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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
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
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
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...
March 8, 2018: Biochemical and Biophysical Research Communications
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
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 which contains tRNA-gRNA polycistron or crRNA-crRNA array...
February 17, 2018: Molecular Plant
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 based on its ability to generate targeted, double-stranded DNA (dsDNA) 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, non-specific ssDNase cleavage is also a property of other type V CRISPR-Cas12 enzymes...
February 15, 2018: Science
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...
February 6, 2018: Aquatic Toxicology
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.
January 29, 2018: Nature Biotechnology
Misha Klein, Behrouz Eslami-Mossallam, Dylan Gonzalez Arroyo, Martin Depken
Due to their specificity, efficiency, and ease of programming, CRISPR-associated nucleases are popular tools for genome editing. On the genomic scale, these nucleases still show considerable off-target activity though, posing a serious obstacle to the development of therapies. Off targeting is often minimized by choosing especially high-specificity guide sequences, based on algorithms that codify empirically determined off-targeting rules. A lack of mechanistic understanding of these rules has so far necessitated their ad hoc implementation, likely contributing to the limited precision of present algorithms...
February 6, 2018: Cell Reports
Hao Yin, Chun-Qing Song, Sneha Suresh, Suet-Yan Kwan, Qiongqiong Wu, Stephen Walsh, Junmei Ding, Roman L Bogorad, Lihua Julie Zhu, Scot A Wolfe, Victor Koteliansky, Wen Xue, Robert Langer, Daniel G Anderson
CRISPR-Cas9 is a versatile RNA-guided genome editing tool. Here we demonstrate that partial replacement of RNA nucleotides with DNA nucleotides in CRISPR RNA (crRNA) enables efficient gene editing in human cells. This strategy of partial DNA replacement retains on-target activity when used with both crRNA and sgRNA, as well as with multiple guide sequences. Partial DNA replacement also works for crRNA of Cpf1, another CRISPR system. We find that partial DNA replacement in the guide sequence significantly reduces off-target genome editing through focused analysis of off-target cleavage, measurement of mismatch tolerance and genome-wide profiling of off-target sites...
March 2018: Nature Chemical Biology
Kulbhushan Chaudhary, Anirudha Chattopadhyay, Dharmendra Pratap
Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system allows biologists to edit genomic DNA of any cell in precise and specific way, entailing great potential for crop improvement, drug development and gene therapy. The system involves a nuclease (Cas9) and a designed guide RNA that are involved in wide range of applications such as genome modification, transcriptional modulation, genomic loci marking and RNA tracking. The limitation of the technique, in view of resistance of thymidine-rich genome to Cas9 cleavage, has now been overcome by the use of Cpf1 nuclease...
January 17, 2018: Biotechnology Letters
Jiyeon Kweon, An-Hee Jang, Da-Eun Kim, Jin Wook Yang, Mijung Yoon, Ha Rim Shin, Jin-Soo Kim, Yongsub Kim
The originally published version of this Article contained an error in the spelling of the author Da-eun Kim, which was incorrectly given as Da-Eun Kim. Furthermore, in Figure 1a, the Cas9 protein was positioned incorrectly during typesetting. These errors have now been corrected in both the PDF and HTML versions of the Article.
January 16, 2018: Nature Communications
Ryan Marshall, Colin S Maxwell, Scott P Collins, Thomas Jacobsen, Michelle L Luo, Matthew B Begemann, Benjamin N Gray, Emma January, Anna Singer, Yonghua He, Chase L Beisel, Vincent Noireaux
CRISPR-Cas systems offer versatile technologies for genome engineering, yet their implementation has been outpaced by ongoing discoveries of new Cas nucleases and anti-CRISPR proteins. Here, we present the use of E. coli cell-free transcription-translation (TXTL) systems to vastly improve the speed and scalability of CRISPR characterization and validation. TXTL can express active CRISPR machinery from added plasmids and linear DNA, and TXTL can output quantitative dynamics of DNA cleavage and gene repression-all without protein purification or live cells...
January 4, 2018: Molecular Cell
Ramya Sundaresan, Hari Priya Parameshwaran, S D Yogesha, Mark Walter Keilbarth, Rakhi Rajan
CRISPR-Cas systems provide bacteria and archaea with sequence-specific protection against invading mobile genetic elements. In the presence of divalent metal ions, Cas9 and Cas12a (formerly Cpf1) proteins target and cleave DNA that is complementary to a cognate guide RNA. The recognition of a protospacer adjacent motif (PAM) sequence in the target DNA by Cas9 and Cas12a is essential for cleavage. This RNA-guided DNA targeting is widely used for gene-editing methods. Here, we show that Francisella tularensis novicida (Fno) Cas12a, FnoCas9, and Streptococcus pyogenes Cas9 (SpyCas9) cleave DNA without a guide RNA in the presence of Mn2+ ions...
December 26, 2017: Cell Reports
Yuchen Liu, Jinghong Han, Zhicong Chen, Hanwei Wu, Hongsong Dong, Guohui Nie
The catalytically dead Cpf1 endonuclease from Acidaminococcus sp. BV3L6 (dAsCpf1) has been used to construct effective transcriptional repressors in bacteria and plants. However, it is still unclear if dAsCpf1 can function in human cells as a transcriptional regulator or a signal conductor. Here, we repurpose the dAsCpf1 system in human cells for a variety of functions, including the activation or repression of gene transcription. Moreover, we construct programmable ligand-controlled dAsCpf1 systems either by coupling crRNAs with engineered riboswitches or by fusing dAsCpf1 proteins with G protein-coupled receptors...
December 13, 2017: Nature Communications
Miguel A Moreno-Mateos, Juan P Fernandez, Romain Rouet, Charles E Vejnar, Maura A Lane, Emily Mis, Mustafa K Khokha, Jennifer A Doudna, Antonio J Giraldez
Cpf1 is a novel class of CRISPR-Cas DNA endonucleases, with a wide range of activity across different eukaryotic systems. Yet, the underlying determinants of this variability are poorly understood. Here, we demonstrate that LbCpf1, but not AsCpf1, ribonucleoprotein complexes allow efficient mutagenesis in zebrafish and Xenopus. We show that temperature modulates Cpf1 activity by controlling its ability to access genomic DNA. This effect is stronger on AsCpf1, explaining its lower efficiency in ectothermic organisms...
December 8, 2017: Nature Communications
Aron Ferenczi, Douglas Euan Pyott, Andromachi Xipnitou, Attila Molnar
The green alga Chlamydomonas reinhardtii is an invaluable reference organism to research fields including algal, plant, and ciliary biology. Accordingly, decades-long standing inefficiencies in targeted nuclear gene editing broadly hinder Chlamydomonas research. Here we report that single-step codelivery of CRISPR/Cpf1 ribonucleoproteins with single-stranded DNA repair templates results in precise and targeted DNA replacement with as much as ∼10% efficiency in C. reinhardtii We demonstrate its use in transgene- and selection-free generation of sequence-specific mutations and epitope tagging at an endogenous locus...
December 19, 2017: Proceedings of the National Academy of Sciences of the United States of America
Hadi Bayat, Mohammad Hossein Modarressi, Azam Rahimpour
Clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein (Cas) is a microbial adaptive immune system. CRISPR-Cas systems are classified into two main classes and six types. Cpf1 is a putative type V (class II) CRISPR effector, which has revolutionized the genome editing approaches through multiple distinct features such as using T-rich protospacer-adjacent motif, applying a short guide RNA lacking trans-activating crRNA, introducing a staggered double-strand break, and possessing RNA processing activity in addition to DNA nuclease activity...
January 2018: Current Microbiology
Jeongbin Park, Sangsu Bae
Summary: Following the type II CRISPR-Cas9 system, type V CRISPR-Cpf1 endonucleases have been found to be applicable for genome editing in various organisms in vivo. However, there are as yet no web-based tools capable of optimally selecting guide RNAs (gRNAs) among all possible genome-wide target sites. Here, we present Cpf1-Database, a genome-wide gRNA library design tool for LbCpf1 and AsCpf1, which have DNA recognition sequences of 5'-TTTN-3' at the 5' ends of target sites. Cpf1-Database provides a sophisticated but simple way to design gRNAs for AsCpf1 nucleases on the genome scale...
November 27, 2017: Bioinformatics
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