scholarly journals Construction of Multiple Guide RNAs in CRISPR/Cas9 Vector Using Stepwise or Simultaneous Golden Gate Cloning: Case Study for Targeting the FAD2 and FATB Multigene in Soybean

Plants ◽  
2021 ◽  
Vol 10 (11) ◽  
pp. 2542
Author(s):  
Won-Nyeong Kim ◽  
Hye-Jeong Kim ◽  
Young-Soo Chung ◽  
Hyun-Uk Kim
Keyword(s):  
Reverse Genetics ◽  
Restriction Enzymes ◽  
Golden Gate ◽  
Knock Out ◽  
Guide Rnas ◽  
Genetics Research ◽  
Multiple Genes ◽  
Cas9 Protein ◽  
Cloning Method ◽  
Golden Gate Cloning

CRISPR/Cas9 is a commonly used technique in reverse-genetics research to knock out a gene of interest. However, when targeting a multigene family or multiple genes, it is necessary to construct a vector with multiple single guide RNAs (sgRNAs) that can navigate the Cas9 protein to the target site. In this protocol, the Golden Gate cloning method was used to generate multiple sgRNAs in the Cas9 vector. The vectors used were pHEE401E_UBQ_Bar and pBAtC_tRNA, which employ a one-promoter/one-sgRNA and a polycistronic-tRNA-gRNA strategy, respectively. Golden Gate cloning was performed with type IIS restriction enzymes to generate gRNA polymers for vector inserts. Four sgRNAs containing the pHEE401E_UBQ_Bar vector and four to six sgRNAs containing the pBAtC_tRNA vector were constructed. In practice, we constructed multiple sgRNAs targeting multiple genes of FAD2 and FATB in soybean using this protocol. These three vectors were transformed into soybeans using the Agrobacterium-mediated method. Using deep sequencing, we confirmed that the T0 generation transgenic soybean was edited at various indel ratios in the predicted target regions of the FAD2 and FATB multigenes. This protocol is a specific guide that allows researchers to easily follow the cloning of multiple sgRNAs into commonly used CRISPR/Cas9 vectors for plants.

scholarly journals Golden Mutagenesis: An efficient multi-sitesaturation mutagenesis approach by Golden Gate cloning with automated primer design

2018 ◽  
Author(s):  
Pascal Püllmann ◽  
Chris Ulpinnis ◽  
Sylvestre Marillonnet ◽  
Ramona Gruetzner ◽  
Steffen Neumann ◽  
...  
Keyword(s):  
Web Application ◽  
Pcr Amplification ◽  
Restriction Enzymes ◽  
Primer Design ◽  
Golden Gate ◽  
Efficient Tool ◽  
Multiple Gene ◽  
Reading Frame ◽  
Cloning Technique ◽  
Golden Gate Cloning

Site-directed methods for the generation of genetic diversity are essential tools in the field of directed enzyme evolution. The Golden Gate cloning technique has been proven to be an efficient tool for a variety of cloning setups. The utilization of restriction enzymes which cut outside of their recognition domain allows the assembly of multiple gene fragments obtained by PCR amplification without altering the open reading frame of the reconstituted gene. We have developed a protocol, termed Golden Muta-genesis that allows the rapid, straightforward, reliable and inexpensive construction of mutagenesis libraries. One to five amino acid positions within a coding sequence could be altered simultaneously using a protocol which can be performed within one day. To facilitate the implementation of this technique, a software library and web application for automated primer design and for the graphical evaluation of the randomization success based on the sequencing results was developed. This allows facile primer design and application of Golden Mutagenesis also for laboratories, which are not specialized in molecular biology.

scholarly journals One-Step Generation of Multiple Gene-Edited Pigs by Electroporation of the CRISPR/Cas9 System into Zygotes to Reduce Xenoantigen Biosynthesis

2021 ◽  
Vol 22 (5) ◽  
pp. 2249
Author(s):  
Fuminori Tanihara ◽  
Maki Hirata ◽  
Nhien Thi Nguyen ◽  
Osamu Sawamoto ◽  
Takeshi Kikuchi ◽  
...  
Keyword(s):  
Gene Editing ◽  
Multiple Gene ◽  
Knock Out ◽  
Editing Efficiency ◽  
Guide Rnas ◽  
Cas9 Protein ◽  
One Step ◽  
Step Generation ◽  
The One

Xenoantigens cause hyperacute rejection and limit the success of interspecific xenografts. Therefore, genes involved in xenoantigen biosynthesis, such as GGTA1, CMAH, and B4GALNT2, are key targets to improve the outcomes of xenotransplantation. In this study, we introduced a CRISPR/Cas9 system simultaneously targeting GGTA1, CMAH, and B4GALNT2 into in vitro-fertilized zygotes using electroporation for the one-step generation of multiple gene-edited pigs without xenoantigens. First, we optimized the combination of guide RNAs (gRNAs) targeting GGTA1 and CMAH with respect to gene editing efficiency in zygotes, and transferred electroporated embryos with the optimized gRNAs and Cas9 into recipient gilts. Next, we optimized the Cas9 protein concentration with respect to the gene editing efficiency when GGTA1, CMAH, and B4GALNT2 were targeted simultaneously, and generated gene-edited pigs using the optimized conditions. We achieved the one-step generation of GGTA1/CMAH double-edited pigs and GGTA1/CMAH/B4GALNT2 triple-edited pigs. Immunohistological analyses demonstrated the downregulation of xenoantigens; however, these multiple gene-edited pigs were genetic mosaics that failed to knock out some xenoantigens. Although mosaicism should be resolved, the electroporation technique could become a primary method for the one-step generation of multiple gene modifications in pigs aimed at improving pig-to-human xenotransplantation.

scholarly journals A Golden Gate-based Plasmid Library for the Rapid Assembly of Biotin Ligase Constructs for Proximity Labelling

2021 ◽  
Author(s):  
Kevin Goslin ◽  
Andrea Finocchio ◽  
Frank Wellmer
Keyword(s):  
Fusion Proteins ◽  
Affinity Purification ◽  
Golden Gate ◽  
Bait Protein ◽  
Biotin Ligase ◽  
Plasmid Library ◽  
Cloning Method ◽  
Transient Interactions ◽  
Golden Gate Cloning

Proximity-labelling has emerged as a powerful tool for the detection of weak and transient interactions between proteins as well as the characterization of subcellular proteomes. One proximity labelling approach makes use of a promiscuous bacterial biotin ligase, termed BioID. Expression of BioID (or of its derivates TurboID and MiniTurbo) fused to a bait protein results in the biotinylation of proximal proteins. These biotinylated proteins can then be isolated by affinity purification using streptavidin-coated beads and identified by mass spectrometry. To facilitate the use of proximity-labelling in plants, we have generated a collection of constructs that can be used for the rapid cloning of TurboID and MiniTurbo fusion proteins using the Golden Gate cloning method. To allow for the use of the constructs in a range of experiments we have designed assembly modules that encode the biotin ligases fused to different linkers as well as different commonly used subcellular localization sequences. We demonstrate the functionality of these vectors through biotinylation assays in tobacco ( Nicotiana benthamiana ) plants .

scholarly journals The Functional Association of ACQOS/VICTR with Salt Stress Resistance in Arabidopsis thaliana Was Confirmed by CRISPR-Mediated Mutagenesis

2021 ◽  
Vol 22 (21) ◽  
pp. 11389
Author(s):  
Sang-Tae Kim ◽  
Minkyung Choi ◽  
Su-Ji Bae ◽  
Jin-Soo Kim
Keyword(s):  
Salt Stress ◽  
Target Genes ◽  
Gene Knockout ◽  
Salt Resistance ◽  
Knock Out ◽  
Chlorophyll Contents ◽  
Guide Rnas ◽  
Osmotic Tolerance ◽  
Multiple Genes ◽  
Acclimation Period

Clustered regularly interspaced palindromic repeat (CRISPR)-mediated mutagenesis has become an important tool in plant research, enabling the characterization of genes via gene knock-out. CRISPR genome editing tools can be applied to generate multi-gene knockout lines. Typically, multiple single-stranded, single guide RNAs (gRNAs) must be expressed in an organism to target multiple genes simultaneously; however, a single gRNA can target multiple genes if the target genes share similar sequences. A gene cluster comprising ACQUIRED OSMOTOLERANCE (ACQOS; AT5G46520) and neighboring nucleotide-binding leucine-rich repeats (NLRs; AT5G46510) is associated with osmotic tolerance. To investigate the role of ACQOS and the tandemly arranged NLR in osmotic tolerance, we introduced small insertion/deletion mutations into two target genes using a single gRNA and obtained transformant plant lines with three different combinations of mutant alleles. We then tested our mutant lines for osmotic tolerance after a salt-stress acclimation period by determining the chlorophyll contents of the mutant seedlings. Our results strongly suggest that ACQOS is directly associated with salt resistance, while the neighboring NLR is not. Here, we confirmed previous findings suggesting the involvement of ACQOS in salt tolerance and demonstrated the usefulness of CRISPR-mediated mutagenesis in validating the functions of genes in a single genetic background.

scholarly journals Saturation mutagenesis genome engineering of infective ΦX174 bacteriophage via unamplified oligo pools and golden gate assembly

2019 ◽  
Author(s):  
Matthew S. Faber ◽  
James T. Van Leuven ◽  
Martina M. Ederer ◽  
Yesol Sapozhnikov ◽  
Zoë L. Wilson ◽  
...  
Keyword(s):  
Amino Acids ◽  
Genome Engineering ◽  
Reverse Genetics ◽  
Viral Evolution ◽  
Single Site ◽  
Saturation Mutagenesis ◽  
Golden Gate ◽  
Genes Encoding ◽  
Site Mutagenesis ◽  
Golden Gate Cloning

Here we present a novel protocol for the construction of saturation single-site—and massive multi-site—mutant libraries of a bacteriophage. We segmented the ΦX174 genome into 14 non-toxic and non-replicative fragments compatible with golden gate assembly. We next used nicking mutagenesis with oligonucleotides prepared from unamplified oligo pools with individual segments as templates to prepare near-comprehensive single-site mutagenesis libraries of genes encoding the F capsid protein (421 amino acids scanned) and G spike protein (172 amino acids scanned). Libraries possessed greater than 99% of all 11,860 programmed mutations. Golden Gate cloning was then used to assemble the complete ΦX174 mutant genome and generate libraries of infective viruses. This protocol will enable reverse genetics experiments for studying viral evolution and, with some modifications, can be applied for engineering of therapeutically relevant bacteriophages with larger genomes.

scholarly journals Joint universal modular plasmids (JUMP): a flexible vector platform for synthetic biology

2021 ◽  
Vol 6 (1) ◽  
Author(s):  
Marcos Valenzuela-Ortega ◽  
Christopher French
Keyword(s):  
Copy Number ◽  
Life Science ◽  
Insertion Site ◽  
Golden Gate ◽  
Common Elements ◽  
Modern Life ◽  
Specific Host ◽  
Modular Cloning ◽  
Golden Gate Cloning ◽  
Selection Of

Abstract Generation of new DNA constructs is an essential process in modern life science and biotechnology. Modular cloning systems based on Golden Gate cloning, using Type IIS restriction endonucleases, allow assembly of complex multipart constructs from reusable basic DNA parts in a rapid, reliable and automation-friendly way. Many such toolkits are available, with varying degrees of compatibility, most of which are aimed at specific host organisms. Here, we present a vector design which allows simple vector modification by using modular cloning to assemble and add new functions in secondary sites flanking the main insertion site (used for conventional modular cloning). Assembly in all sites is compatible with the PhytoBricks standard, and vectors are compatible with the Standard European Vector Architecture (SEVA) as well as BioBricks. We demonstrate that this facilitates the construction of vectors with tailored functions and simplifies the workflow for generating libraries of constructs with common elements. We have made available a collection of vectors with 10 different microbial replication origins, varying in copy number and host range, and allowing chromosomal integration, as well as a selection of commonly used basic parts. This design expands the range of hosts which can be easily modified by modular cloning and acts as a toolkit which can be used to facilitate the generation of new toolkits with specific functions required for targeting further hosts.

scholarly journals A modular cloning toolkit for genome editing in plants

2019 ◽  
Author(s):  
Florian Hahn ◽  
Andrey Korolev ◽  
Laura Sanjurjo Loures ◽  
Vladimir Nekrasov
Keyword(s):  
Genome Editing ◽  
Higher Order ◽  
Plant Science ◽  
Golden Gate ◽  
Coding Sequences ◽  
Guide Rna ◽  
Guide Rnas ◽  
Science Community ◽  
Modular Cloning ◽  
Multiple Expression

AbstractBackgroundCRISPR/Cas has recently become a widely used genome editing tool in various organisms, including plants. Applying CRISPR/Cas often requires delivering multiple expression units into plant and hence there is a need for a quick and easy cloning procedure. The modular cloning (MoClo), based on the Golden Gate (GG) method, has enabled development of cloning systems with standardised genetic parts, e.g. promoters, coding sequences or terminators, that can be easily interchanged and assembled into expression units, which in their own turn can be further assembled into higher order multigene constructs.ResultsHere we present an expanded cloning toolkit that contains ninety-nine modules encoding a variety of CRISPR/Cas-based nucleases and their corresponding guide RNA backbones. Among other components, the toolkit includes a number of promoters that allow expression of CRISPR/Cas nucleases (or any other coding sequences) and their guide RNAs in monocots and dicots. As part of the toolkit, we present a set of modules that enable quick and facile assembly of tRNA-sgRNA polycistronic units without a PCR step involved. We also demonstrate that our tRNA-sgRNA system is functional in wheat protoplasts.ConclusionsWe believe the presented CRISPR/Cas toolkit is a great resource that will contribute towards wider adoption of the CRISPR/Cas genome editing technology and modular cloning by researchers across the plant science community.

scholarly journals Rapid Evaluation of CRISPR Guides and Donors for Engineering Mice

Genes ◽  
2020 ◽  
Vol 11 (6) ◽  
pp. 628
Author(s):  
Elena McBeath ◽  
Jan Parker-Thornburg ◽  
Yuka Fujii ◽  
Neeraj Aryal ◽  
Chad Smith ◽  
...  
Keyword(s):  
Genomic Dna ◽  
Sanger Sequencing ◽  
High Efficiency ◽  
Genetically Engineered ◽  
Rapid Evaluation ◽  
Knock Out ◽  
Guide Rna ◽  
Guide Rnas ◽  
Genetically Engineered Mice ◽  
The Cost

Although the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/ CRISPR associated protein 9 (Cas9) technique has dramatically lowered the cost and increased the speed of generating genetically engineered mice, success depends on using guide RNAs and donor DNAs which direct efficient knock-out (KO) or knock-in (KI). By Sanger sequencing DNA from blastocysts previously injected with the same CRISPR components intended to produce the engineered mice, one can test the effectiveness of different guide RNAs and donor DNAs. We describe in detail here a simple, rapid (three days), inexpensive protocol, for amplifying DNA from blastocysts to determine the results of CRISPR point mutation KIs. Using it, we show that (1) the rate of KI seen in blastocysts is similar to that seen in mice for a given guide RNA/donor DNA pair, (2) a donor complementary to the variable portion of a guide integrated in a more all-or-none fashion, (3) donor DNAs can be used simultaneously to integrate two different mutations into the same locus, and (4) by placing silent mutations about every 6 to 10 bp between the Cas9 cut site and the desired mutation(s), the desired mutation(s) can be incorporated into genomic DNA over 30 bp away from the cut at the same high efficiency as close to the cut.

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