Standard CRISPR-mediated gene disruption strategies rely on Cas9-induced DNA double-strand breaks (DSBs). Here, we show that CRISPR-dependent base editing efficiently inactivates genes by precisely ...converting four codons (CAA, CAG, CGA, and TGG) into STOP codons without DSB formation. To facilitate gene inactivation by induction of STOP codons (iSTOP), we provide access to a database of over 3.4 million single guide RNAs (sgRNAs) for iSTOP (sgSTOPs) targeting 97%–99% of genes in eight eukaryotic species, and we describe a restriction fragment length polymorphism (RFLP) assay that allows the rapid detection of iSTOP-mediated editing in cell populations and clones. To simplify the selection of sgSTOPs, our resource includes annotations for off-target propensity, percentage of isoforms targeted, prediction of nonsense-mediated decay, and restriction enzymes for RFLP analysis. Additionally, our database includes sgSTOPs that could be employed to precisely model over 32,000 cancer-associated nonsense mutations. Altogether, this work provides a comprehensive resource for DSB-free gene disruption by iSTOP.
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•CRISPR-mediated base editing allows the conversion of four codons into STOP codons•Induction of STOP codons (iSTOP) can be monitored using restriction enzymes•97%–99% of genes in eight eukaryotic species are targetable by iSTOP•iSTOP can be employed to model over 32,000 cancer-associated nonsense mutations
Billon et al. describe a CRISPR-based approach to inactivate genes by directly converting four codons into STOP codons. The induction of STOP codons (iSTOP) can be rapidly monitored using restriction enzymes. iSTOP can be employed to inactivate eukaryotic genes on a genome-wide scale and model cancer-associated nonsense mutations.
Csm, a type III-A CRISPR-Cas interference complex, is a CRISPR RNA (crRNA)-guided RNase that also possesses target RNA-dependent DNase and cyclic oligoadenylate (cOA) synthetase activities. However, ...the structural features allowing target RNA-binding-dependent activation of DNA cleavage and cOA generation remain unknown. Here, we report the structure of Csm in complex with crRNA together with structures of cognate or non-cognate target RNA bound Csm complexes. We show that depending on complementarity with the 5′ tag of crRNA, the 3′ anti-tag region of target RNA binds at two distinct sites of the Csm complex. Importantly, the interaction between the non-complementary anti-tag region of cognate target RNA and Csm1 induces a conformational change at the Csm1 subunit that allosterically activates DNA cleavage and cOA generation. Together, our structural studies provide crucial insights into the mechanistic processes required for crRNA-meditated sequence-specific RNA cleavage, RNA target-dependent non-specific DNA cleavage, and cOA generation.
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•Crystal structure of the type III-A CRISPR-RNA-guided surveillance complex•Cryo-EM structures of cognate and non-cognate target RNA-bound Csm complexes•Complementary and non-complementary 3′ anti-tag binds at two distinct sites•Target RNA binding allosterically activates ssDNA cleavage and cOA synthesis
Structures of the type III-A CRISPR-Csm complex in the presence of target RNAs reveal the molecular basis for a unique mode of self/non-self discrimination.
Targeted integration of transgenes can be achieved by strategies based on homologous recombination （HR）, mi- crohomology-mediated end joining （MMEJ） or non-homologous end joining （NHEJ）. The more ...generally used HR is inefficient for achieving gene integration in animal embryos and tissues, because it occurs only during cell division, although MMEJ and NHEJ can elevate the efficiency in some systems. Here we devise a homology-mediated end joining （HMEJ）-based strategy, using CRISPR/Cas9-mediated cleavage of both transgene donor vector that contains guide RNA target sites and -800 bp of homology arms, and the targeted genome. We found no significant improve- ment of the targeting efficiency by the HMEJ-based method in either mouse embryonic stem cells or the neuroblas- toma cell line, N2a, compared to the HR-based method. However, the HMEJ-based method yielded a higher knock- in efficiency in HEK293T cells, primary astrocytes and neurons. More importantly, this approach achieved transgene integration in mouse and monkey embryos, as well as in hepatocytes and neurons in vivo, with an efficiency much greater than HR-, NHEJ- and MMEJ-based strategies. Thus, the HMEJ-based strategy may be useful for a variety of applications, including gene editing to generate animal models and for targeted gene therapies.
Controlling and enhancing CRISPR systems Shivram, Haridha; Cress, Brady F; Knott, Gavin J ...
Nature chemical biology,
01/2021, Letnik:
17, Številka:
1
Journal Article
Recenzirano
Odprti dostop
Many bacterial and archaeal organisms use clustered regularly interspaced short palindromic repeats-CRISPR associated (CRISPR-Cas) systems to defend themselves from mobile genetic elements. These ...CRISPR-Cas systems are classified into six types based on their composition and mechanism. CRISPR-Cas enzymes are widely used for genome editing and offer immense therapeutic opportunity to treat genetic diseases. To realize their full potential, it is important to control the timing, duration, efficiency and specificity of CRISPR-Cas enzyme activities. In this Review we discuss the mechanisms of natural CRISPR-Cas regulatory biomolecules and engineering strategies that enhance or inhibit CRISPR-Cas immunity by altering enzyme function. We also discuss the potential applications of these CRISPR regulators and highlight unanswered questions about their evolution and purpose in nature.
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Topical drug delivery in the oral mucosa has its set of challenges due to the unique anatomical and physiological features of the oral cavity. As such, the outcomes of local ...pharmacological treatments in oral disorders can fail due to unsuccessfully drug delivery. Oral mucositis, a severe inflammatory and ulcerative side effect of oncological treatments, is one of such diseases. Although the damaged tissue is within reach, no approved topical drug treatment is available. Several strategies based on its physiopathology have been implemented and clinically used. Even so, results tend to lack or be insufficient to improve patient’s quality of life. The use of corticosteroids has been employed in such strategies due to their strong anti-inflammatory action. Typically, these are administrated in simple liquid formulations, where the drug is dispersed or solubilized, lacking the ability to maintain local concentration. In this work, we propose the development of a biocompatible delivery system with boosted abilities of retention and control release of budesonide, a corticosteroid with an elevated ratio of topical anti-inflammatory to systemic action. Through spray-drying, polymeric particles of Chitosan and Eudragit® E PO were produced and characterized for the vectorization of this drug.
•SARS-CoV-2 has infected millions people worldwide with numerous deaths since December 2019.•Type V and VI CRISPR enzymes are efficient agents for diagnosis and combating single-stranded RNA viruses ...such as SARS-CoV-2.•DETECTR and SHERLOCK represent rapid, specific and efficient diagnostic platforms for point of care detection of SARS-CoV-2.•PAC-MAN is a SARS-CoV-2 plausible therapeutic approach which pave the way to use CRISPR systems as robust antivirals.
Type V and VI CRISPR enzymes are RNA-guided, DNA and RNA-targeting effectors that allow specific gene knockdown. Cas12 and Cas13 are CRISPR proteins that are efficient agents for diagnosis and combating single-stranded RNA (ssRNA) viruses. The programmability of these proteins paves the way for the detection and degradation of RNA viruses by targeting RNAs complementary to its CRISPR RNA (crRNA). Approximately two-thirds of viruses causing diseases contain ssRNA genomes. The Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) has caused the outbreak of the coronavirus disease 2019 (COVID-19), which has infected more than 88 million people worldwide with near 2 million deaths since December 2019. Thus, accurate and rapid diagnostic and therapeutic tools are essential for early detection and treatment of this widespread infectious disease. For us, the CRISPR based platforms seem to be a plausible new approach for an accurate detection and treatment of SARS-CoV-2.
In this review, we talk about Cas12 and Cas13 CRISPR systems and their applications in diagnosis and treatment of RNA virus mediated diseases. In continue, the SARS-CoV-2 pathogenicity, and its conventional diagnostics and antivirals will be discussed. Moreover, we highlight novel CRISPR based diagnostic platforms and therapies for COVID-19. We also discuss the challenges of diagnostic CRISPR based platforms as well as clarifying the proposed solution for high efficient selective in vivo delivery of CRISPR components into SARS-CoV-2-infected cells.
Phages use anti-CRISPR proteins to deactivate the CRISPR-Cas system. The mechanisms for the inhibition of type I and type II systems by anti-CRISPRs have been elucidated. However, it has remained ...unknown how the type V CRISPR-Cas12a (Cpf1) system is inhibited by anti-CRISPRs. Here we identify the anti-CRISPR protein AcrVA5 and report the mechanisms by which it inhibits CRISPR-Cas12a. Our structural and biochemical data show that AcrVA5 functions as an acetyltransferase to modify Moraxella bovoculi (Mb) Cas12a at Lys635, a residue that is required for recognition of the protospacer-adjacent motif. The AcrVA5-mediated modification of MbCas12a results in complete loss of double-stranded DNA (dsDNA)-cleavage activity. In contrast, the Lys635Arg mutation renders MbCas12a completely insensitive to inhibition by AcrVA5. A cryo-EM structure of the AcrVA5-acetylated MbCas12a reveals that Lys635 acetylation provides sufficient steric hindrance to prevent dsDNA substrates from binding to the Cas protein. Our study reveals an unprecedented mechanism of CRISPR-Cas inhibition and suggests an evolutionary arms race between phages and bacteria.
Abstract
The development of the CRISPR-Cas9 technology has provided a simple yet powerful system for genome editing. Current gRNA design tools serve as an important platform for the efficient ...application of the CRISPR systems. However, most of the existing tools are black-box models that suffer from limitations, such as variable performance and unclear mechanism of decision making. Here, we introduce CRISPRedict, an interpretable gRNA efficiency prediction model for CRISPR-Cas9 gene editing. Its strength lies in the fact that it can accurately predict efficient guide RNAs—with equivalent performance to state-of-the-art tools—while being a simple linear model. Implemented as a user-friendly web server, CRISPRedict offers (i) quick and accurate predictions across various experimental conditions (e.g. U6/T7 transcription); (ii) regression and classification models for scoring gRNAs and (iii) multiple visualizations to explain the obtained results. Given its performance, interpretability, and versatility, we expect that it will assist researchers in the gRNA design process and facilitate genome editing research. CRISPRedict is available for use at http://www.crispredict.org/.
Graphical Abstract
Graphical Abstract
CRISPRedict is an interpretable gRNA efficiency prediction model for CRISPR-Cas9 gene editing. It can accurately predict efficient gRNAs—with equivalent performance to state-of-the-art tools—while being a simple linear model.
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