Construction and transformation of CRISPR/Cas9 genome editing vector of Flammulina filiformis G protein-coupled receptor gene
Mycosystema,2019,Vol 38,No. 03
【Abstract】 Two putative G protein-coupled receptor genes Fvgpcr1 and Fvgpcr2 in Flammulina filiformis were obtained by amino acid homology alignment (Blast P) and transcriptome data analysis of the fungus at hypha and primordium stages before and after cold induction. Four expression vectors including pCAMBIA0390-hph-Fvcas9-Fvgpcr1-sgRNA1/sgRNA2 and pCAMBIA0390-hph-Fvcas9-Fvgpcr2-sgRNA1/sgRNA2 were constructed for gene disruption of G-protein coupled receptor genes Fvgpcr1 and Fvgpcr2. The expression vector pCAMBIA0390-hph-Fvcas9-Fvgpcr-sgRNA was introduced into the mycelium of F. filiformis by Agrobacterium tumefaciens-mediated transformation (ATMT). Transformants harvested from hygromycin and cefotaxime rescreening were identified by PCR, RT-qPCR, and Western hybridization. Results showed that the expression vectors pCAMBIA0390-hphFvcas9-Fvgpcr-sgRNA1 and pCAMBIA0390-hph-Fvcas9-Fvgpcr-sgRNA2 were successfully integrated into the genome of F. filiformis and the FvCas9 protein was successfully expressed. However, the Fvgpcr deletion mutant was not obtained. This study used the ATMT method to construct a CRISPR/Cas9 gene disruption system in F. filiformis, which is of great significance for the subsequent gene disruption.
Acta Agronomica Sinica,2019,Vol 45,No. 06
【Abstract】 Clustered Regularly Interspaced Short Palindromic Repeat and Cas9 (CRISPR/Cas9), a new generation of genomeediting technology, is widely applied among bacteria, yeast, animals and plants, however, the typical CRISRP/Cas9 cannot recognize the NGA proto-spacer-motif (PAM), which limits its application. In order to broaden the applications of CRIPSR/Cas9 system, we modified the Streptococcus pyogenes Cas9 (SpCas9) sequence by the PCR site-direct mutagenesis, which encodes V (1135), Q (1335), and R (1337), to make the CRIPSR/Cas9-VQR able to recognize the NGA PAM motif. We also constructed a binary expression vector of CRISRP/Cas9-VQR with maize ubiquitin as the promoter to drive the Cas9-VQR, optimizing SpCas9-codon, adding conserved nuclear localization signal sequence, and increasing the conserved 3' UTR sequence of monocots, and using OsU6 transcripts of sRNA. CRISPR/Cas9-VQR could recognize the NGA motif and cut targeted sequence in vivo. We assembled the Cas9-VQR protein with the s RNAs in vitro. The Cas9-VQR could cleave the targeted fragments with about 5%–70% of mutation efficiency. In the transformation of rice, we detected about 27.50%–70.50% of mutation ratio, with an average of 46.23%. This system broadens the CRISPR/Cas9 applications in crops, especially in these with higher PAM locus of NGA.
Different SlU6 Promoters Cloning and Establishment of CRISPR/Cas9 Mediated Gene Editing System in Tomato
Scientia Agricultura Sinica,2018,Vol 51,No. 02
【Abstract】 [Objective] U6 promoter is a vital element for the transcription of sgRNA in the CRISPR/Cas9 system. It is necessary to clone some endogenous U6 promoters with high transcriptional activity and construct CRISPR/Cas9 vector, which can provide a strong technical basis for functional genomics and molecular breeding in tomato. [Method] Four different tomato U6 promoters were cloned by first round of PCR amplification from tomato cultivar Zhongshu 4. Each U6 promoter with two different length was truncated and used to construct plant expression vector carried SlU6 promoter:: GUS, respectively. The eight GUS fusion expression vectors were transformed into tomato leaves by agroinfiltration. According to the degree of GUS staining, the promoter with high transcriptional activity was selected to construct the CRISPR/Cas9 gene editing vector with target sequence from powdery mildew-related gene MLO1 and EDR1. These gene editing vectors were transformed into tomato protoplast by PEG method. The mutation of endogenous target genes in each transformed tomato protoplast was analyzed by a restriction enzyme PCR (RE/PCR) assay. Finally, the types of endogenous gene mutation were analyzed by sequencing. The efficiency of the CRISPR/Cas9 system based on tomato endogenous U6 promoter was verified by the frequency distribution map of mutant loci. [Result] 4 kinds and 8 different lengths of tomato U6 promoters were obtained by two rounds of PCR. Their lengths were 452, 202, 448, 206, 433, 190, 448 and 218 bp, respectively. After sequences analysis, the results showed that the four tomato U6 promoters also contained the USE motif and TATA box which were found in Arabidopsis U6 promoters. The construction of GUS fusion expression vectors driven by corresponding truncated tomato U6 promoters were done and transformed into tomato leaves. The GUS histochemical staining showed that the transformed tomato leaves were dyed blue, which indicated that all 8 SlU6 promoters had transcriptional activity. The SlU6-2P4 promoter was chosen to drive sgRNA transcription and construct the CRISPR/Cas9 system with target sequence from MLO1 and EDR1 respectively. The result showed that endogenous SlU6-2P4 promoter could drive sgRNA transcription and gene MLO1 was edited successfully in tomato. The sequence analysis revealed that all types of gene mutations were base substitution and the hotspot of mutation only existed in the target region of endogenous gene. [Conclusion] Four kinds of SlU6 promoters with high transcription efficiency were obtained from tomato. The established CRISPR/Cas9 system based on SlU6-2 promoter could successfully achieve the editing of endogenous genes in tomato.
Scientia Agricultura Sinica,2018,Vol 51,No. 04
【Abstract】 [Objective] Porcine reproductive and respiratory syndrome (PRRS), commonly known as “blue ear disease”, is a highly fatal infectious disease with porcine reproductive and respiratory syndrome virus (PRRSV) being the causative pathogen. PRRSV causes major economic losses in the pork industry world-wide. The genetic variability of PRRSV is high and an ideal vaccine to prevent the occurrence of this disease is not available. Cluster of differentiation 163 (CD163) is the important receptor for the entry of PRRSV into the porcine alveolar macrophage (PAM) cells. The aim of this study was to generate CD163 gene edited Large White pigs by using the CRISPR/Cas9 and somatic cell nuclear transfer (SCNT) techniques. [Method] CRISPR/Cas9 vector was constructed for editing the exon 7 of the porcine CD163 gene. The constructed vectors were transfected into pig fetal fibroblasts to obtain gene edited positive cell colonies. The CD163 gene edited fibroblasts and in vitro matured porcine oocytes were employed as nuclear donors and nuclear receptors respectively to obtain reconstructed embryos. For obtaining CD163 gene edited pigs the reconstructed embryos were transferred into recipient sows and performing the subsequent propagation experiment. [Result] The designed gRNA could effectively recognize the intended site. Genotyping analysis of cloned cell showed that 21 colonies had mutations in the CD163 gene, of which 14 colonies had either a monoallelic mutation or a biallelic heterozygous mutation, and 7 colonies had a biallelic homozygous mutation. Through SCNT, we successfully obtained CD163 biallelic edited Large White pigs. Successful breeding allowed us to obtain F 1 generation CD163 gene edited piglets, and they were all in good health. It was anticipated that more F 1 piglets would be produced soon. [Conclusion] The CD163 biallelic edited Large White pigs that did not harbor a drug resistant gene in their genome were produced and they could thus safely and quickly serve as a gene donor for breeding of PRRSV resistant pigs.
Microbiology China,2018,Vol 45,No. 08
【Abstract】 [ Background] Genome editing method coupling the CRISPR (Clustered regularly interspaced short palindromic repeats) system with λ-Red recombination technology has become an important access to Escherichia coli genomic editing. Recently, there have been several genomic editing strategies in E. coli based on CRISPR system. However, these methods often require many processes, such as the elimination of single plasmid or multi-fragment assembly, and are still inefficient, tedious and time-consuming. [ Objective] Establish a fast, continuous and efficient CRISPR genome editing method based on several different temperature-sensitive plasmids in E. coli, and improve the editing efficiency of CRISPR in E. coli. [ Methods] The pTarget plasmid in traditional CRISPR method was modified to an RK2 ts-type plasmid with better temperature sensitivity. Two plasmids with different resistance markers (pTW-A/S) were constructed and used alternately to eliminate false positives. And this realized the elimination of plasmids and next round of gene integration simultaneously. [ Results] At the same temperature, RK2 ts-type plasmid was eliminated more easily than pSC101 ts-type plasmid, and could selectively eliminate pTW-A/S plasmid. Moreover, it eliminated pTW-A/S plasmid and transformed other plasmids and target fragments during the next round of gene integration simultaneously. The efficiency of gene knock-out/integration reached 100%. Then, we used this method to construct BP03 by modifying gene BspanD and aspA on strain BP01 continuously, leading to successfully increased production of β-alanine. [ Conclusion] A novel, efficient, convenient, continuous and flexible CRISPR/Cas9-mediated genome editing strategy has been established in E. coli. This coordinate use of multiple temperature-sensitive plasmids method solves the problem of incomplete elimination of pTarget plasmids in traditional CRISPR systems, and it also avoids the low connection efficiency in process of large plasmids construction. Therefore, it shortens the experimental hours and provides a powerful tool for the construction of metabolic engineering strains.
Chinese Journal of Biotechnology,2017,Vol 33,No. 03
【Abstract】 Synthetic biology is an emerging discipline, which aims at creating artificial lives or remolding the present organisms to generate new features. To achieve these goals, synthetic biologists need to design and synthesize new genes, pathways, modules or even whole genomes. As these enabling technologies (e.g., gene synthesis, DNA assembly and genome editing) are very important for the progress of synthetic biology, we will focus on the development of these technologies in this review.
Chinese Journal of Biotechnology,2017,Vol 33,No. 10
【Abstract】 Breakthroughs of genome-editing in recent years have paved the way to develop new therapeutic strategies. These genome-editing tools mainly include Zinc-finger nucleases (ZFNs), Transcription activator-like effector nucleases (TALENs), and clustered regulatory interspaced short palindromic repeat (CRISPR)/Cas-based RNA-guided DNA endonucleases. However, off-target effects are still the major issue in genome editing, and limit the application in gene therapy. Here, we summarized the cause and compared different detection methods of off-targets.
Chinese Journal of Biotechnology,2017,Vol 33,No. 10
【Abstract】 The development of genome editing techniques based on CRISPR (Clustered regularly interspaced short palindromic repeats) -Cas9 system has revolutionized biomedical researches. It can be utilized to edit genome sequence in almost any organisms including Caenorhabditis elegans, one of the most convenient and classic genetic model animals. The application of CRISPR-Cas9 mediated genome editing in C. elegans promotes the functional analysis of gene and proteins under many physiological conditions. In this mini-review, we summarized the development of CRISPR-Cas9 mediated genome editing in C. elegans.
Chinese Journal of Biotechnology,2017,Vol 33,No. 10
【Abstract】 Targeted genome editing technology is an important tool to study the function of genes and to modify organisms at the genetic level. Recently, CRISPR-Cas (clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins) system has emerged as an efficient tool for specific genome editing in animals and plants. CRISPR-Cas system uses CRISPR-associated endonuclease and a guide RNA to generate double-strand breaks at the target DNA site, subsequently leading to genetic modifications. CRISPR-Cas system has received widespread attention for manipulating the genomes with simple, easy and high specificity. This review summarizes recent advances of diverse applications of the CRISPR-Cas toolkit in plant research and crop breeding, including expanding the range of genome editing, precise editing of a target base, and efficient DNA-free genome editing technology. This review also discusses the potential challenges and application prospect in the future, and provides a useful reference for researchers who are interested in this field.
Scientia Agricultura Sinica,2016,Vol 49,No. 07
【Abstract】 In this review, the steps of development of the CRISPR/Cas9 genome editing system and its applications in various plant genomes were highlighted. The CRISPR/Cas9 genome editing technology originates from the prokaryotic adaptive immune systems that confer resistance to foreign genetic elements such as plasmids and phages. The natural CRISPR/Cas systems show extensive structural and functional diversity. Based on the Cas protein contents and amino acid sequences, the natural CRISPR/Cas systems have been classified into three major classes, Type I, TypeⅡ and Type III. The TypeⅡCRISPR/Cas system is the engineered one for targeted genome editing purpose in most of cases so far, as it needs optimization of the Cas expression and design of the sg RNA only. In 2013, the first applications of CRISPR/Cas9 genome editing technology in plants were published. Since then, the CRISPR/Cas9 system has been used in various plant species for targeted genome editing. Like ZFNs and TALENs, CRISPR/Cas9 system uses engineered nuclease to generate double-strand breaks(DSBs) on the targeted DNA site, and subsequently to stimulate cellular DNA repair mechanisms by exploiting either NHEJ pathway or HDR pathway to generate small insertions/deletions/genome modifications. CRISPR/Cas9 technology allows researchers to perform targeted mutagenesis on target genes of different crops, precisely and easily changing the sequences and functions of particular genes at exact chromosomal locations in different plant genomes. Compared with ZFNs and TALENs technologies, CRISPR/Cas9 genome editing system is based on RNA-guided engineered nucleases, and is easier to manipulate. Furthermore, CRISPR/Cas9 is capable of introducing DSBs at multiple sites. The potential of multiplexing provides practical advantages over ZFNs and TALENs technologies, to edit multiple target genes in the same pathway simultaneously. Due to the practical advantages of CRISPR/Cas9 over the other genome editing technologies, it establishes a prosperous outlook in gene discovery and trait development in crop genetic improvement and breeding studies. In this review, the possible applications of CRISPR/Cas9 genome editing technique in various aspects of plant genetics and breeding were also discussed, except the targeted genome editing. CRISPR/Cas9 genome editing technology is another stepping stone in utilizing genetic manipulation in genetic studies and breeding, after genetic modification. Unlike genetic modification, CRISPR/Cas9 genome editing technique generates phenotypic variation that is indistinguishable from that obtained through natural means or conventional mutagenesis. This ambiguity challenges the current GMO regulatory definitions, and provides a potential barrier for further use of CRISPR/Cas9 genome editing technique in crop genetics and breeding.
Chinese Journal of Biotechnology,2015,Vol 31,No. 06
【Abstract】 Genome editing refers to the experimental methods to targeted modify specific loci in the genomic DNA sequence. In recent years, engineered endonucleases, including ZFN, TALEN and CRISPR/Cas, have been developed as a new-generation genome editing technique, and greatly improved the feasibility of gene function analyses, gene therapy, etc.Here, we briefly summarize the basic principle, developmental process and applications of this technology.