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https://www.readbyqxmd.com/read/29765043/programmable-sequential-mutagenesis-by-inducible-cpf1-crrna-array-inversion
#1
Ryan D Chow, Hyunu Ray Kim, Sidi Chen
Mutations and genetic alterations are often sequentially acquired in various biological and pathological processes, such as development, evolution, and cancer. Certain phenotypes only manifest with precise temporal sequences of genetic events. While multiple approaches have been developed to model the effects of mutations in tumorigenesis, few recapitulate the stepwise nature of cancer evolution. Here we describe a flexible sequential mutagenesis system, Cpf1-Flip, with inducible inversion of a single crRNA array (FlipArray), and demonstrate its application in stepwise mutagenesis in murine and human cells...
May 15, 2018: Nature Communications
https://www.readbyqxmd.com/read/29735714/real-time-observation-of-dna-target-interrogation-and-product-release-by-the-rna-guided-endonuclease-crispr-cpf1-cas12a
#2
Digvijay Singh, John Mallon, Anustup Poddar, Yanbo Wang, Ramreddy Tippana, Olivia Yang, Scott Bailey, Taekjip Ha
CRISPR-Cas9, which imparts adaptive immunity against foreign genomic invaders in certain prokaryotes, has been repurposed for genome-engineering applications. More recently, another RNA-guided CRISPR endonuclease called Cpf1 (also known as Cas12a) was identified and is also being repurposed. Little is known about the kinetics and mechanism of Cpf1 DNA interaction and how sequence mismatches between the DNA target and guide-RNA influence this interaction. We used single-molecule fluorescence analysis and biochemical assays to characterize DNA interrogation, cleavage, and product release by three Cpf1 orthologs...
May 7, 2018: Proceedings of the National Academy of Sciences of the United States of America
https://www.readbyqxmd.com/read/29710373/transforming-plant-biology-and-breeding-with-crispr-cas9-cas12-and-cas13
#3
REVIEW
Patrick Schindele, Felix Wolter, Holger Puchta
Currently, biology is revolutionized by ever growing applications of the CRISPR/Cas system. As discussed in this Review, new avenues have opened up for plant research and breeding by the use of the sequence-specific DNases Cas9 and Cas12 (formerly named Cpf1) and, more recently, the RNase Cas13 (formerly named C2c2). Although double strand break-induced gene editing based on error-prone non-homologous end joining has been applied to obtain new traits, such as powdery mildew resistance in wheat or improved pathogen resistance and increased yield in tomato, improved technologies based on CRISPR/Cas for programmed change of plant genomes via homologous recombination have recently been developed...
April 30, 2018: FEBS Letters
https://www.readbyqxmd.com/read/29678175/gene-repression-via-multiplex-grna-strategy-in-y-lipolytica
#4
Jin-Lai Zhang, Yang-Zi Peng, Duo Liu, Hong Liu, Ying-Xiu Cao, Bing-Zhi Li, Chun Li, Ying-Jin Yuan
BACKGROUND: The oleaginous yeast Yarrowia lipolytica is a promising microbial cell factory due to their biochemical characteristics and native capacity to accumulate lipid-based chemicals. To create heterogenous biosynthesis pathway and manipulate metabolic flux in Y. lipolytica, numerous studies have been done for developing synthetic biology tools for gene regulation. CRISPR interference (CRISPRi), as an emerging technology, has been applied for specifically repressing genes of interest...
April 20, 2018: Microbial Cell Factories
https://www.readbyqxmd.com/read/29650467/chemically-modified-cpf1-crispr-rnas-mediate-efficient-genome-editing-in-mammalian-cells
#5
Moira A McMahon, Thazha P Prakash, Don W Cleveland, C Frank Bennett, Meghdad Rahdar
CRISPR-based gene editing is a powerful technology for engineering mammalian genomes. It holds the potential as a therapeutic, although much-needed in vivo delivery systems have yet to be established. Here, using the Cpf1-crRNA (CRISPR RNA) crystal structure as a guide, we synthesized a series of systematically truncated and chemically modified crRNAs, and identify positions that are amenable to modification while retaining gene-editing activity. Modified crRNAs were designed with the same modifications that provide protection against nucleases and enable wide distribution in vivo...
March 6, 2018: Molecular Therapy: the Journal of the American Society of Gene Therapy
https://www.readbyqxmd.com/read/29622802/design-and-assessment-of-engineered-crispr-cpf1-and-its-use-for-genome-editing
#6
Bin Li, Chunxi Zeng, Yizhou Dong
Cpf1, a CRISPR endonuclease discovered in Prevotella and Francisella 1 bacteria, offers an alternative platform for CRISPR-based genome editing beyond the commonly used CRISPR-Cas9 system originally discovered in Streptococcus pyogenes. This protocol enables the design of engineered CRISPR-Cpf1 components, both CRISPR RNAs (crRNAs) to guide the endonuclease and Cpf1 mRNAs to express the endonuclease protein, and provides experimental procedures for effective genome editing using this system. We also describe quantification of genome-editing activity and off-target effects of the engineered CRISPR-Cpf1 in human cell lines using both T7 endonuclease I (T7E1) assay and targeted deep sequencing...
May 2018: Nature Protocols
https://www.readbyqxmd.com/read/29617431/membrane-permeabilizing-amphiphilic-peptide-delivers-recombinant-transcription-factor-and-crispr-cas9-cpf1-ribonucleoproteins-in-hard-to-modify-cells
#7
Thomas Del'Guidice, Jean-Pascal Lepetit-Stoffaes, Louis-Jean Bordeleau, Joannie Roberge, Vanessa Théberge, Coraline Lauvaux, Xavier Barbeau, Jessica Trottier, Vibhuti Dave, Denis-Claude Roy, Bruno Gaillet, Alain Garnier, David Guay
Delivery of recombinant proteins to therapeutic cells is limited by a lack of efficient methods. This hinders the use of transcription factors or Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) ribonucleoproteins to develop cell therapies. Here, we report a soluble peptide designed for the direct delivery of proteins to mammalian cells including human stem cells, hard-to-modify primary natural killer (NK) cells, and cancer cell models. This peptide is composed of a 6x histidine-rich domain fused to the endosomolytic peptide CM18 and the cell penetrating peptide PTD4...
2018: PloS One
https://www.readbyqxmd.com/read/29610531/small-molecules-promote-crispr-cpf1-mediated-genome-editing-in-human-pluripotent-stem-cells
#8
Xiaojie Ma, Xi Chen, Yan Jin, Wenyan Ge, Weiyun Wang, Linghao Kong, Junfang Ji, Xing Guo, Jun Huang, Xin-Hua Feng, Junfen Fu, Saiyong Zhu
Human pluripotent stem cells (hPSCs) have potential applications in biological studies and regenerative medicine. However, precise genome editing in hPSCs remains time-consuming and labor-intensive. Here we demonstrate that the recently identified CRISPR-Cpf1 can be used to efficiently generate knockout and knockin hPSC lines. The unique properties of CRISPR-Cpf1, including shorter crRNA length and low off-target activity, are very attractive for many applications. In particular, we develop an unbiased drug-selection-based platform feasible for high-throughput screening in hPSCs and this screening system enables us to identify small molecules VE-822 and AZD-7762 that can promote CRISPR-Cpf1-mediated precise genome editing...
April 3, 2018: Nature Communications
https://www.readbyqxmd.com/read/29598153/seamless-genetic-conversion-of-smn2-to-smn1-via-crispr-cpf1-and-single-stranded-oligodeoxynucleotides-in-spinal-muscular-atrophy-patient-specific-ipscs
#9
Miaojin Zhou, Zhiqing Hu, Liyan Qiu, Tao Zhou, Mai Feng, Qian Hu, Baitao Zeng, Zhuo Li, Qianru Sun, Yong Wu, Xionghao Liu, Lingqian Wu, Desheng Liang
Spinal muscular atrophy (SMA) is a kind of neuromuscular disease characterized by progressive motor neuron loss in the spinal cord. It is caused by mutations in the survival motor neuron 1 (SMN1) gene. SMN1 has a paralogous gene, survival motor neuron 2 (SMN2), in humans that is present in almost all SMA patients. The generation and genetic correction of SMA patient specific induced pluripotent stem cells (iPSCs) is a viable, autologous therapeutic strategy for the disease. Here, we generated c-Myc-free and non-integrating iPSCs from the urine cells of an SMA patient using an episomal iPSC reprogramming vector and designed a unique crRNA that does not have similar sequences (≤3 mismatches) anywhere in the human reference genome...
March 29, 2018: Human Gene Therapy
https://www.readbyqxmd.com/read/29580925/macbeth-multiplex-automated-corynebacterium-glutamicum-base-editing-method
#10
Yu Wang, Ye Liu, Jiao Liu, Yanmei Guo, Liwen Fan, Xiaomeng Ni, Xiaomei Zheng, Meng Wang, Ping Zheng, Jibin Sun, Yanhe Ma
CRISPR/Cas9 or Cpf1-introduced double strand break dramatically decreases bacterial cell survival rate, which hampers multiplex genome editing in bacteria. In addition, the requirement of a foreign DNA template for each target locus is labor demanding and may encounter more GMO related regulatory hurdle in industrial applications. Herein, we developed a multiplex automated Corynebacterium glutamicum base editing method (MACBETH) using CRISPR/Cas9 and activation-induced cytidine deaminase (AID), without foreign DNA templates, achieving single-, double-, and triple-locus editing with efficiencies up to 100%, 87...
March 24, 2018: Metabolic Engineering
https://www.readbyqxmd.com/read/29575326/the-crispr-cas-revolution-reaches-the-rna-world-cas13-a-new-swiss-army-knife-for-plant-biologists
#11
Felix Wolter, Holger Puchta
Application of the bacterial CRISPR/Cas systems to eukaryotes is revolutionizing biology. Cas9 and Cas12 (previously called Cpf1) are widely used as DNA nucleases for inducing site specific DNA breaks for different kinds of genome engineering applications, and in their mutated forms as DNA binding proteins to modify gene expression. Moreover, histone modifications, as well as cytosine methylation or base editing, were achieved with these systems in plants. Recently, with the discovery of the nuclease Cas13a (previously called C2c2), molecular biologists have obtained a system that enables sequence specific cleavage of single stranded RNA molecules...
March 25, 2018: Plant Journal: for Cell and Molecular Biology
https://www.readbyqxmd.com/read/29553573/base-editing-with-a-cpf1-cytidine-deaminase-fusion
#12
Xiaosa Li, Ying Wang, Yajing Liu, Bei Yang, Xiao Wang, Jia Wei, Zongyang Lu, Yuxi Zhang, Jing Wu, Xingxu Huang, Li Yang, Jia Chen
The targeting range of CRISPR-Cas9 base editors (BEs) is limited by their G/C-rich protospacer-adjacent motif (PAM) sequences. To overcome this limitation, we developed a CRISPR-Cpf1-based BE by fusing the rat cytosine deaminase APOBEC1 to a catalytically inactive version of Lachnospiraceae bacterium Cpf1. The base editor recognizes a T-rich PAM sequence and catalyzes C-to-T conversion in human cells, while inducing low levels of indels, non-C-to-T substitutions and off-target editing.
March 19, 2018: Nature Biotechnology
https://www.readbyqxmd.com/read/29534717/genome-editing-and-transcriptional-repression-in-pseudomonas-putida-kt2440-via-the-type-ii-crispr-system
#13
Jun Sun, Qingzhuo Wang, Yu Jiang, Zhiqiang Wen, Lirong Yang, Jianping Wu, Sheng Yang
BACKGROUND: The soil bacterium Pseudomonas putida KT2440 is a "generally recognized as safe"-certified strain with robust property and versatile metabolism. Thus, it is an ideal candidate for synthetic biology, biodegradation, and other biotechnology applications. The known genome editing approaches of Pseudomonas are suboptimal; thus, it is necessary to develop a high efficiency genome editing tool. RESULTS: In this study, we established a fast and convenient CRISPR-Cas9 method in P...
March 13, 2018: Microbial Cell Factories
https://www.readbyqxmd.com/read/29530750/exploiting-endogenous-crispr-cas-system-for-multiplex-genome-editing-in-clostridium-tyrobutyricum-and-engineer-the-strain-for-high-level-butanol-production
#14
Jie Zhang, Wenming Zong, Wei Hong, Zhong-Tian Zhang, Yi Wang
Although CRISPR-Cas9/Cpf1 have been employed as powerful genome engineering tools, heterologous CRISPR-Cas9/Cpf1 are often difficult to introduce into bacteria and archaea due to their severe toxicity. Since most prokaryotes harbor native CRISPR-Cas systems, genome engineering can be achieved by harnessing these endogenous immune systems. Here, we report the exploitation of Type I-B CRISPR-Cas of Clostridium tyrobutyricum for genome engineering. In silico CRISPR array analysis and plasmid interference assay revealed that TCA or TCG at the 5'-end of the protospacer was the functional protospacer adjacent motif (PAM) for CRISPR targeting...
March 9, 2018: Metabolic Engineering
https://www.readbyqxmd.com/read/29526756/structural-insights-into-the-apo-structure-of-cpf1-protein-from-francisella-novicida
#15
Kyungjin Min, Hyunjun Yoon, Inseong Jo, Nam-Chul Ha, Kyeong Sik Jin, Jin-Soo Kim, Hyung Ho Lee
Clustered regularly interspaced short palindromic repeats (CRISPRs) from Prevotella and Francisella 1 (Cpf1) are RNA-guided endonucleases that produce cohesive double-stranded breaks in DNA by specifically recognizing thymidine-rich protospacer-adjacent motif (PAM) sequences. Cpf1 is emerging as a powerful genome-editing tool. Despite previous structural studies on various Cpf1 proteins, the apo-structure of Cpf1 remains unknown. In the present study, we determined the solution structure of the Cpf1 protein from Francisella novicida (FnCpf1) with and without CRISPR RNA (crRNA) using small-angle X-ray scattering, providing the insights into the apo-structure of FnCpf1...
April 15, 2018: Biochemical and Biophysical Research Communications
https://www.readbyqxmd.com/read/29472270/crispr-rnas-trigger-innate-immune-responses-in-human-cells
#16
Sojung Kim, Taeyoung Koo, Hyeon-Gun Jee, Hee-Yeon Cho, Gyeorae Lee, Dong-Gyun Lim, Hyoung Shik Shin, Jin-Soo Kim
Here, we report that CRISPR guide RNAs (gRNAs) with a 5'-triphosphate group (5'-ppp gRNAs) produced via in vitro transcription trigger RNA-sensing innate immune responses in human and murine cells, leading to cytotoxicity. 5'-ppp gRNAs in the cytosol are recognized by DDX58, which in turn activates type I interferon responses, causing up to ∼80% cell death. We show that the triphosphate group can be removed by a phosphatase in vitro and that the resulting 5'-hydroxyl gRNAs in complex with Cas9 or Cpf1 avoid innate immune responses and can achieve targeted mutagenesis at a frequency of 95% in primary human CD4+ T cells...
February 22, 2018: Genome Research
https://www.readbyqxmd.com/read/29462720/engineering-introns-to-express-rna-guides-for-cas9-and-cpf1-mediated-multiplex-genome-editing
#17
Dan Ding, Kaiyuan Chen, Yuedan Chen, Hong Li, Kabin Xie
The clustered regularly interspaced short palindromic repeat (CRISPR) system has emerged as the revolutionary platform for DNA targeting. This system uses a site-specific RNA guide to direct a CRISPR effector (e.g., Cas9 and Cpf1) to a DNA target. Here, we elaborate a general strategy to simultaneously express multiple guide RNAs (gRNA) and CRISPR RNAs (crRNA) from introns of Cas9 and Cpf1. This method utilizes the endogenous tRNA processing system or crRNA processing activity of Cpf1 to cleave the spliced intron that contains tRNA-gRNA polycistron or crRNA-crRNA array...
April 2, 2018: Molecular Plant
https://www.readbyqxmd.com/read/29449511/crispr-cas12a-target-binding-unleashes-indiscriminate-single-stranded-dnase-activity
#18
Janice S Chen, Enbo Ma, Lucas B Harrington, Maria Da Costa, Xinran Tian, Joel M Palefsky, Jennifer A Doudna
CRISPR-Cas12a (Cpf1) proteins are RNA-guided enzymes that bind and cut DNA as components of bacterial adaptive immune systems. Like CRISPR-Cas9, Cas12a has been harnessed for genome editing on the basis of its ability to generate targeted, double-stranded DNA breaks. Here we show that RNA-guided DNA binding unleashes indiscriminate single-stranded DNA (ssDNA) cleavage activity by Cas12a that completely degrades ssDNA molecules. We find that target-activated, nonspecific single-stranded deoxyribonuclease (ssDNase) cleavage is also a property of other type V CRISPR-Cas12 enzymes...
April 27, 2018: Science
https://www.readbyqxmd.com/read/29433082/acute-exposure-to-chlorpyrifos-induces-reversible-changes-in-health-parameters-of-nile-tilapia-oreochromis-niloticus
#19
Eman Zahran, Engy Risha, Walaa Awadin, Dušan Palić
Chlorpyrifos (CPF) is one of the most common insecticides found in freshwater ecosystems, and has been detected in agricultural and fishery products worldwide. This study focused on comprehensive panel of hematological, immunotoxic and pathology changes in Nile tilapia (Oreochromis niloticus) during and after exposure to CPF at 15 μg/L (0.043 μM) (1/10 LC50 , group CPF1), or 75 μg/L (0.21 μM) (1/2 LC50 , group CPF2) for 14 days, followed by 2 weeks recovery. Different endpoints were used to determine effects of CPF on fish health: hematological parameters; antioxidant levels in liver and gills; innate immune parameters; expression levels of pro-inflammatory cytokine genes at mRNA level in anterior kidney and spleen; and histopathological assessment of gills, liver, and kidney tissues...
April 2018: Aquatic Toxicology
https://www.readbyqxmd.com/read/29431740/deep-learning-improves-prediction-of-crispr-cpf1-guide-rna-activity
#20
Hui Kwon Kim, Seonwoo Min, Myungjae Song, Soobin Jung, Jae Woo Choi, Younggwang Kim, Sangeun Lee, Sungroh Yoon, Hyongbum Henry Kim
We present two algorithms to predict the activity of AsCpf1 guide RNAs. Indel frequencies for 15,000 target sequences were used in a deep-learning framework based on a convolutional neural network to train Seq-deepCpf1. We then incorporated chromatin accessibility information to create the better-performing DeepCpf1 algorithm for cell lines for which such information is available and show that both algorithms outperform previous machine learning algorithms on our own and published data sets.
March 2018: Nature Biotechnology
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