event is to integrate the Cas9 and the DNA repair protein genes RecET, and the second RMCE event enables the integration of customized sgRNA and a repair template. Following this workflow, we achieved precise genome editing in four different gammaproteobacterial species. We also show that the inserted landing pad and the entire editing machinery can be removed scarlessly after editing. We report here the construction of a single landing pad transposon and demonstrate its functionality across multiple species. The modular design of the landing pad and accessory vectors allows design and assembly of genome editing platforms for other organisms in a similar way. We believe this approach will greatly expand the list of bacteria amenable to genetic manipulation and provides the means to advance our understanding of the microbial world.
| S-EPMC7641437 | biostudies-literature
Project description:The SNP within intron 3 of the porcine IGF2 gene (G3072A) plays an important role for muscle growth and fat deposition in pigs. In this study, the StCas9 derived from Streptococcus thermophilus together with the Drosha-mediated sgRNA-shRNA structure were combined to boost the G to A base editing on the IGF2 SNP site, which we called "SNP editing." The codon-humanized StCas9 as we previously reported was firstly compared with the prevalently used SpCas9 derived from Streptococcus pyogenes using our idiomatic surrogate report assay, and the StCas9 demonstrated a comparable targeting activity. On the other hand, by combining shRNA with sgRNA, simultaneous gene silencing and genome targeting can be achieved. Thus, the novel IGF2.sgRNA-LIG4.shRNA-IGF2.sgRNA structure was constructed to enhance the sgRNA/Cas9-mediated HDR-based IGF2 SNP editing by silencing the LIG4 gene, which is a key molecule of the HDR's competitive NHEJ pathway. The sgRNA-shRNA/StCas9 all-in-one expression vector and the IGF2.sgRNA/StCas9 as control were separately used to transfect porcine PK15 cells together with an ssODNs donor for the IGF2 SNP editing. The editing events were detected by the RFLP assay, Sanger sequencing as well as Deep-sequencing, and the Deep-sequencing results finally demonstrated a significant higher HDR-based editing efficiency (16.38%) for our sgRNA-shRNA/StCas9 strategy. In short, we achieved effective IGF2 SNP editing by using the combined sgRNA-shRNA/StCas9 strategy, which will facilitate the further production of base-edited animals and perhaps extend for the gene therapy for the base correction of some genetic diseases.
| S-EPMC6482158 | biostudies-literature
Project description:Gene editing constitutes a novel approach for precisely correcting disease-causing gene mutations. Frameshift mutations in COL7A1 causing recessive dystrophic epidermolysis bullosa are amenable to open reading frame restoration by non-homologous end joining repair-based approaches. Efficient targeted deletion of faulty COL7A1 exons in polyclonal patient keratinocytes would enable the translation of this therapeutic strategy to the clinic. In this study, using a dual single-guide RNA (sgRNA)-guided Cas9 nuclease delivered as a ribonucleoprotein complex through electroporation, we have achieved very efficient targeted deletion of COL7A1 exon 80 in recessive dystrophic epidermolysis bullosa (RDEB) patient keratinocytes carrying a highly prevalent frameshift mutation. This ex vivo non-viral approach rendered a large proportion of corrected cells producing a functional collagen VII variant. The effective targeting of the epidermal stem cell population enabled long-term regeneration of a properly adhesive skin upon grafting onto immunodeficient mice. A safety assessment by next-generation sequencing (NGS) analysis of potential off-target sites did not reveal any unintended nuclease activity. Our strategy could potentially be extended to a large number of COL7A1 mutation-bearing exons within the long collagenous domain of this gene, opening the way to precision medicine for RDEB.
| S-EPMC6520462 | biostudies-literature