scholarly journals Disabling Cas9 by an anti-CRISPR DNA mimic

2017 ◽  
Author(s):  
Jiyung Shing ◽  
Fuguo Jiang ◽  
Jun-Jie Liu ◽  
Nicholas L. Bray ◽  
Benjamin J. Rauch ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Gene Editing ◽  
Adaptive Immune System ◽  
Binding Mode ◽  
Human Cells ◽  
Guide Rna ◽  
Protospacer Adjacent Motif ◽  
Cas9 Protein ◽  
Potential Applications ◽  
Natural Inhibitors

CRISPR-Cas9 gene editing technology is derived from a microbial adaptive immune system, where bacteriophages are often the intended target. Natural inhibitors of CRISPR-Cas9 enable phages to evade immunity and show promise in controlling Cas9-mediated gene editing in human cells. However, the mechanism of CRISPR-Cas9 inhibition is not known and the potential applications for Cas9 inhibitor proteins in mammalian cells has not fully been established. We show here that the anti-CRISPR protein AcrIIA4 binds only to assembled Cas9-single guide RNA (sgRNA) complexes and not to Cas9 protein alone. A 3.9 Å resolution cryo-EM structure of the Cas9-sgRNA-AcrIIA4 complex revealed that the surface of AcrIIA4 is highly acidic and binds with 1:1 stoichiometry to a region of Cas9 that normally engages the DNA protospacer adjacent motif (PAM). Consistent with this binding mode, order-of-addition experiments showed that AcrIIA4 interferes with DNA recognition but has no effect on pre-formed Cas9-sgRNA-DNA complexes. Timed delivery of AcrIIA4 into human cells as either protein or expression plasmid allows on-target Cas9-mediated gene editing while reducing off-target edits. These results provide a mechanistic understanding of AcrIIA4 function and demonstrate that inhibitors can modulate the extent and outcomes of Cas9-mediated gene editing.

scholarly journals Genome Editing in Zebrafish by ScCas9 Recognizing NNG PAM

Cells ◽  
2021 ◽  
Vol 10 (8) ◽  
pp. 2099
Author(s):  
Yunxing Liu ◽  
Fang Liang ◽  
Zijiong Dong ◽  
Song Li ◽  
Jianmin Ye ◽  
...  
Keyword(s):  
Genome Editing ◽  
Streptococcus Pyogenes ◽  
Gene Editing ◽  
Zebrafish Genome ◽  
Human Cells ◽  
Ribonucleoprotein Complex ◽  
Guide Rna ◽  
Protospacer Adjacent Motif ◽  
Low Activity

The CRISPR/Cas9 system has been widely used for gene editing in zebrafish. However, the required NGG protospacer adjacent motif (PAM) of Streptococcus pyogenes Cas9 (SpCas9) notably restricts the editable range of the zebrafish genome. Recently, Cas9 from S. canis (ScCas9), which has a more relaxed 5′-NNG-3′ PAM, was reported to have activities in human cells and plants. However, the editing ability of ScCas9 has not been tested in zebrafish. Here we characterized and optimized the activity of ScCas9 in zebrafish. Delivered as a ribonucleoprotein complex, ScCas9 can induce mutations in zebrafish. Using the synthetic modified crRNA:tracrRNA duplex instead of in vitro-transcribed single guide RNA, the low activity at some loci were dramatically improved in zebrafish. As far as we know, our work is the first report on the evaluation of ScCas9 in animals. Our work optimized ScCas9 as a new nuclease for targeting relaxed NNG PAMs for zebrafish genome editing, which will further improve genome editing in zebrafish.

scholarly journals Various Aspects of a Gene Editing System—CRISPR–Cas9

2020 ◽  
Vol 21 (24) ◽  
pp. 9604
Author(s):  
Edyta Janik ◽  
Marcin Niemcewicz ◽  
Michal Ceremuga ◽  
Lukasz Krzowski ◽  
Joanna Saluk-Bijak ◽  
...  
Keyword(s):  
Genome Editing ◽  
Gene Editing ◽  
Genome Engineering ◽  
High Efficiency ◽  
Adaptive Immune System ◽  
Zinc Finger Nucleases ◽  
Transcription Activator ◽  
Guide Rna ◽  
Adaptive Immune ◽  
Cas9 Protein

The discovery of clustered, regularly interspaced short palindromic repeats (CRISPR) and their cooperation with CRISPR-associated (Cas) genes is one of the greatest advances of the century and has marked their application as a powerful genome engineering tool. The CRISPR–Cas system was discovered as a part of the adaptive immune system in bacteria and archaea to defend from plasmids and phages. CRISPR has been found to be an advanced alternative to zinc-finger nucleases (ZFN) and transcription activator-like effector nucleases (TALEN) for gene editing and regulation, as the CRISPR–Cas9 protein remains the same for various gene targets and just a short guide RNA sequence needs to be altered to redirect the site-specific cleavage. Due to its high efficiency and precision, the Cas9 protein derived from the type II CRISPR system has been found to have applications in many fields of science. Although CRISPR–Cas9 allows easy genome editing and has a number of benefits, we should not ignore the important ethical and biosafety issues. Moreover, any tool that has great potential and offers significant capabilities carries a level of risk of being used for non-legal purposes. In this review, we present a brief history and mechanism of the CRISPR–Cas9 system. We also describe on the applications of this technology in gene regulation and genome editing; the treatment of cancer and other diseases; and limitations and concerns of the use of CRISPR–Cas9.

scholarly journals Exploiting DNA Endonucleases to Advance Mechanisms of DNA Repair

Biology ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 530
Author(s):  
Marlo K. Thompson ◽  
Robert W. Sobol ◽  
Aishwarya Prakash
Keyword(s):  
Dna Repair ◽  
Mammalian Cells ◽  
Genome Stability ◽  
Gene Editing ◽  
Adaptive Immune System ◽  
Zinc Finger Nucleases ◽  
Specific Gene ◽  
Target Sequence ◽  
Transcription Activator ◽  
Low Cytotoxicity

The earliest methods of genome editing, such as zinc-finger nucleases (ZFN) and transcription activator-like effector nucleases (TALENs), utilize customizable DNA-binding motifs to target the genome at specific loci. While these approaches provided sequence-specific gene-editing capacity, the laborious process of designing and synthesizing recombinant nucleases to recognize a specific target sequence, combined with limited target choices and poor editing efficiency, ultimately minimized the broad utility of these systems. The discovery of clustered regularly interspaced short palindromic repeat sequences (CRISPR) in Escherichia coli dates to 1987, yet it was another 20 years before CRISPR and the CRISPR-associated (Cas) proteins were identified as part of the microbial adaptive immune system, by targeting phage DNA, to fight bacteriophage reinfection. By 2013, CRISPR/Cas9 systems had been engineered to allow gene editing in mammalian cells. The ease of design, low cytotoxicity, and increased efficiency have made CRISPR/Cas9 and its related systems the designer nucleases of choice for many. In this review, we discuss the various CRISPR systems and their broad utility in genome manipulation. We will explore how CRISPR-controlled modifications have advanced our understanding of the mechanisms of genome stability, using the modulation of DNA repair genes as examples.

scholarly journals Application of CRISPR/Cas9 technology in wild apple (Malus sieverii) for paired sites gene editing

Plant Methods ◽  
2021 ◽  
Vol 17 (1) ◽  
Author(s):  
Yan Zhang ◽  
Ping Zhou ◽  
Tohir A. Bozorov ◽  
Daoyuan Zhang
Keyword(s):  
Abiotic Stress ◽  
Human Activities ◽  
Genetic Improvement ◽  
Gene Editing ◽  
New Technologies ◽  
Tianshan Mountains ◽  
Biotic And Abiotic Stress ◽  
Guide Rna ◽  
Cas9 Protein ◽  
Albino Phenotype

Abstract Background Xinjiang wild apple is an important tree of the Tianshan Mountains, and in recent years, it has undergone destruction by many biotic and abiotic stress and human activities. It is necessary to use new technologies to research its genomic function and molecular improvement. The clustered regulatory interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) system has been successfully applied to genetic improvement in many crops, but its editing capability varies depending on the different combinations of the synthetic guide RNA (sgRNA) and Cas9 protein expression devices. Results In this study, we used 2 systems of vectors with paired sgRNAs targeting to MsPDS. As expected, we successfully induced the albino phenotype of calli and buds in both systems. Conclusions We conclude that CRISPR/Cas9 is a powerful system for editing the wild apple genome and expands the range of plants available for gene editing.

scholarly journals A potential clue for the application of prime editing in tomato, a dicot plant

2021 ◽  
Author(s):  
Tien Van Vu ◽  
Jihae Kim ◽  
Swati Das ◽  
Jae-Yean Kim
Keyword(s):  
High Frequency ◽  
Mammalian Cells ◽  
Genome Engineering ◽  
Crop Improvement ◽  
Plant Genome ◽  
Guide Rna ◽  
Genome Modification ◽  
Potential Applications ◽  
Targeted Deep Sequencing ◽  
Synthetic Sequence

Precision genome editing is highly desired for crop improvement. The recently emerged CRISPR/Cas technology offers great potential applications in precision plant genome engineering. A prime editing (PE) approach combining a reverse transcriptase (RT) with a Cas9 nickase and a priming extended guide RNA has shown a high frequency for precise genome modification in mammalian cells and several plant species. However, the applications of the PE approach in dicot plants are still limited and inefficient. We designed and tested prime editors for precision editing of a synthetic sequence in a transient assay and for desirable alleles of 10 loci in tomato by stable transformation. However, our data obtained by targeted deep sequencing also revealed inefficient PE activity in both the tobacco and tomato systems. Further assessment of the activities of the PE components uncovered potential reasons for the inefficiency of the PE complexes. These data could also help explain the recent successes of some prime editors in plants using improved expression systems. Our work provides an important clue for the application of the PE approach in crop improvement.

scholarly journals RNA-CLAMP Enables Photo-activated Control of CRISPR-Cas9 Gene Editing by Site-specific Intramolecular Cross-linking of the sgRNA

2021 ◽  
Author(s):  
Dongyang Zhang ◽  
Shuaijiang Jin ◽  
Luping Liu ◽  
Ember Tota ◽  
Zijie Li ◽  
...  
Keyword(s):  
Dna Cleavage ◽  
Mammalian Cells ◽  
Gene Editing ◽  
Cross Linking ◽  
Stimuli Responsive ◽  
Spatiotemporal Resolution ◽  
Site Specific ◽  
Guide Rna ◽  
Dna Cleavage Activity ◽  
Versatile Tool

AbstractHere we introduce RNA-CLAMP, a technology which enables site-specific and enzymatic cross-linking (clamping) of two selected stem loops within an RNA of interest. Intramolecular clamping of the RNA can disrupt normal RNA function, whereas subsequent photo-cleavage of the crosslinker restores activity. We applied the RNA-CLAMP technique to the single guide RNA of the CRISPR-Cas9 gene editing system. By clamping two stem loops of the single-guide RNA (sgRNA) with a photo-cleavable cross-linker, gene editing was completely silenced. Visible light irradiation cleaved the crosslinker and restored gene editing with high spatiotemporal resolution. Furthermore, by designing two photo-cleavable linkers which are responsive to different wavelength of lights, we achieved multiplexed photo-activation of gene editing in mammalian cells. Notably, although the Cas9-sgRNA RNP is not capable of DNA cleavage activity upon clamping, it maintained the capability to bind to the target DNA. The RNA-CLAMP enabled photo-activated CRISPR-Cas9 gene editing platform offers clean background, free choice of activation wavelength and multiplexing capability. We believe that this technology to precisely and rapidly control gene editing will serve as a versatile tool in the future development of stimuli responsive gene editing technologies. Beyond gene editing, RNA-CLAMP provides a site-specific tool for manipulating the internal structure of functional RNAs.

scholarly journals Random-PE: an efficient integration of random sequences into mammalian genome by prime editing

2021 ◽  
Vol 2 (1) ◽  
Author(s):  
Yaoge Jiao ◽  
Lifang Zhou ◽  
Rui Tao ◽  
Yanhong Wang ◽  
Yun Hu ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Mammalian Cells ◽  
Fine Tuning ◽  
Random Sequences ◽  
Strand Breaks ◽  
Guide Rna ◽  
Simple Strategy ◽  
Protospacer Adjacent Motif ◽  
Efficient Integration

AbstractPrime editing (PE) enables efficiently targeted introduction of multiple types of small-sized genetic change without requiring double-strand breaks or donor templates. Here we designed a simple strategy to introduce random DNA sequences into targeted genomic loci by prime editing, which we named random prime editing (Random-PE). In our strategy, the prime editing guide RNA (pegRNA) was engineered to harbor random sequences between the primer binding sequence (PBS) and homologous arm (HA) of the reverse transcriptase templates. With these pegRNAs, we achieved efficient targeted insertion or substitution of random sequences with different lengths, ranging from 5 to 10, in mammalian cells. Importantly, the diversity of inserted sequences is well preserved. By fine-tuning the design of random sequences, we were able to make simultaneously insertions or substitutions of random sequences in multiple sites, allowing in situ evolution of multiple positions in a given protein. Therefore, these results provide a framework for targeted integration of random sequences into genomes, which can be redirected for manifold applications, such as in situ protospacer adjacent motif (PAM) library construction, enhancer screening, and DNA barcoding.

scholarly journals Functional Identification of the Xanthomonas oryzae pv. oryzae Type I-C CRISPR-Cas System and Its Potential in Gene Editing Application

2021 ◽  
Vol 12 ◽  
Author(s):  
Qibing Liu ◽  
Siwei Wang ◽  
Juying Long ◽  
Zhuoyue Chen ◽  
Bing Yang ◽  
...  
Keyword(s):  
Pseudomonas Syringae ◽  
Gene Editing ◽  
Xanthomonas Oryzae ◽  
Functional Study ◽  
Target Sequence ◽  
Type I ◽  
Functional Identification ◽  
Guide Rna ◽  
Protospacer Adjacent Motif ◽  
New Vision

The type I clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system is one of five adaptive immune systems and exists widely in bacteria and archaea. In this study, we showed that Xanthomonas oryzae pv. oryzae (Xoo) possesses a functional CRISPR system by engineering constructs mimicking its CRISPR cassette. CRISPR array analysis showed that the TTC at the 5′-end of the target sequence is a functional protospacer-adjacent motif (PAM) of CRISPR. Guide RNA (gRNA) deletion analysis identified a minimum of 27-bp spacer that was required to ensure successful self-target killing in PXO99A strain. Mutants with deletion of individual Cas genes were constructed to analyze the effects of Cas proteins on mature CRISPR RNA (crRNA), processing intermediates and DNA interference. Results showed that depleting each of the three genes, cas5d, csd1, and csd2 inactivated the pre-crRNA processing, whereas inactivation of cas3 impaired in processing pre-crRNA. Furthermore, the Xoo CRISPR/Cas system was functional in Pseudomonas syringae pv. tomato. Collectively, our results would contribute to the functional study of CRISPR/Cas system of Xoo, and also provide a new vision on the use of bacterial endogenous systems as a convenient tool for gene editing.

scholarly journals CRISPR/dCas9-Based Systems: Mechanisms and Applications in Plant Sciences

Plants ◽  
2021 ◽  
Vol 10 (10) ◽  
pp. 2055
Author(s):  
Chou Khai Soong Karlson ◽  
Siti Nurfadhlina Mohd-Noor ◽  
Nadja Nolte ◽  
Boon Chin Tan
Keyword(s):  
Genetically Modified Organisms ◽  
Crop Improvement ◽  
Gene Activation ◽  
Delivery Methods ◽  
Guide Rna ◽  
Functional Studies ◽  
Protospacer Adjacent Motif ◽  
Efficient Delivery ◽  
Potential Applications ◽  
Transcriptional Modulation

RNA-guided genomic transcriptional regulation tools, namely clustered regularly interspaced short palindromic repeats interference (CRISPRi) and CRISPR-mediated gene activation (CRISPRa), are a powerful technology for gene functional studies. Deriving from the CRISPR/Cas9 system, both systems consist of a catalytically dead Cas9 (dCas9), a transcriptional effector and a single guide RNA (sgRNA). This type of dCas9 is incapable to cleave DNA but retains its ability to specifically bind to DNA. The binding of the dCas9/sgRNA complex to a target gene results in transcriptional interference. The CRISPR/dCas9 system has been explored as a tool for transcriptional modulation and genome imaging. Despite its potential applications and benefits, the challenges and limitations faced by the CRISPR/dCas9 system include the off-target effects, protospacer adjacent motif (PAM) sequence requirements, efficient delivery methods and the CRISPR/dCas9-interfered crops being labeled as genetically modified organisms in several countries. This review highlights the progression of CRISPR/dCas9 technology as well as its applications and potential challenges in crop improvement.

scholarly journals Enhanced Cas12a multi-gene regulation using a CRISPR array separator

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Jens P Magnusson ◽  
Antonio Ray Rios ◽  
Lingling Wu ◽  
Lei S Qi
Keyword(s):  
Mammalian Cells ◽  
Gene Editing ◽  
Gc Content ◽  
Guide Rna ◽  
Naturally Occurring ◽  
Crispr Array ◽  
Endogenous Genes ◽  
Simultaneous Activation ◽  
Type V ◽  
Array Performance

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.

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