• Korean Journal of Agricultural Science
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• 제47권4호
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• pp.903-920
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• 2020

Since its first demonstration as a practical genome editing tool in the early 2010s, the use of clustered regularly interspaced short palindromic repeat (CRISPR) along with the endonuclease Cas9 (CRISPR/Cas9) has become an essential choice for generating targeted mutations. Due to its relative simplicity and cost-effectiveness compared to other molecular scissors, i.e., zinc finger nuclease (ZFN) and transcription activator-like effector nuclease (TALEN), the CRISPR/Cas9 system has been shown to have a massive influence on genetic studies regardless of the biological kingdom. Although the system is in the process of being established, numerous protocols have already been released for the system and there have been various topics of CRISPR related papers published each year in ever-increasing manner. Here, we will briefly introduce CRISPR/Cas9 system and discuss the variants of the CRISPR system. Also, their applications to crop improvement will be dealt with mainly ornamental crops among horticultural crops other than Arabidopsis as a model plant. Finally, some issues on the barriers restraining the use of CRISPR system on floricultural crops, the prospect of CRISPR system as a DNA-free genome editing tool with efficient facilitators and finally, the future perspectives on the CRISPR system will be described.

• Journal of Biomedical Engineering Research
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• 제42권3호
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• pp.125-142
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• 2021

The POCT (point-of-care test) sensing that has been a fast-developing field is expected to be a next generation technology in health care. The POCT sensors for the detection of proteins, small molecules and especially nucleic acids have lately attracted considerable attention. According to the World Health Organization (WHO), the POCT methods are required to follow the ASSURED guidelines (Affordable, Sensitive, Specific, User- friendly, Robust and rapid, Equipment-free, Deliverable to all people who need the test). Recently, several CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) based diagnostic techniques using the sensitive gene recognition function of CRISPR have been reported. CRISPR/Cas (Cas, CRISPR associated protein) systems based detection technology is the most innovative gene analysis technology that is following the ASSURED guidelines. It is being re-emerged as a powerful diagnostic tool that can detect nucleic acids due to its characteristics that enable rapid, sensitive and specific analyses of nucleic acid. The first CRISPR-based diagnosis begins with the discovery of the additional function of Cas13a. The enzymatic cleavage occurs when the conjugate of Cas protein and CRISPR RNA (crRNA) detect a specific complementary sequence of the target sequence. Enzymatic cleavage occurs on not only the target sequence, but also all surrounding non-target single-stranded RNAs. This discovery was immediately utilized as a biosensor, and numerous sensor studies using CRISPR have been reported since then. In this review, the concept of CRISPR, the characteristics of the Cas protein required for CRISPR diagnosis, the current research trends of CRISPR diagnostic technology, and some aspects to be improved in the future are covered.

• BMB Reports
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• 제56권2호
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• pp.102-107
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• 2023

Genome editing using CRISPR-associated technology is widely used to modify the genomes rapidly and efficiently on specific DNA double-strand breaks (DSBs) induced by Cas9 endonuclease. However, despite swift advance in Cas9 engineering, structural basis of Cas9-recognition and cleavage complex remains unclear. Proper assembly of this complex correlates to effective Cas9 activity, leading to high efficacy of genome editing events. Here, we develop a CRISPR/Cas9-RAD51 plasmid constitutively expressing RAD51, which can bind to single-stranded DNA for DSB repair. We show that the efficiency of CRISPR-mediated genome editing can be significantly improved by expressing RAD51, responsible for DSB repair via homologous recombination (HR), in both gene knock-out and knock-in processes. In cells with CRISPR/Cas9-RAD51 plasmid, expression of the target genes (cohesin SMC3 and GAPDH) was reduced by more than 1.9-fold compared to the CRISPR/Cas9 plasmid for knock-out of genes. Furthermore, CRISPR/Cas9-RAD51 enhanced the knock-in efficiency of DsRed donor DNA. Thus, the CRISPR/Cas9-RAD51 system is useful for applications requiring precise and efficient genome edits not accessible to HR-deficient cell genome editing and for developing CRISPR/Cas9-mediated knockout technology.

• Journal of the Korean Magnetic Resonance Society
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• 제25권3호
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• pp.39-44
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• 2021

The CRISPR-Cas system provides adaptive immunity for bacteria and archaea against invading phages and foreign plasmids. In the Class 1 CRISPR-Cas system, multi-subunit Cas proteins assemble with crRNA to bind to DNA targets. To disarm the bacterial defense system, bacteriophages evolved anti-CRISPR (Acr) proteins that actively inhibit the host CRISPR-Cas function. Here we report the backbone resonance assignments of AcrIF7 protein that inhibits the type I-F CRISPR-Cas system of Pseudomonas aeruginosa using triple-resonance nuclear magnetic resonance spectroscopy. We employed various computational methods to predict the structure and binding interface of AcrIF7, and assessed the model with experimental data. AcrIF7 binds to Cas8f protein via flexible loop regions to inhibit target DNA binding, suggesting that conformational heterogeneity is important for the Cas-Acr interaction.

• Clinical and Experimental Pediatrics
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• 제64권6호
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• pp.269-279
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• 2021

Clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9 (CRISPR-Cas9) is an ancient prokaryotic defense system that precisely cuts foreign genomic DNA under the control of a small number of guide RNAs. The CRISPR-Cas9 system facilitates efficient double-stranded DNA cleavage that has been recently adopted for genome editing to create or correct inherited genetic mutations causing disease. Congenital heart disease (CHD) is generally caused by genetic mutations such as base substitutions, deletions, and insertions, which result in diverse developmental defects and remains a leading cause of birth defects. Pediatric CHD patients exhibit a spectrum of cardiac abnormalities such as septal defects, valvular defects, and abnormal chamber development. CHD onset occurs during the prenatal period and often results in early lethality during childhood. Because CRISPR-Cas9-based genome editing technology has gained considerable attention for its potential to prevent and treat diseases, we will review the CRISPR-Cas9 system as a genome editing tool and focus on its therapeutic application for CHD.

• BMB Reports
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• 제57권1호
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• pp.12-18
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• 2024

Due to the development of CRISPR technology, the era of effective editing of target genes has arrived. However, the off-target problem that occurs when recognizing target DNA due to the inherent nature of CRISPR components remains the biggest task to be overcome in the future. In this review, the principle of inducing such unintended off-target editing is analyzed from the structural aspect of CRISPR, and the methodology that has been developed to reduce off-target editing until now is summarized.

• Journal of Microbiology and Biotechnology
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• 제27권2호
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• pp.207-218
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• 2017

Clustered regulatory interspaced short palindromic repeats (CRISPR) in association with CRISPR-associated protein (Cas) is an adaptive immune system, playing a pivotal role in the defense of bacteria and archaea. Ease of handling and cost effectiveness make the CRISPR-Cas system an ideal programmable nuclease tool. Recent advances in understanding the CRISPR-Cas system have tremendously improved its efficiency. For instance, it is possible to recapitulate the chronicle CRISPR-Cas from its infancy and inaugurate a developed version by generating novel variants of Cas proteins, subduing off-target effects, and optimizing of innovative strategies. In summary, the CRISPR-Cas system could be employed in a number of applications, including providing model systems, rectification of detrimental mutations, and antiviral therapies.

• BMB Reports
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• 제57권1호
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• pp.40-49
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• 2024

Prokaryotes encode clustered regularly interspaced short palindromic repeat (CRISPR) arrays and CRISPR-associated (Cas) genes as an adaptive immune machinery. CRISPR-Cas systems effectively protect hosts from the invasion of foreign enemies, such as bacteriophages and plasmids. During a process called 'adaptation', non-self-nucleic acid fragments are acquired as spacers between repeats in the host CRISPR array, to establish immunological memory. The highly conserved Cas1-Cas2 complexes function as molecular recorders to integrate spacers in a time course manner, which can subsequently be expressed as crRNAs complexed with Cas effector proteins for the RNA-guided interference pathways. In some of the RNA-targeting type III systems, Cas1 proteins are fused with reverse transcriptase (RT), indicating that RT-Cas1-Cas2 complexes can acquire RNA transcripts for spacer acquisition. In this review, we summarize current studies that focus on the molecular structure and function of the RT-fused Cas1-Cas2 integrase, and its potential applications as a directional RNA-recording tool in cells. Furthermore, we highlight outstanding questions for RT-Cas1-Cas2 studies and future directions for RNA-recording CRISPR technologies.

• BMB Reports
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• 제54권2호
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• pp.98-105
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• 2021

The clustered regularly interspaced short palindromic repeats (CRISPR) system is a family of DNA sequences originally discovered as a type of acquired immunity in prokaryotes such as bacteria and archaea. In many CRISPR systems, the functional ribonucleoproteins (RNPs) are composed of CRISPR protein and guide RNAs. They selectively bind and cleave specific target DNAs or RNAs, based on sequences complementary to the guide RNA. The specific targeted cleavage of the nucleic acids by CRISPR has been broadly utilized in genome editing methods. In the process of genome editing of eukaryotic cells, CRISPR-mediated DNA double-strand breaks (DSB) at specific genomic loci activate the endogenous DNA repair systems and induce mutations at the target sites with high efficiencies. Two of the major endogenous DNA repair machineries are non-homologous end joining (NHEJ) and homology-directed repair (HDR). In case of DSB, the two repair pathways operate in competition, resulting in several possible outcomes including deletions, insertions, and substitutions. Due to the inherent stochasticity of DSB-based genome editing methods, it was difficult to achieve defined single-base changes without unanticipated random mutation patterns. In order to overcome the heterogeneity in DSB-mediated genome editing, novel methods have been developed to incorporate precise single-base level changes without inducing DSB. The approaches utilized catalytically compromised CRISPR in conjunction with base-modifying enzymes and DNA polymerases, to accomplish highly efficient and precise genome editing of single and multiple bases. In this review, we introduce some of the advances in single-base level CRISPR genome editing methods and their applications.

• BMB Reports
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• 제57권1호
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• pp.2-11
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• 2024

Advancements in gene and cell therapy have resulted in novel therapeutics for diseases previously considered incurable or challenging to treat. Among the various contributing technologies, genome editing stands out as one of the most crucial for the progress in gene and cell therapy. The discovery of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) and the subsequent evolution of genetic engineering technology have markedly expanded the field of target-specific gene editing. Originally studied in the immune systems of bacteria and archaea, the CRISPR system has demonstrated wide applicability to effective genome editing of various biological systems including human cells. The development of CRISPR-based base editing has enabled directional cytosine-to-thymine and adenine-to-guanine substitutions of select DNA bases at the target locus. Subsequent advances in prime editing further elevated the flexibility of the edit multiple consecutive bases to desired sequences. The recent CRISPR technologies also have been actively utilized for the development of in vivo and ex vivo gene and cell therapies. We anticipate that the medical applications of CRISPR will rapidly progress to provide unprecedented possibilities to develop novel therapeutics towards various diseases.