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Shaoya Li, Jingying Li, Yubing He, Meilian Xu, Jiahui Zhang, Wenming Du, Yunde Zhao, Lanqin Xia
One of the main obstacles to gene replacement in plants is efficient delivery of a donor repair template (DRT) into the nucleus for homology-directed DNA repair (HDR) of double-stranded DNA breaks. Production of RNA templates in vivo for transcript-templated HDR (TT-HDR) could overcome this problem, but primary transcripts are often processed and transported to the cytosol, rendering them unavailable for HDR. We show that coupling CRISPR-Cpf1 (CRISPR from Prevotella and Francisella 1) to a CRISPR RNA (crRNA) array flanked with ribozymes, along with a DRT flanked with either ribozymes or crRNA targets, produces primary transcripts that self-process to release the crRNAs and DRT inside the nucleus...
March 18, 2019: Nature Biotechnology
Yuanyue Shan, Xiaoming Zhou, Ru Huang, Da Xing
MicroRNAs (miRNAs) are short noncoding RNAs that post-transcriptionally regulate gene expression. It has been proved that the aberrant expression of miRNAs is related to disease and miRNAs can serve as potential biomarkers for early tumor diagnosis. Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas13a is recently discovered CRISPR-RNA (crRNA) guided RNA manipulation tools. The recognition of target RNA can morphologically activate the robust non-specific trans ribonuclease activity of Cas13a...
March 15, 2019: Analytical Chemistry
MaryClare F Rollins, Saikat Chowdhury, Joshua Carter, Sarah M Golden, Heini M Miettinen, Andrew Santiago-Frangos, Dominick Faith, C Martin Lawrence, Gabriel C Lander, Blake Wiedenheft
Bacteria and archaea have evolved sophisticated adaptive immune systems that rely on CRISPR RNA (crRNA)-guided detection and nuclease-mediated elimination of invading nucleic acids. Here, we present the cryo-electron microscopy (cryo-EM) structure of the type I-F crRNA-guided surveillance complex (Csy complex) from Pseudomonas aeruginosa bound to a double-stranded DNA target. Comparison of this structure to previously determined structures of this complex reveals a ∼180-degree rotation of the C-terminal helical bundle on the "large" Cas8f subunit...
March 1, 2019: Molecular Cell
Syed Zawar Shah, Anum Rehman, Hira Nasir, Azka Asif, Bakhtawar Tufail, Muhammad Usama, Basit Jabbar
BACKGROUND: The current era of genome engineering has been revolutionized by the evolution of a bacterial adaptive immune system, CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) into a radical technology that is making an expeditious progress in its mechanism, function and applicability.. METHODS: A systematic literature review study was carried out with the help of all available information and online resources.. RESULTS: In this review, we intend to elucidate different aspects of CRISPR in the light of current advancements...
January 2019: Journal of Ayub Medical College, Abbottabad: JAMC
Hyo Young Kim, Seong Jae Kang, Yongmoon Jeon, Jinsu An, Jihyun Park, Hee Jae Lee, Jeong-Eun Jang, JongSeong Ahn, Duhee Bang, Hak Suk Chung, Cherlhyun Jeong, Dae-Ro Ahn
We demonstrated that 19 out of 20 RNA residues in the guide region of crRNA can be replaced with DNA residues with high GC-contents. The cellular activity of the chimeric crRNAs to disrupt the target gene was comparable to that of the native crRNA.
March 7, 2019: Chemical Communications: Chem Comm
Yufeng Liu, Hongpan Xu, Chang Liu, Lijun Peng, Haroon Khan, Lunbiao Cui, Rui Huang, Chao Wu, Sisi Shen, Su Wang, Wenbiao Liang, Zhiyang Li, Benbo Xu, Nongyue He
It is urgent to find an avian influenza A H7N9 detection simple method which is suitable for on-site detection. The Cas13a protein just likes a nanomachine, when specifically bound to target RNA by single-stranded RNA (crRNA), changes its protein structure and produces RNase activity, which degrades RNA non-specifically. Harnessing Cas13a, the paper aims to establish an underlying on-site H7N9 virus nucleic acid detection method. LwCas13a protein nanomachine was expressed in a prokaryotic expression system and purified by nickel column...
April 1, 2019: Journal of Biomedical Nanotechnology
Irmantas Mogila, Migle Kazlauskiene, Skaiste Valinskyte, Giedre Tamulaitiene, Gintautas Tamulaitis, Virginijus Siksnys
The type III-A Csm complex of Streptococcus thermophilus (StCsm) provides immunity against invading nucleic acids through the coordinated action of three catalytic domains: RNase (Csm3), ssDNase (Cas10-HD), and cyclic oligoadenylates synthase (Cas10-Palm). The matured StCsm complex is composed of Cas10:Csm2:Csm3:Csm4:Csm5 subunits and 40-nt CRISPR RNA (crRNA). We have carried out gene disruptions for each subunit and isolated deletion complexes to reveal the role of individual subunits in complex assembly and function...
March 5, 2019: Cell Reports
Minghui Guo, Kaiming Zhang, Yuwei Zhu, Grigore D Pintilie, Xiaoyu Guan, Shanshan Li, Michael F Schmid, Zhuo Ma, Wah Chiu, Zhiwei Huang
The type III CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated genes) systems are bacterially encoded adaptive immune systems for defense against invading nucleic acids. They accomplish this task through the coordinated cleavage of invading substrates of single-stranded RNA and DNA (ssDNA and ssRNA) by the Csm (type III-A) or Cmr (type III-B) effector complexes. The ssRNA is complementarily bound to the CRISPR RNA (crRNA). However, the structural basis for the DNase and RNase activation of the Csm nucleoprotein complex is largely unknown...
February 27, 2019: Cell Research
Bryan W Dorsey, Lei Huang, Alfonso Mondragón
Clustered regularly interspaced short palindromic repeats (CRISPR) and their associated Cas proteins provide an immune-like response in many prokaryotes against extraneous nucleic acids. CRISPR-Cas systems are classified into different classes and types. Class 1 CRISPR-Cas systems form multi-protein effector complexes that includes a guide RNA (crRNA) used to identify the target for destruction. Here we present crystal structures of Staphylococcus epidermidis Type III-A CRISPR subunits Csm2 and Csm3 and a 5...
February 13, 2019: Nucleic Acids Research
Fei Teng, Jing Li, Tongtong Cui, Kai Xu, Lu Guo, Qingqin Gao, Guihai Feng, Chuanyuan Chen, Dali Han, Qi Zhou, Wei Li
CRISPR-Cas12a/Cpf1, a single RNA-guided endonuclease system, provides a promising tool for genome engineering. However, only three Cas12a orthologs have been employed for mammalian genome editing, and the editing efficiency as well as targeting coverage still requires improvements. Here, we harness six novel Cas12a orthologs for genome editing in human and mouse cells, some of which utilize simple protospacer adjacent motifs (PAMs) that remarkably increase the targeting range in the genomes. Moreover, we identify optimized CRISPR RNA (crRNA) scaffolds that can increase the genome editing efficiency of Cas12a...
February 5, 2019: Genome Biology
Phong T Phan, Michael Schelling, Chaoyou Xue, Dipali G Sashital
Type I, II, and V CRISPR-Cas systems are RNA-guided dsDNA targeting defense mechanisms found in bacteria and archaea. During CRISPR interference, Cas effectors use CRISPR-derived RNAs (crRNAs) as guides to bind complementary sequences in foreign dsDNA, leading to the cleavage and destruction of the DNA target. Mutations within the target or in the protospacer adjacent motif can reduce the level of CRISPR interference, although the level of defect is dependent on the type and position of the mutation, as well as the guide sequence of the crRNA...
2019: Methods in Enzymology
Mu-Sen Liu, Shanzhong Gong, Helen-Hong Yu, David W Taylor, Kenneth A Johnson
Bacterial adaptive immune systems employ clustered regularly interspaced short palindromic repeats (CRISPR) along with their CRISPR-associated genes (Cas) to form CRISPR RNA (crRNA)-guided surveillance complexes, which target foreign nucleic acids for destruction. Cas9 is unique in that it is composed of a single polypeptide that utilizes both a crRNA and a trans-activating crRNA (tracrRNA) or a single guide RNA to create double-stranded breaks in sequences complementary to the RNA via the HNH and RuvC nuclease domains...
2019: Methods in Enzymology
Yibei Xiao, Ailong Ke
Type I CRISPR-Cas, the most prevalent CRISPR system, features a sequential target searching and degradation process. First, the multisubunit surveillance complex Cascade recognizes the matching dsDNA target flanked by protospacer adjacent motif (PAM), promotes the heteroduplex formation between CRISPR RNA (crRNA) and the target strand (TS) DNA, and displaces the nontarget strand (NTS) DNA, resulting in R-loop formation. The helicase-nuclease fusion enzyme Cas3 is then specifically recruited to Cascade/R-loop, nicks, and processively degrades the DNA target...
2019: Methods in Enzymology
Christophe Rouillon, Januka S Athukoralage, Shirley Graham, Sabine Grüschow, Malcolm F White
Type III CRISPR effector complexes utilize a bound CRISPR RNA (crRNA) to detect the presence of RNA from invading mobile genetic elements in the cell. This RNA binding results in the activation of two enzymatic domains of the Cas10 subunit-the HD nuclease domain, which degrades DNA, and PALM/cyclase domain. The latter synthesizes cyclic oligoadenylate (cOA) molecules by polymerizing ATP, and cOA acts as a second messenger in the cell, switching on the antiviral response by activating host ribonucleases and other proteins...
2019: Methods in Enzymology
Kwang-Hyun Park, Yan An, Eui-Jeon Woo
The CRISPR-Cas system is the prokaryotic immune response that destroys invading foreign nucleic acids. Based on the architecture and distinct mechanism of targeting, the CRISPR-Cas system is classified into six types (I-VI). The Csm complex belongs to the type III system and consists of five subunits (Cas10 and Csm2-5) and a crRNA. The Csm complex targets RNA and RNA-dependent single-strand DNA. Here, we present a protocol for in vitro reconstitution of a Csm complex from a hyperthermophilic archaeon Thermococcus onnurineus NA1 (ToCsm complex)...
2019: Methods in Enzymology
Bartosz Turkowyd, Hanna Müller-Esparza, Vanessa Climenti, Niklas Steube, Ulrike Endesfelder, Lennart Randau
Type I CRISPR-Cas systems utilize small CRISPR RNA (crRNA) molecules to scan DNA strands for target regions. Different crRNAs are bound by several CRISPR-associated (Cas) protein subunits that form the stable ribonucleoprotein complex Cascade. The Cascade-mediated DNA surveillance process requires a sufficient degree of base-complementarity between crRNA and target sequences and relies on the recognition of small DNA motifs, termed protospacer adjacent motifs. Recently, super-resolution microscopy and single-particle tracking methods have been developed to follow individual protein complexes in live cells...
2019: Methods in Enzymology
Daijiro Takeshita, Momoe Sato, Hideko Inanaga, Tomoyuki Numata
Clustered regularly interspaced short palindromic repeat (CRISPR) loci and CRISPR-associated (Cas) genes encode CRISPR RNAs (crRNA) and Cas proteins, respectively, which play important roles in the adaptive immunity system (CRISPR-Cas system) in prokaryotes. The crRNA and Cas proteins form ribonucleoprotein effector complexes to capture and degrade invading genetic materials with base complementarity to the crRNA guide sequences. The Csm complex, a type III-A effector complex, comprises five Cas proteins (Csm1-Csm5) and a crRNA, which co-transcriptionally degrades invading DNA and RNA...
January 10, 2019: Journal of Molecular Biology
Xu Tang, Qiurong Ren, Lijia Yang, Yu Bao, Zhaohui Zhong, Yao He, Shishi Liu, Caiyan Qi, Binglin Liu, Yan Wang, Simon Sretenovic, Yingxiao Zhang, Xuelian Zheng, Tao Zhang, Yiping Qi, Yong Zhang
CRISPR-Cas9 and Cas12a are two powerful genome editing systems. Expression of CRISPR in plants is typically achieved with a mixed dual promoter system, in which Cas protein is expressed by a Pol II promoter and a guide RNA is expressed by a species-specific Pol III promoter such as U6 or U3. To achieve coordinated expression and compact vector packaging, it is desirable to express both CRISPR components under a single Pol II promoter. Previously, we demonstrated a first-generation single transcript unit (STU)-Cas9 system, STU-Cas9-RZ, which is based on hammerhead ribozyme for processing single guide RNAs (sgRNAs)...
December 24, 2018: Plant Biotechnology Journal
Bin Li, Chunxi Zeng, Wenqing Li, Xinfu Zhang, Xiao Luo, Weiyu Zhao, Chengxiang Zhang, Yizhou Dong
Previously, researchers discovered a series of anti-CRISPR proteins that inhibit CRISPR-Cas activity, such as Cas9 and Cpf1 (Cas12a). Herein, we constructed crRNA variants consisting of chemically modified DNA-crRNA and RNA-crRNA duplexes and identified that phosphorothioate (PS)-modified DNA-crRNA duplex completely blocked the function of Cpf1. More important, without prehybridization, these PS-modified DNA oligonucleotides showed the ability to suppress DNA double-strand breaks induced by two Cpf1 orthologs, AsCpf1 and LbCpf1...
December 18, 2018: Cell Reports
Konrad Schwefel, Stefanie Spiegler, Sabine Ameling, Christiane D Much, Robin A Pilz, Oliver Otto, Uwe Völker, Ute Felbor, Matthias Rath
CCM3, originally described as PDCD10, regulates blood-brain barrier integrity and vascular maturation in vivo. CCM3 loss-of-function variants predispose to cerebral cavernous malformations (CCM). Using CRISPR/Cas9 genome editing, we here present a model which mimics complete CCM3 inactivation in cavernous endothelial cells (ECs) of heterozygous mutation carriers. Notably, we established a viral- and plasmid-free crRNA:tracrRNA:Cas9 ribonucleoprotein approach to introduce homozygous or compound heterozygous loss-of-function CCM3 variants into human ECs and studied the molecular and functional effects of long-term CCM3 inactivation...
December 13, 2018: Journal of Cellular and Molecular Medicine
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