Efficient expression of multiple guide RNAs for CRISPR/Cas genome editing

Hsieh‐Feng, Vicki; Yang, Yinong

doi:10.1007/s42994-019-00014-w

Cited by 17 publications

(7 citation statements)

References 110 publications

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“…SgRNA is a special sequence with a length of about 20 bp and its 3′ end is close to the PAM sequence. It can complement and pair with the target site of genomic DNA as well as can also perform imperfect complementary pairing (off-target) with thousands of other sequences in the genome (Kleinstiver et al 2015;Hsieh-Feng and Yang 2020). Therefore, when designing sgRNA of genomic target sites, we need to find PAM sequences in the Table 3 The quantitative detection of the iron load produced by E. coli…”

Section: Discussionmentioning

confidence: 99%

“…The CRISPR/Cas9 system composed of clustered regularly interspaced short palindromic repeats (CRISPR) and its affiliated Cas9 protein that exists on some bacteria and most archaic (Wiedenheft 2013 ). When foreign genes invade bacteria and archaic, they identify the invading DNA and insert it between the leader and repeat sequences of the CRISPR sequence, while the protospacer adjacent motif (PAM) to help Cas9 nuclease finds the cleavage site of the invading DNA, Cas9 protein then cleaves the foreign DNA to degrade it to protect the bacteria from harm (Hsieh-Feng and Yang 2020 ). Subsequently, the system was artificially modified with CRISPR/Cas9 gene editing technology (Demirci et al 2018 ), which is simple, efficient and accurate compared to zinc finger nuclease (ZFN), transcriptional activator-like effector nuclease (TALENS) and homologous recombination technology (Urnov et al 2010 ).…”

Section: Introductionmentioning

confidence: 99%

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New insights into the construction of wild-type Saba pig-derived Escherichia coli irp2 gene deletion strains

et al. 2021

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To construct wild-type E. coli irp2 gene deletion strains, CRISPR/Cas9 gene editing technology was used, and the difficulty and key points of gene editing of wild-type strains were analyzed. Based on the resistance of the CRISPR/Cas9 system expression vector, 4 strains of 41 E. coli strains isolated from Saba pigs were selected as the target strains for the deletion of the irp2 gene, which were sensitive to both ampicillin and kanamycin. Then, CRISPR/Cas9 technology was combined with homologous recombination technology to construct recombinant vectors containing Cas9, sgRNA and donor sequences to knock out the irp2 gene. Finally, the absence of the irp2 gene in E. coli was further verified by iron uptake assays, iron carrier production assays and growth curve measurements. The results showed that three of the selected strains showed single base mutations and deletions (Δirp2-1, Δirp2-2 and Δirp2-3). The deletion of the irp2 gene reduced the ability of E. coli to take up iron ions and produce iron carriers, but not affect the growth characteristics of E. coli. It is shown that the CRISPR/Cas9 knock-out system constructed in this study can successfully knock out the irp2 gene of the wild-type E. coli. Our results providing new insights into genome editing in wild-type strains, which enable further functional studies of the irp2 gene in wild-type E. coli.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

New insights into the construction of wild-type Saba pig-derived Escherichia coli irp2 gene deletion strains

et al. 2021

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“…Precise processing and release of individual gRNAs from a polycistronic transcript is the key to the success of a multiplex CRISPR system. The polycistronic mRNA containing multiple [123][124][125]. Among these gRNA processing systems, tRNA and Csy4 systems appear more effective for Cas9 and HH-HDV for Cas12a [113,126,127].…”

Section: Open Accessmentioning

confidence: 99%

Construct design for CRISPR/Cas-based genome editing in plants

Hassan

Zhang

Yuan

et al. 2021

Trends in Plant Science

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CRISPR construct design is a key step in the practice of genome editing, which includes identification of appropriate Cas proteins, design and selection of guide RNAs (gRNAs), and selection of regulatory elements to express gRNAs and Cas proteins. Here, we review the choices of CRISPR-based genome editors suited for different needs in plant genome editing applications. We consider the technical aspects of gRNA design and the associated computational tools. We also discuss strategies for the design of multiplex CRISPR constructs for highthroughput manipulation of complex biological processes or polygenic traits. We provide recommendations for different elements of CRISPR constructs and discuss the remaining challenges of CRISPR construct optimization in plant genome editing. Genome editing and associated technologiesGenome editing can be defined as a targeted intervention of genetic materials (i.e., DNA or RNA) in living organisms to deliberately alter their sequences. Although genome editing can target both DNA and RNA, here we only review DNA editing. DNA editing mainly relies on the introduction of in vivo DNA double-stranded breaks (DSBs) induced by the engineered sequence-specific nucleases (SSNs) programmed to recognize predefined sites in a genome. The induced DSBs are then repaired by cellular DNA repair mechanisms, namely non-homologous end-joining (NHEJ) and homologydirected repair (HDR) (Figure 1). The repair of DSBs by NHEJ results in mutation at the break site, largely via imprecise sequence insertions or deletions (indels), disrupting the native structure and function of the targeted sequences (e.g., genes, promoters). In addition, NHEJ can mediate targeted sequence insertion or replacement when a suitable DNA fragment is provided [1]. By contrast, repair by HDR can precisely introduce predefined sequences carried by a donor DNA template (Figure 1).The SSNs, with the capacity to introduce DSB in DNA, are referred to as the key elements in genome editing technologies and include meganucleases [2], zinc finger nucleases (ZFNs) [3], transcription activator-like effector nucleases (TALENs) [4], and clustered regularly interspaced short palindromic repeat (CRISPR) systems [5][6][7][8]. Unlike ZFNs and TALENs, which rely on protein-DNA interaction to define target specificity, CRISPR systems use RNA-DNA interaction to guide the DNA targeting and cleavage, making it a simple, efficient, and inexpensive technology for genetic manipulation. CRISPR systems have now become the leading genome editing technology and have been applied in a wide variety of plant species. Efficient genome editing has been achieved in many dicot and monocot species using diverse CRISPR-Cas systems for fundamental research and crop improvement and the application of CRISPR-Cas technology in plants has been increased dramatically over the past few years [9][10][11][12].Three classes of CRISPR technology are currently available for editing plant genomes [10,13]. These are CRISPR-Cas nucleases, base editors, and prime editors. CRISPR-Cas...

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“…Several methods for simultaneously expressing multiple gRNAs targeting different sites are available. Multiple gRNAs can be expressed using a different promoter and terminator for each gRNA, or by expressing RNA-cleaving enzymes such as 5′-end hammerhead and 3′-end hepatitis delta virus dual ribozyme, CRISPR-associated RNA endoribonuclease Csy4 from Pseudomonas aeruginosa , or tRNA processing enzymes [ 20 , 25 , 26 , 27 , 28 , 29 , 30 ]. Furthermore, PAM positioning must be considered when designing multiplexed experiments involving sgRNAs targeting multiple specific sites.…”

Section: Introductionmentioning

confidence: 99%

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

Choi

Bae

2021

IJMS

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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.

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Efficient expression of multiple guide RNAs for CRISPR/Cas genome editing

Cited by 17 publications

References 110 publications

New insights into the construction of wild-type Saba pig-derived Escherichia coli irp2 gene deletion strains

New insights into the construction of wild-type Saba pig-derived Escherichia coli irp2 gene deletion strains

Construct design for CRISPR/Cas-based genome editing in plants

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

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