scholarly journals CRISPR as a Diagnostic Tool

Biomolecules ◽  
2021 ◽  
Vol 11 (8) ◽  
pp. 1162
Author(s):  
Seohyun Kim ◽  
Sangmin Ji ◽  
Hye Ran Koh
Keyword(s):  
Diagnostic Tool ◽  
Specific Binding ◽  
Diagnostic Methods ◽  
Specific Gene ◽  
Guide Rna ◽  
Engineering Technique ◽  
Target Dna ◽  
Target Binding ◽  
Genetic Engineering Technique ◽  
Disease Related Gene

Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas system has recently gained growing attention as a diagnostic tool due to its capability of specific gene targeting. It consists of Cas enzymes and a guide RNA (gRNA) that can cleave the target DNA or RNA based on the sequence of the gRNA, making it an attractive genetic engineering technique. In addition to the target-specific binding and cleavage, the trans-cleavage activity was reported for some Cas proteins, including Cas12a and Cas13a, which is to cleave the surrounding single-stranded DNA or RNA upon the target binding of Cas-gRNA complex. All these activities of the CRISPR-Cas system are based on its target-specific binding, making it applied to develop diagnostic methods by detecting the disease-related gene as well as microRNAs and the genetic variations such as single nucleotide polymorphism and DNA methylation. Moreover, it can be applied to detect the non-nucleic acids target such as proteins. In this review, we cover the various CRISPR-based diagnostic methods by focusing on the activity of the CRISPR-Cas system and the form of the target. The CRISPR-based diagnostic methods without target amplification are also introduced briefly.

scholarly journals Structural basis for Cas9 off-target activity

2021 ◽  
Author(s):  
Martin Pacesa ◽  
Chun-Han Lin ◽  
Antoine Clery ◽  
Katja Bargsten ◽  
Matthew J. Irby ◽  
...  
Keyword(s):  
Rational Design ◽  
Target Prediction ◽  
Structural Basis ◽  
Guide Rna ◽  
Guide Rnas ◽  
Target Dna ◽  
Bona Fide ◽  
Dna Specificity ◽  
Target Binding ◽  
Target Activity

The target DNA specificity of the CRISPR-associated genome editor nuclease Cas9 is determined by complementarity to a 20-nucleotide segment in its guide RNA. However, Cas9 can bind and cleave partially complementary off-target sequences, which raises safety concerns for its use in clinical applications. Here we report crystallographic structures of Cas9 bound to bona fide off-target substrates, revealing that off-target binding is enabled by a range of non- canonical base pairing interactions and preservation of base stacking within the guide-off-target heteroduplex. Off-target sites containing single-nucleotide deletions relative to the guide RNA are accommodated by base skipping rather than RNA bulge formation. Additionally, PAM-distal mismatches result in duplex unpairing and induce a conformational change of the Cas9 REC lobe that perturbs its conformational activation. Together, these insights provide a structural rationale for the off-target activity of Cas9 and contribute to the improved rational design of guide RNAs and off-target prediction algorithms.

scholarly journals Molecular mechanism of target site selection and remodeling by type V CRISPR-associated transposons

2021 ◽  
Author(s):  
Irma Querques ◽  
Michael Schmitz ◽  
Seraina Oberli ◽  
Christelle Chanez ◽  
Martin Jinek
Keyword(s):  
Zinc Finger Protein ◽  
Complex Structure ◽  
Specific Gene ◽  
Type I ◽  
Site Specific ◽  
Guide Rna ◽  
Aaa Atpase ◽  
Transposon Insertion ◽  
Target Dna ◽  
Type V

Although the canonical function of CRISPR-Cas systems is to provide adaptive immunity against mobile genetic elements, type I-F, I-B and V-K systems have been adopted by Tn7-like transposons to direct RNA-guided transposon insertion. Type V-K CRISPR-associated transposons rely on the activities of the pseudonuclease Cas12k, the transposase TnsB, the AAA+ ATPase TnsC and the zinc-finger protein TniQ. However, the molecular and structural details of RNA-directed DNA transposition have remained elusive. Here we report cryo-electron microscopic structures of a Cas12k-guide RNA-target DNA complex and a DNA-bound, polymeric TnsC filament. The Cas12k complex structure reveals an intricate guide RNA architecture and critical interactions mediating RNA-guided target DNA recognition. The assembly of the TnsC helical filament is ATP-dependent and accompanied by structural remodeling of the bound DNA duplex. In vivo transposition assays corroborate key features of the structures, and biochemical experiments further show that TniQ restricts TnsC polymerization, while the TnsB transposase interacts directly with TnsC filaments to trigger their disassembly upon ATP hydrolysis. Together, these results suggest a mechanistic model whereby RNA-directed target selection by Cas12k primes TnsC polymerization and DNA remodeling, generating a recruitment platform for TnsB to catalyze site-specific transposon insertion. The present work advances our mechanistic understanding of the cross-talk between CRISPR effectors and the transposition machinery and will inform design efforts to harness CRISPR-associated transposons as programmable site-specific gene insertion tools for genome engineering applications.

scholarly journals Hepatic Venous Duplex as an Alternative Non- invasive Diagnostic Tool for Diagnosis of Pulmonary Hypertension

2020 ◽  
Vol 16 (2) ◽  
pp. 86-91
Author(s):  
Md Harisul Hoque ◽  
SM Mustafa Zaman ◽  
Khurshid Ahmed ◽  
Sajal Krisna Banerjee ◽  
Md Faisal Ibne Kabir ◽  
...  
Keyword(s):  
Pulmonary Hypertension ◽  
Diagnostic Tool ◽  
Right Heart Catheterization ◽  
Diagnostic Methods ◽  
Heart Catheterization ◽  
Right Heart ◽  
Non Invasive ◽  
Close Proximity ◽  
The Right ◽  
Practical Demonstration

Pulmonary hypertension is a hemodynamic disorder defined by abnormally high pulmonary artery pressure that affects the arteries in your lungs and the right side of your heart. In this study, hepatic venous duplex will be done to diagnose and quantify the PH. So that Patients can avoid unnecessary invasive right heart catheterization. This practical demonstration is the key to enrich our experience and knowledge in the field of PH. Objectives of this study was to assess PH status by Hepatic venous Duplex (HVD) as well by right heart catheterization and to compare them. This study was conducted in the Department of Cardiology, BSMMU, Shahbagh, Dhaka extending from July 2018 to December 2019. Total 100 (One hundred) subjects were enrolled in this study. It was an Observational study and includes the subjects between 18 years to 45 years of age. Results of this study shows very close proximity to that of Right heart catheterization. Hemodynamic changes in Hepatic venous duplex study could be used as an alternative diagnostic tool for evaluating moderate to severe pulmonary hypertension. This method could counteract the weakness of the currently used diagnostic methods and improve the accuracy of assessing pulmonary hypertension when combined with other methods. University Heart Journal Vol. 16, No. 2, Jul 2020; 86-91

scholarly journals Mechanism of R-loop formation and conformational activation of Cas9

2021 ◽  
Author(s):  
Martin Pacesa ◽  
Martin Jinek
Keyword(s):  
Dna Cleavage ◽  
Structural Basis ◽  
Seed Region ◽  
Loop Formation ◽  
Base Pairs ◽  
Guide Rna ◽  
Target Strand ◽  
Target Dna ◽  
Dna Strand ◽  
Dna Substrate

Cas9 is a CRISPR-associated endonuclease capable of RNA-guided, site-specific DNA cleavage. The programmable activity of Cas9 has been widely utilized for genome editing applications. Despite extensive studies, the precise mechanism of target DNA binding and on-/off-target discrimination remains incompletely understood. Here we report cryo-EM structures of intermediate binding states of Streptococcus pyogenes Cas9 that reveal domain rearrangements induced by R-loop propagation and PAM-distal duplex positioning. At early stages of binding, the Cas9 REC2 and REC3 domains form a positively charged cleft that accommodates the PAM-distal duplex of the DNA substrate. Target hybridisation past the seed region positions the guide-target heteroduplex into the central binding channel and results in a conformational rearrangement of the REC lobe. Extension of the R-loop to 16 base pairs triggers the relocation of the HNH domain towards the target DNA strand in a catalytically incompetent conformation. The structures indicate that incomplete target strand pairing fails to induce the conformational displacements necessary for nuclease domain activation. Our results establish a structural basis for target DNA-dependent activation of Cas9 that advances our understanding of its off-target activity and will facilitate the development of novel Cas9 variants and guide RNA designs with enhanced specificity and activity.

scholarly journals Computational design of probes to detect bacterial genomes by multivalent binding

2020 ◽  
Vol 117 (16) ◽  
pp. 8719-8726 ◽  
Author(s):  
Tine Curk ◽  
Chris A. Brackley ◽  
James D. Farrell ◽  
Zhongyang Xing ◽  
Darshana Joshi ◽  
...  
Keyword(s):  
Genomic Dna ◽  
Bacterial Infections ◽  
Bacterial Genome ◽  
Computational Design ◽  
Diagnostic Methods ◽  
Detection Sensitivity ◽  
Target Sequence ◽  
Inappropriate Use ◽  
Single Target ◽  
Target Dna

Rapid methods for diagnosis of bacterial infections are urgently needed to reduce inappropriate use of antibiotics, which contributes to antimicrobial resistance. In many rapid diagnostic methods, DNA oligonucleotide probes, attached to a surface, bind to specific nucleotide sequences in the DNA of a target pathogen. Typically, each probe binds to a single target sequence; i.e., target–probe binding is monovalent. Here we show using computer simulations that the detection sensitivity and specificity can be improved by designing probes that bind multivalently to the entire length of the pathogen genomic DNA, such that a given probe binds to multiple sites along the target DNA. Our results suggest that multivalent targeting of long pieces of genomic DNA can allow highly sensitive and selective binding of the target DNA, even if competing DNA in the sample also contains binding sites for the same probe sequences. Our results are robust to mild fragmentation of the bacterial genome. Our conclusions may also be relevant for DNA detection in other fields, such as disease diagnostics more broadly, environmental management, and food safety.

scholarly journals Enhancement of target specificity of CRISPR–Cas12a by using a chimeric DNA–RNA guide

2020 ◽  
Vol 48 (15) ◽  
pp. 8601-8616 ◽  
Author(s):  
Hanseop Kim ◽  
Wi-jae Lee ◽  
Yeounsun Oh ◽  
Seung-Hun Kang ◽  
Junho K Hur ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Genome Engineering ◽  
Target Genes ◽  
Target Sequence ◽  
Energy Potential ◽  
Guide Rna ◽  
Sequence Recognition ◽  
Protospacer Adjacent Motif ◽  
Effective Manner ◽  
Target Dna

Abstract The CRISPR–Cas9 system is widely used for target-specific genome engineering. CRISPR–Cas12a (Cpf1) is one of the CRISPR effectors that controls target genes by recognizing thymine-rich protospacer adjacent motif (PAM) sequences. Cas12a has a higher sensitivity to mismatches in the guide RNA than does Cas9; therefore, off-target sequence recognition and cleavage are lower. However, it tolerates mismatches in regions distant from the PAM sequence (TTTN or TTN) in the protospacer, and off-target cleavage issues may become more problematic when Cas12a activity is improved for therapeutic purposes. Therefore, we investigated off-target cleavage by Cas12a and modified the Cas12a (cr)RNA to address the off-target cleavage issue. We developed a CRISPR–Cas12a that can induce mutations in target DNA sequences in a highly specific and effective manner by partially substituting the (cr)RNA with DNA to change the energy potential of base pairing to the target DNA. A model to explain how chimeric (cr)RNA guided CRISPR–Cas12a and SpCas9 nickase effectively work in the intracellular genome is suggested. Chimeric guide-based CRISPR- Cas12a genome editing with reduced off-target cleavage, and the resultant, increased safety has potential for therapeutic applications in incurable diseases caused by genetic mutations.

Characterization of Tissue-Specific HIF-1 and HIF-2 Regulatory Elements of the Erythropoietin Gene

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4763-4763
Author(s):  
Donghoon Yoon ◽  
Hyojin Kim ◽  
Minyoung Jang ◽  
Jihyun Song ◽  
Gregory E Arnold ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Specific Binding ◽  
Regulatory Element ◽  
Regulatory Elements ◽  
Specific Gene ◽  
Mrna Levels ◽  
Specific Element ◽  
Α Subunit ◽  
Tissue Specific

Abstract Hypoxia regulates erythropoiesis and other essential processes via hypoxia-inducible transcription factors (HIFs). HIFs are heterodimers that consist of an α subunit (3 isotypes with significant homology; HIF-1α, HIF-2α, HIF-3α), and a common b-subunit; HIF-1 and HIF-2, in some instances exhibiting tissue- and gene-specific gene regulation. Erythropoietin (EPO) was the first identified HIF-1 target gene with the defined HIF-1 binding sequence. However, subsequent works suggested that HIF-2 also regulates EPO transcription and that there are other regulatory elements of EPO gene (i.e. Kidney Inducible Element KIE, Negative Regulatory Element NRE, and Negative Regulatory Liver specific Element NRLE). In silico analysis of the human EPO genome found two additional potential HIF-binding elements in the KIE and NRE regions. The comparative analysis of phylogenically conserved sequences of human, mouse, dog, and rat Epo genes further refined these mouse Epo gene HIF-binding elements as mKIE, mNRE1, mNRE2, and mNRLE2. We treated mice in hypoxia chamber (8% O2) and monitored changes of Epo mRNA levels in liver, kidney, brain, spleen, and bone marrow. All tested tissues increased Epo transcription during hypoxia. Bone marrow, spleen, kidney, and brain showed a peak of induction of Epo transcript at 3 hours of hypoxia treatment, while liver reached the highest level at 6 hours. Mice were sacrificed and organs were harvested, and in vivo chromatin immunoprecipitation (ChIPs) was performed with antibodies against HIF-1α and HIF- 2α and tissue-specific binding regions were defined. The results from these studies are summarized below. HIF-1 mKIE rnNRE mNRE2 mNRLE2 Norm Hyp Norm Hyp Norm Hyp Norm Hyp Liver − + − − + − ? ? Kidney − + − − + − + − Brain − + − − − + − + BM − + − − − − − + Splsen − + − − − − − + HIF-2 mKIE mNRE mNRE2 mNRLE2 Norm Hyp Norm Hyp Norm Hyp Norm Hyp “+” denotes presence and “-” absence of binding of HIF-1 and HIF-2, “?” – indicates inconclusive results. “Norm” - normoxia, “Hyp” - hypoxia. Liver − + − − − + − + Kidney + − − − + − ? ? Brain − − − − − − − + BM − − − − − − + − Spleen − + − − − − − + In conclusion, we demonstrate the differential hypoxia-induced binding of HIF-1 and HIF-2 at different HIF binding elements in the tissues known to express Epo. Further studies will be required to define the function of these HIF-1 and HIF-2 binding elements in tissue specific Epo expression and their role in health and disease.

scholarly journals A mouse model for adult cardiac-specific gene deletion with CRISPR/Cas9

2015 ◽  
Vol 113 (2) ◽  
pp. 338-343 ◽  
Author(s):  
Kelli J. Carroll ◽  
Catherine A. Makarewich ◽  
John McAnally ◽  
Douglas M. Anderson ◽  
Lorena Zentilin ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Gene Deletion ◽  
Disease Modeling ◽  
Embryonic Lethality ◽  
Null Alleles ◽  
Specific Gene ◽  
Specific Expression ◽  
Guide Rna ◽  
Cardiac Gene ◽  
Genomic Editing

Clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas)9 genomic editing has revolutionized the generation of mutant animals by simplifying the creation of null alleles in virtually any organism. However, most current approaches with this method require zygote injection, making it difficult to assess the adult, tissue-specific functions of genes that are widely expressed or which cause embryonic lethality when mutated. Here, we describe the generation of cardiac-specific Cas9 transgenic mice, which express high levels of Cas9 in the heart, but display no overt defects. In proof-of-concept experiments, we used Adeno-Associated Virus 9 (AAV9) to deliver single-guide RNA (sgRNA) that targets the Myh6 locus exclusively in cardiomyocytes. Intraperitoneal injection of postnatal cardiac-Cas9 transgenic mice with AAV9 encoding sgRNA against Myh6 resulted in robust editing of the Myh6 locus. These mice displayed severe cardiomyopathy and loss of cardiac function, with elevation of several markers of heart failure, confirming the effectiveness of this method of adult cardiac gene deletion. Mice with cardiac-specific expression of Cas9 provide a tool that will allow rapid and accurate deletion of genes following a single injection of AAV9-sgRNAs, thereby circumventing embryonic lethality. This method will be useful for disease modeling and provides a means of rapidly editing genes of interest in the heart.

scholarly journals The GATA-4 transcription factor transactivates the cardiac muscle-specific troponin C promoter-enhancer in nonmuscle cells.

1994 ◽  
Vol 14 (11) ◽  
pp. 7517-7526 ◽  
Author(s):  
H S Ip ◽  
D B Wilson ◽  
M Heikinheimo ◽  
Z Tang ◽  
C N Ting ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcription Factor ◽  
Transcription Factors ◽  
Cardiac Muscle ◽  
Binding Site ◽  
Cardiac Myocytes ◽  
Specific Binding ◽  
Troponin C ◽  
Specific Gene ◽  
Cardiac Muscle Cells

The unique contractile phenotype of cardiac myocytes is determined by the expression of a set of cardiac muscle-specific genes. By analogy to other mammalian developmental systems, it is likely that the coordinate expression of cardiac genes is controlled by lineage-specific transcription factors that interact with promoter and enhancer elements in the transcriptional regulatory regions of these genes. Although previous reports have identified several cardiac muscle-specific transcriptional elements, relatively little is known about the lineage-specific transcription factors that regulate these elements. In this report, we demonstrate that the slow/cardiac muscle-specific troponin C (cTnC) enhancer contains a specific binding site for the lineage-restricted zinc finger transcription factor GATA-4. This GATA-4-binding site is required for enhancer activity in primary cardiac myocytes. Moreover, the cTnC enhancer can be transactivated by overexpression of GATA-4 in non-cardiac muscle cells such as NIH 3T3 cells. In situ hybridization studies demonstrate that GATA-4 and cTnC have overlapping patterns of expression in the hearts of postimplantation mouse embryos and that GATA-4 gene expression precedes cTnC expression. Indirect immunofluorescence reveals GATA-4 expression in cultured cardiac myocytes from neonatal rats. Taken together, these results are consistent with a model in which GATA-4 functions to direct tissue-specific gene expression during mammalian cardiac development.

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