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Tim Künne, Yifan Zhu, Fausia da Silva, Nico Konstantinides, Rebecca E McKenzie, Ryan N Jackson, Stan Jj Brouns
Prokaryotes use primed CRISPR adaptation to update their memory bank of spacers against invading genetic elements that have escaped CRISPR interference through mutations in their protospacer target site. We previously observed a trend that nucleotide-dependent mismatches between crRNA and the protospacer strongly influence the efficiency of primed CRISPR adaptation. Here we show that guanine-substitutions in the target strand of the protospacer are highly detrimental to CRISPR interference and interference-dependent priming, while cytosine-substitutions are more readily tolerated...
August 11, 2018: Nucleic Acids Research
Guilhem Faure, Sergey A Shmakov, Kira S Makarova, Yuri I Wolf, Alexandra B Crawley, Rodolphe Barrangou, Eugene V Koonin
Trans-activating CRISPR (tracr) RNA is a distinct RNA species that interacts with the CRISPR (cr) RNA to form the dual guide (g) RNA in type II and subtype V-B CRISPR-Cas systems. The tracrRNA-crRNA interaction is essential for pre-crRNA processing as well as target recognition and cleavage. The tracrRNA consists of an antirepeat, which forms an imperfect hybrid with the repeat in the crRNA, and a distal region containing a Rho-independent terminator. Exhaustive comparative analysis of the sequences and predicted structures of the Class 2 CRISPR guide RNAs shows that all these guide RNAs share distinct structural features, in particular, the nexus stem-loop that separates the repeat-antirepeat hybrid from the distal portion of the tracrRNA and the conserved GU pair at that end of the hybrid...
August 14, 2018: RNA Biology
Kuan-Chung Su, Mary-Jane Tsang, Neil Emans, Iain M Cheeseman
A key goal for cell biological analyses is to assess the phenotypes that result from eliminating a target gene. For the past 25 years, the predominant strategy utilized in human tissue culture cells has been RNAi-mediated protein depletion. However, RNAi suffers well-documented off-target effects as well as incomplete and reversible protein depletion. The implementation of CRISPR/Cas9-based DNA cleavage has revolutionized the capacity to conduct functional studies in human cells. However, this approach is still under-utilized for conducting visual phenotypic analyses, particularly for essential genes that require conditional strategies to eliminate their gene products...
August 9, 2018: Molecular Biology of the Cell
Dan Wang, Dejun Ma, Jingxin Han, Linghao Kong, Luyuan Li, Zhen Xi
To achieve multisite targeting-based DNA cleavage simultaneously, we designed two kinds of CRISPR RNA arrays by fusing four single sgRNAs or crRNAs with uncleavable RNA linkers (CRISPRay). CRISPRay could operate on four adjacent target sites to cleave target DNA in a collaborative manner. Two CRISPR RNA arrays demonstrated robust inactivation to the firefly luciferase gene in living cells. In vitro DNA cleavage and DNA sequencing also validated that sgRNA arrays directed SpCas9 nuclease to cut target DNA at four cleavage sites simultaneously while crRNA array-guided FnCpf1 nuclease showed target-activated, non-specific DNase activity on both target DNA and non-target DNA at random sites...
August 7, 2018: Chembiochem: a European Journal of Chemical Biology
Jie Zhang, Wei Hong, Wenming Zong, Pixiang Wang, Yi Wang
CRISPR-Cpf1 is a type V CRISPR system that has recently been exploited for genome engineering purposes. Compared to the well-known Streptococcus pyogenes CRISPR-Cas9 system, the effector protein Cpf1 recognizes T-rich protospacer-adjacent motif (PAM) instead of G-rich PAM (used by CRISPR-Cas9), which could offer a substantial expansion of the existing genetic toolbox for genome editing. In this study, we report the implementation of the Acidaminococcus sp. Cpf1 (AsCpf1) for markerless genome engineering in Clostridium beijerinckii, a prominent species for biosolvent production through the well-known Acetone-Butanol-Ethanol (ABE) pathway...
August 3, 2018: Journal of Biotechnology
Mario Hupfeld, Despoina Trasanidou, Livia Ramazzini, Jochen Klumpp, Martin J Loessner, Samuel Kilcher
CRISPR-Cas systems provide bacteria with adaptive immunity against invading DNA elements including bacteriophages and plasmids. While CRISPR technology has revolutionized eukaryotic genome engineering, its application to prokaryotes and their viruses remains less well established. Here we report the first functional CRISPR-Cas system from the genus Listeria and demonstrate its native role in phage defense. LivCRISPR-1 is a type II-A system from the genome of L. ivanovii subspecies londoniensis that uses a small, 1078 amino acid Cas9 variant and a unique NNACAC protospacer adjacent motif...
July 27, 2018: Nucleic Acids Research
Seung Min Kim, Sang Chul Shin, Eunice EunKyeong Kim, Sang-Heon Kim, Kwideok Park, Seung Ja Oh, Mihue Jang
Cas9 ribonucleoprotein (RNP)-mediated delivery has emerged as an ideal approach for in vivo applications. However, the delivery of Cas9 RNPs requires electroporation or lipid- or cationic-reagent-mediated transfection. Here, we developed a carrier-free Cas9 RNP delivery system for robust gene editing in vivo. For simultaneous delivery of Cas9 and a guide RNA into target cells without the aid of any transfection reagents, we established a multifunctional Cas9 fusion protein (Cas9-LMWP) that forms a ternary complex with synthetic crRNA:tracrRNA hybrids in a simple procedure...
July 26, 2018: ACS Nano
Xuhua Zhang, Linping Xu, Ruihua Fan, Quanli Gao, Yunfeng Song, Xiaodong Lyu, Jiangtao Ren, Yongping Song
Cpf1, an RNA-guided DNA endonuclease that belongs to a new class II CRISPR system, has recently been harnessed for genome editing. Herein, we report an RNase-resistant caged truncated pre-tRNA-like crRNA (catRNA) that confers precise and efficient gene editing with the Lachnospiraceae bacterium Cpf1 (LbCpf1) and enables the reprogramming of catalytically dead LbCpf1 (dCpf1) lacking DNA endonuclease activity into a transcriptional modulator. Specific gene knock-outs and knock-ins were increased 3.2-fold and 4...
2018: Cell Discovery
Wolfgang R Hess, Shengwei Hou, Manuel Brenes-Álvarez, Viktoria Reimann, Omer S Alkhnbashi, Rolf Backofen, Alicia M Muro-Pastor
Novel CRISPR-Cas systems possess substantial potential for genome editing and manipulation of gene expression. The types and numbers of CRISPR-Cas systems vary substantially between different organisms. Some filamentous cyanobacteria harbor >40 different putative CRISPR repeat-spacer cassettes, while the number of cas gene instances is much lower. Here we addressed the types and diversity of CRISPR-Cas systems and of CRISPR-like repeat-spacer arrays in 171 publicly available genomes of multicellular cyanobacteria...
July 11, 2018: RNA Biology
Kawanda Foster, Joshua Kalter, Walter Woodside, Rebecca M Terns, Michael P Terns
CRISPR-Cas systems provide prokaryotes with RNA-based adaptive immunity against viruses and plasmids. A unique feature of Type III CRISPR-Cas systems is that they selectively target transcriptionally-active invader DNA, and can cleave both the expressed RNA transcripts and source DNA. The Type III-A effector crRNP (CRISPR RNA-Cas protein complex), which contains Cas proteins Csm1-5, recognizes and degrades invader RNA and DNA in a crRNA-guided, manner. Interestingly, Type III-A systems also employ Csm6, an HEPN family ribonuclease that does not stably associate with the Type III-A effector crRNP, but nevertheless contributes to defense via mechanistic details that are still being determined...
August 1, 2018: RNA Biology
Aamir Mir, Julia F Alterman, Matthew R Hassler, Alexandre J Debacker, Edward Hudgens, Dimas Echeverria, Michael H Brodsky, Anastasia Khvorova, Jonathan K Watts, Erik J Sontheimer
RNA-based drugs depend on chemical modifications to increase potency and to decrease immunogenicity in vivo. Chemical modification will likely improve the guide RNAs involved in CRISPR-Cas9-based therapeutics as well. Cas9 orthologs are RNA-guided microbial effectors that cleave DNA. Here, we explore chemical modifications at all positions of the crRNA guide and tracrRNA cofactor. We identify several heavily modified versions of crRNA and tracrRNA that are more potent than their unmodified counterparts. In addition, we describe fully chemically modified crRNAs and tracrRNAs (containing no 2'-OH groups) that are functional in human cells...
July 6, 2018: Nature Communications
Lei Li, Keke Wei, Guosong Zheng, Xiaocao Liu, Shaoxin Chen, Weihong Jiang, Yinhua Lu
Streptomyces has strong capability to produce a large number of bioactive natural products and remains invaluable sources for the discovery of novel drug leads. Although the Streptococcus pyogenes ( Sp ) CRISPR-Cas9-assisted genome editing tool has been developed for rapid genetic engineering in Streptomyces , it has a number of limitations, including the toxicity of Sp Cas9 expression in some important industrial Streptomyces strains and the need for complex expression constructs when targeting multiple genomic loci...
July 6, 2018: Applied and Environmental Microbiology
Wenjing Wei, Shuai Zhang, Joy Fleming, Ying Chen, Zihui Li, Shanghua Fan, Yi Liu, Wei Wang, Ting Wang, Ying Liu, Baiguang Ren, Ming Wang, Jianjian Jiao, Yuanyuan Chen, Ying Zhou, Yafeng Zhou, Shoujin Gu, Xiaoli Zhang, Li Wan, Tao Chen, Lin Zhou, Yong Chen, Xian-En Zhang, Chuanyou Li, Hongtai Zhang, Lijun Bi
Clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas) systems are prokaryotic adaptive immune systems against invading nucleic acids. CRISPR locus variability has been exploited in evolutionary and epidemiological studies of Mycobacterium tuberculosis, the causative agent of tuberculosis, for over 20 yr, yet the biological function of this type III-A system is largely unexplored. Here, using cell biology and biochemical, mutagenic, and RNA-seq approaches, we show it is active in invader defense and has features atypical of type III-A systems: mature CRISPR RNA (crRNA) in its crRNA-CRISPR/Cas protein complex are of uniform length (∼71 nt) and appear not to be subject to 3'-end processing after Cas6 cleavage of repeat RNA 8 nt from its 3' end...
July 6, 2018: FASEB Journal: Official Publication of the Federation of American Societies for Experimental Biology
Keunsub Lee, Yingxiao Zhang, Benjamin P Kleinstiver, Jimmy A Guo, Martin J Aryee, Jonah Miller, Aimee Malzahn, Scott Zarecor, Carolyn J Lawrence-Dill, J Keith Joung, Yiping Qi, Kan Wang
CRISPR/Cas9 and Cas12a (Cpf1) nucleases are two of the most powerful genome editing tools in plants. In this work, we compared their activities by targeting maize glossy2 gene coding region that has overlapping sequences recognized by both nucleases. We introduced constructs carrying SpCas9-guide RNA (gRNA) and LbCas12a-CRISPR RNA (crRNA) into maize inbred B104 embryos using Agrobacterium-mediated transformation. On-target mutation analysis showed that 90%-100% of the Cas9-edited T0 plants carried indel mutations and 63%-77% of them were homozygous or biallelic mutants...
July 4, 2018: Plant Biotechnology Journal
Shaoya Li, Jingying Li, Jiahui Zhang, Wenming Du, Jindong Fu, Suhas Sutar, Yunde Zhao, Lanqin Xia
The recently developed CRISPR/Cpf1 system expands the range of genome editing and is emerging as an alternative powerful tool for both plant functional genomics and crop improvement. Cpf1-crRNA produces double strand DNA breaks (DSBs) with long 5' protruding ends, which may facilitate the pairing and insertion of repair templates through homology-directed repair (HDR) for targeted gene replacement and introduction of the desired DNA elements at specific gene loci for crop improvement. However, the potential mechanism underlying HDR of DSBs generated by Cpf1-crRNA remains to be investigated and the inherent low efficiency of HDR and poor availability of exogenous donor DNA as repair templates strongly impede the use of HDR for precise genome editing in crop plants...
June 28, 2018: Journal of Experimental Botany
Mitchell O'Connell
Prokaryotic adaptive immune systems use CRISPRs (Clustered Regularly Interspaced Short Palindromic Repeats) and CRISPR associated (Cas) proteins for RNA-guided cleavage of foreign genetic elements. The focus of this review, Type VI CRISPR-Cas systems, include a single protein known as Cas13 (formerly C2c2), that when assembled with a crRNA forms a crRNA-guided RNA-targeting effector complex. Type VI CRISPR-Cas systems can be divided into four subtypes (A-D) based on Cas13 phylogeny. All Cas13 proteins studied to date possess two enzymatically distinct ribonuclease activities that are required for optimal interference...
June 22, 2018: Journal of Molecular Biology
Shiraz A Shah, Omer S Alkhnbashi, Juliane Behler, Wenyuan Han, Qunxin She, Wolfgang R Hess, Roger A Garrett, Rolf Backofen
A study was undertaken to identify conserved proteins that are encoded adjacent to cas gene cassettes of Type III CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats - CRISPR associated) interference modules. Type III modules have been shown to target and degrade dsDNA, ssDNA and ssRNA and are frequently intertwined with cofunctional accessory genes, including genes encoding CRISPR-associated Rossman Fold (CARF) domains. Using a comparative genomics approach, and defining a Type III association score accounting for coevolution and specificity of flanking genes, we identified and classified 39 new Type III associated gene families...
June 19, 2018: RNA Biology
Huihui Sun, Fanfan Li, Jie Liu, Fayu Yang, Zhenhai Zeng, Xiujuan Lv, Mengjun Tu, Yeqing Liu, Xianglian Ge, Changbao Liu, Junzhao Zhao, Zongduan Zhang, Jia Qu, Zongming Song, Feng Gu
Cpf1 has been harnessed as a tool for genome manipulation in various species because of its simplicity and high efficiency. Our recent study demonstrated that FnCpf1 could be utilized for human genome editing with notable advantages for target sequence selection due to the flexibility of the protospacer adjacent motif (PAM) sequence. Multiplex genome editing provides a powerful tool for targeting members of multigene families, dissecting gene networks, modeling multigenic disorders in vivo, and applying gene therapy...
June 15, 2018: Molecular Therapy: the Journal of the American Society of Gene Therapy
Diego Bernal-Bernal, Javier Abellón-Ruiz, Antonio A Iniesta, Elena Pajares-Martínez, Eva Bastida-Martínez, Marta Fontes, S Padmanabhan, Montserrat Elías-Arnanz
Expression of CRISPR-Cas systems is a prerequisite for their defensive role against invading genetic elements. Yet, much remains unknown about how this crucial step is regulated. We describe a new mechanism controlling CRISPR-cas expression, which requires an extracytoplasmic function (ECF) σ factor (DdvS), its membrane-bound anti-σ (DdvA) and a global regulatory complex (CarD-CarG). Transcriptomic analyses revealed that the DdvS/CarD/CarG-dependent regulon comprises a type III-B CRISPR-Cas system in Myxococcus xanthus...
June 9, 2018: Nucleic Acids Research
Xiao Zhao, Liang Liu, Jiayan Lang, Keman Cheng, Yongwei Wang, Xueyan Li, Jian Shi, Yanli Wang, Guangjun Nie
Mutant KRAS is a known driver oncogene in pancreatic cancer. However, this protein remains an "undruggable" therapeutic target. Inhibiting mutated KRAS expression at the mRNA level is a potentially effective strategy. Recently, a novel CRISPR-Cas effector, Cas13a has been reported to specifically knock down mRNA expression under the guidance of a single CRISPR-RNA in mammalian cells. Here we demonstrate that the CRISPR-Cas13a system can be engineered for targeted therapy of mutant KRAS in pancreatic cancer...
September 1, 2018: Cancer Letters
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