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Sonali Majumdar, Michael P Terns
Diverse CRISPR-Cas immune systems protect archaea and bacteria from viruses and other mobile genetic elements. All CRISPR-Cas systems ultimately function by sequence-specific destruction of invading complementary nucleic acids. However, each CRISPR system uses compositionally distinct crRNP [CRISPR (cr) RNA/Cas protein] immune effector complexes to recognize and destroy invasive nucleic acids by unique molecular mechanisms. Previously, we found that Type I-A (Csa) effector crRNPs from Pyrococcus furiosus function in vivo to eliminate invader DNA...
October 3, 2018: Extremophiles: Life Under Extreme Conditions
Li Lin, Xiubin He, Tianyuan Zhao, Lingkai Gu, Yeqing Liu, Xiaoyu Liu, Hongyan Liu, Fayu Yang, Mengjun Tu, Lianchao Tang, Xianglian Ge, Changbao Liu, Junzhao Zhao, Zongming Song, Jia Qu, Feng Gu
FnCpf1-mediated genome-editing technologies have enabled a broad range of research and medical applications. Recently, we reported that FnCpf1 possesses activity in human cells and recognizes a more compatible PAM (protospacer adjacent motif, 5'-KYTV-3'), compared with the other two commonly used Cpf1 enzymes (AsCpf1 and LbCpf1), which requires a 5'-TTTN-3' PAM. However, due to the efficiency and fidelity, FnCpf1-based clinical and basic applications remain a challenge. The direct repeat (DR) sequence is one of the key elements for FnCpf1-mediated genome editing...
September 1, 2018: Molecular Therapy: the Journal of the American Society of Gene Therapy
Mollie S Schubert, Edward Cedrone, Barry Neun, Mark A Behlke, Marina A Dobrovolskaia
Objectives: CRISPR/Cas9 is currently the primary tool used for genome editing in mammalian cells. To cleave and alter genomic DNA, both the Cas9 nuclease and a guide RNA ( gRNA ) must be present in the nucleus. One preferred method of introducing these reagents is direct transfection of a recombinant Cas9 protein complexed with a synthetic gRNA as a ribonucleoprotein (RNP) complex. It is well established from prior work in RNA interference that synthetic RNAs can induce a type I interferon (IFN) response that can limit the application of such methods both in vitro and in vivo...
2018: Journal of cytokine biology
Morito Kurata, Natalie K Wolf, Walker S Lahr, Madison T Weg, Mitchell G Kluesner, Samantha Lee, Kai Hui, Masano Shiraiwa, Beau R Webber, Branden S Moriarity
The CRISPR/Cas9 system is an RNA guided nuclease system that evolved as a mechanism of adaptive immunity in bacteria. This system has been adopted for numerous genome engineering applications in research and recently, therapeutics. The CRISPR/Cas9 system has been largely implemented by delivery of Cas9 as protein, RNA, or plasmid along with a chimeric crRNA-tracrRNA guide RNA (gRNA) under the expression of a pol III promoter, such as U6. Using this approach, multiplex genome engineering has been achieved by delivering several U6-gRNA plasmids targeting multiple loci...
2018: PloS One
Zhen-Hai Li, Min Liu, Xiao-Mei Lyu, Feng-Qing Wang, Dong-Zhi Wei
In this study, we focused on the applicability of CRISPR/Cpf1 in genome simplification of Saccharomyces cerevisiae and established a CRISPR/Cpf1 assisted method for rapid markerless large fragment deletion to facilitate laboratory evolution of geome of S. cerevisiae by rational genetic engineering. This method uses a Cpf1 expression plasmid and a crRNA array expression plasmid. The DNA fragment between two DSBs generated by CRISPR/Cpf1 can be cut off from the chromosome, along with re-ligation of the genomic endpoints of the DSBs...
September 9, 2018: Journal of Basic Microbiology
Su Bin Moon, Jeong Mi Lee, Jeong Gu Kang, Nan-Ee Lee, Dae-In Ha, Do Yon Kim, Sun Hee Kim, Kwangsun Yoo, Daesik Kim, Jeong-Heon Ko, Yong-Sam Kim
Genome editing has been harnessed through the development of CRISPR system, and the CRISPR from Prevotella and Francisella 1 (Cpf1) system has emerged as a promising alternative to CRISPR-Cas9 for use in various circumstances. Despite the inherent multiple advantages of Cpf1 over Cas9, the adoption of Cpf1 has been unsatisfactory because of target-dependent insufficient indel efficiencies. Here, we report an engineered CRISPR RNA (crRNA) for highly efficient genome editing by Cpf1, which includes a 20-base target-complementary sequence and a uridinylate-rich 3'-overhang...
September 7, 2018: Nature Communications
Lisa Nickel, Andrea Ulbricht, Omer S Alkhnbashi, Konrad U Förstner, Liam Cassidy, Katrin Weidenbach, Rolf Backofen, Ruth A Schmitz
The clustered regularly interspaced short palindromic repeat (CRISPR) system is a prokaryotic adaptive defense system against foreign nucleic acids. In the methanoarchaeon Methanosarcina mazei Gö1, two types of CRISPR-Cas systems are present (type I-B and type III-C). Both loci encode a Cas6 endonuclease, Cas6b-IB and Cas6b-IIIC, typically responsible for maturation of functional short CRISPR RNAs (crRNAs). To evaluate potential cross cleavage activity, we biochemically characterized both Cas6b proteins regarding their crRNA binding behavior and their ability to process pre-crRNA from the respective CRISPR array in vivo...
September 13, 2018: RNA Biology
Hyo Min Park, Hui Liu, Joann Wu, Anthony Chong, Vanessa Mackley, Christof Fellmann, Anirudh Rao, Fuguo Jiang, Hunghao Chu, Niren Murthy, Kunwoo Lee
Engineering of the Cpf1 crRNA has the potential to enhance its gene editing efficiency and non-viral delivery to cells. Here, we demonstrate that extending the length of its crRNA at the 5' end can enhance the gene editing efficiency of Cpf1 both in cells and in vivo. Extending the 5' end of the crRNA enhances the gene editing efficiency of the Cpf1 RNP to induce non-homologous end-joining and homology-directed repair using electroporation in cells. Additionally, chemical modifications on the extended 5' end of the crRNA result in enhanced serum stability...
August 17, 2018: Nature Communications
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. Since the early 1990s, the predominant strategy utilized in human tissue culture cells has been RNA interference (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 underutilized for conducting visual phenotypic analyses, particularly for essential genes that require conditional strategies to eliminate their gene products...
October 1, 2018: Molecular Biology of the Cell
Dan Wang, Dejun Ma, Jingxin Han, Linghao Kong, Lu-Yuan Li, Zhen Xi
To achieve multisite-targeting-based DNA cleavage simultaneously, we designed two kinds of CRISPR RNA arrays by fusing four single guide RNAs (sgRNAs for Cas9 or crRNAs for Cpf1) with uncleavable RNA linkers (CRISPRay). The CRISPRay could operate on four adjacent target sites to cleave target DNA in a collaborative manner. Two CRISPR RNA arrays demonstrated robust inactivation of the firefly luciferase gene in living cells. In vitro DNA cleavage and DNA sequencing also verified that sgRNA arrays directed SpCas9 nuclease to cut target DNA at four cleavage sites simultaneously whereas crRNA-array-guided FnCpf1 nuclease showed target-activated, nonspecific DNase activity on both target DNA and nontarget 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...
October 20, 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...
August 28, 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
Shengwei Hou, Manuel Brenes-Álvarez, Viktoria Reimann, Omer S Alkhnbashi, Rolf Backofen, Alicia M Muro-Pastor, Wolfgang R Hess
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...
August 15, 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 a strong capability for producing a large number of bioactive natural products and remains invaluable as a source for the discovery of novel drug leads. Although the Streptococcus pyogenes 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...
September 15, 2018: Applied and Environmental Microbiology
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