CRISPR/Cas9 시스템을 이용한 돼지 수정란에서의 cd163 발현 효율 분석

박미령(Mi-Ryung Park),이민국(Min Gook Lee),이보람(Bo Ram Lee),옥선아(Sun A Ock),변승준(Sung June Byun) 한국산학기술학회 2023 한국산학기술학회논문지 Vol.24 No.4

유전자 편집 돼지는 농업과 의료 분야에서 제공될 수 있어 폭넓게 각광 받고 있다. 최근 CRISPR/Cas9 시스템이 적용됨에 따라 genome editing이 효율적으로 향상되었다. 돼지 수정란 내 CRISPR/Cas9을 세포질내 미세주입으로 site specific mutations을 가능할 수 있게 하였다. 본 연구에서는 돼지 수정란 내 cd163 gRNA와 CRISPR/Cas9 components를 이용하여 도입한 후 그 효율성을 비교 분석하였다. CRISPR/Cas9 protein과 cd163 gRNA를 수정란 내 주입하여 발달율을 비교 분석한 결과 대조구 (90.6%)와 미세주입 그룹 (78.9% and 85.2%)에서 유의적 차이는 인정되지 않았다. 또한, 배반포 발달율을 비교 분석 한 결과 CRISPR/Cas9 protein과 cd163 gRNA 주입 그룹 (19.9% and 19.6%)로 대조구와 (21.5%) 유사하게 나타났다. 각각의 배반포를 이용하여 cd163 유전자의 targeted modification을 분석하였다. 그 결과 cd163(10+134) gRNA를 주입한 그룹의 경우(22.7%) cd163(10) 주입한 그룹보다(12.9%) 유의적으로 높은 효율을 나타내었다. 유전자 변형은 4bp deletion 일어난 것부터 72bp insertions 패턴까지 다양한 패턴으로 나타나는 것을 확인할 수 있었다. 이러한 결과를 바탕으로 CRISPR/Cas9 system을 돼지 수정란 내 미세주입 함으로써 유전자 편집 돼지 생산에 응용할 수 있을 것으로 사료된다. Gene editing (GE) in pig production can have a wide impact by increasing the availability of gene-edited pigs for agriculture and biomedicine. Recent applications of the clustered regularly interspaced short palindromic repeats (CRISPR)/ CRISPR-associated protein 9 (Cas9) system hold promise for improving the efficacy of gene editing. The cytoplasmic microinjection of the CRISPR/Cas9 system enables the induction of site-specific mutations in porcine zygotes. In this study, we examined the efficiency of the CRISPR/Cas9 protein and cluster of differentiation 163 (cd163) guide RNA (gRNA) components for introduction into zygotes by cytoplasmic microinjection. The cleavage rates of the CRISPR/Cas9 protein and cd163 gRNA injected groups (78.9% and 85.2%) were statistically similar to that of the control group (90.6%). Moreover, the blastocyst formation rates of the CRISPR/Cas9 protein and cd163 gRNA injected groups (19.9% and 19.6%) were also statistically similar to that of the control group (21.5%). When individual blastocysts were genotyped, we observed targeted modification of the genes in the subsequent blastocysts. In the samples of 10 ng/ul, each of the CRISPR/Cas9 protein and the cd163 (10+134) gRNA injected group (22.7%) was significantly higher (p<0.05) than that in the 10 ng/ul samples each of CRISPR/Cas9 protein and cd163(10) gRNA injected group (12.9%). Various types of indel mutations, including 4 bp deletion to 72 bp insertions, were detected in the mutant blastocysts. These results suggest that the CRISPR/Cas9 technology can be applied to produce gene-edited pigs by direct zygote injection.

Highly efficient DSB-free base editing for streptomycetes with CRISPR-BEST

Tong, Yaojun,Whitford, Christopher M.,Robertsen, Helene L.,Blin, Kai,Jørgensen, Tue S.,Klitgaard, Andreas K.,Gren, Tetiana,Jiang, Xinglin,Weber, Tilmann,Lee, Sang Yup National Academy of Sciences 2019 PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF Vol.116 No.41

<P><B>Significance</B></P><P>Although CRISPR-Cas9 tools dramatically simplified the genetic manipulation of actinomycetes, significant concerns of genome instability caused by the DNA double-strand breaks (DSBs) and common off-target effects remain. To address these concerns, we developed CRISPR-BEST, a DSB-free and high-fidelity single-nucleotide–resolution base editing system for streptomycetes and validated its use by determining editing properties and genome-wide off-target effects. Furthermore, our CRISPR-BEST toolkit supports Csy4-based multiplexing to target multiple genes of interest in parallel. We believe that our CRISPR-BEST approach is a significant improvement over existing genetic manipulation methods to engineer streptomycetes, especially for those strains that cannot be genome-edited using normal DSB-based genome editing systems, such as CRISPR-Cas9.</P><P>Streptomycetes serve as major producers of various pharmacologically and industrially important natural products. Although CRISPR-Cas9 systems have been developed for more robust genetic manipulations, concerns of genome instability caused by the DNA double-strand breaks (DSBs) and the toxicity of Cas9 remain. To overcome these limitations, here we report development of the DSB-free, single-nucleotide–resolution genome editing system CRISPR-BEST (CRISPR-Base Editing SysTem), which comprises a cytidine (CRISPR-cBEST) and an adenosine (CRISPR-aBEST) deaminase-based base editor. Specifically targeted by an sgRNA, CRISPR-cBEST can efficiently convert a C:G base pair to a T:A base pair and CRISPR-aBEST can convert an A:T base pair to a G:C base pair within a window of approximately 7 and 6 nucleotides, respectively. CRISPR-BEST was validated and successfully used in different <I>Streptomyces</I> species. Particularly in nonmodel actinomycete <I>Streptomyces collinus</I> Tu¨365, CRISPR-cBEST efficiently inactivated the 2 copies of <I>kirN</I> gene that are in the duplicated kirromycin biosynthetic pathways simultaneously by STOP codon introduction. Generating such a knockout mutant repeatedly failed using the conventional DSB-based CRISPR-Cas9. An unbiased, genome-wide off-target evaluation indicates the high fidelity and applicability of CRISPR-BEST. Furthermore, the system supports multiplexed editing with a single plasmid by providing a Csy4-based sgRNA processing machinery. To simplify the protospacer identification process, we also updated the CRISPy-web (https://crispy.secondarymetabolites.org), and now it allows designing sgRNAs specifically for CRISPR-BEST applications.</P>

Characterization of distinct mutation patterns by CRISPR-Cas9 and CRISPR-Cpf1 genome editing systems

Taegeun Bae,Woo Chang Hwang,Dohyeon Lee,송길태,Junseok W Hur,허준호 대한독성 유전단백체 학회 2019 Molecular & cellular toxicology Vol.15 No.4

Backgrounds: The differences of DNA mutation patterns of genome editing by CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas9 and CRISPR-Cpf1 is not completely understood. Methods: CRISPR-Cas9 or CRISPR-Cpf1 is applied for genome editing at the exact same four genomic locations in human embryonic kidney 293T (HEK293T) cells. The mutation patterns of the target genomic loci were analyzed by targeted deep sequencing. Results: The mutation patterns of CRISPR-Cas9 and CRISPR-Cpf1 showed prominent differences. CRISPR- Cpf1 mediated genome engineering almost always resulted in deletion, while CRISPR-Cas9 showed remarkably high rate of insertion mutation of 1 base pair. The deletion patterns of CRISPR-Cas9 and CRISPR- Cpf1 were also different. The deletion patterns of CRISPR-Cas9 were composed of diverse and evenly distributed deletion patterns, while the mutation patterns of CRISPR-Cpf1 were composed of fewer major patterns. Conclusion: Genome editing by CRISPR-Cas9 and CRISPR-Cpf1 shows different characteristics.

CRISPR technologies for bacterial systems: Current achievements and future directions

Choi, K.R.,Lee, S.Y. Pergamon Press ; Elsevier Science Ltd 2016 BIOTECHNOLOGY ADVANCES Vol.34 No.7

<P>Throughout the decades of its history, the advances in bacteria-based bio-industries have coincided with great leaps in strain engineering technologies. Recently unveiled clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated proteins (Cas) systems are now revolutionizing biotechnology as well as biology. Diverse technologies have been derived from CRISPR/Cas systems in bacteria, yet the applications unfortunately have not been actively employed in bacteria as extensively as in eukaryotic organisms. A recent trend of engineering less explored strains in industrial microbiology metabolic engineering, synthetic biology, and other related disciplines is demanding facile yet robust tools, and various CRISPR technologies have potential to cater to the demands. Here, we briefly review the science in CRISPR/Cas systems and the milestone inventions that enabled numerous CRISPR technologies. Next, we describe CRISPR/Cas-derived technologies for bacterial strain development, including genome editing and gene expression regulation applications. Then, other CRISPR technologies possessing great potential for industrial applications are described, including typing and tracking of bacterial strains, virome identification, vaccination of bacteria, and advanced antimicrobial approaches. For each application, we note our suggestions for additional improvements as well. In the same context, replication of CRISPR/Cas-based chromosome imaging technologies developed originally in eukaryotic systems is introduced with its potential impact on studying bacterial chromosomal dynamics. Also, the current patent status of CRISPR technologies is reviewed. Finally, we provide some insights to the future of CRISPR technologies for bacterial systems by proposing complementary techniques to be developed for the use of CRISPR technologies in even wider range of applications. (C) 2016 Published by Elsevier Inc.</P>

CRISPR/Cas 진단의 원리와 현황

박지웅,강봉근,신화희,신준근,Park, Jeewoong,Kang, Bong Keun,Shin, Hwa Hui,Shin, Jun Geun 대한의용생체공학회 2021 의공학회지 Vol.42 No.3

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.

식물 유전체 편집을 위한 CRISPR/Cas9의 적용

이만보(Man Bo Lee),서용원(Yong Weon Seo) 고려대학교 생명자원연구소 2017 생명자원연구 Vol.25 No.-

초 록 최근 유전자 편집 기술을 Streptococcus pyogenes에서 유래한 CRISPR/Cas9 system이 주도하고 있다. 바이러스 침입에 대항하는 면역체계인 CRISPR/Cas9 system을 활용하여 목표 유전자에 돌연변이를 유발하거나 목표 유전자를 원하는 염기서열로 편집할 수 있다. CRISPR/Cas9 기술은 목표 유전자의 변화로 인한 생물학적 표현형 변화 연구에 적용되며 기능 유전학 연구에 핵심적인 역할을 할 수 있다. 나아가 인간 질병 modeling, 유전질환 치료, 신약 개발, 식물 바이오 매스 증대, 종자 생산량 증산 등 다양한 영역에서 활용 가능하다. CRISPR/Cas9 system의 CrRNA과 tracrRNA은 결합하여 sgRNA로서 역할을 하고 목표 유전자 특이성 및 Cas9 단백질의 인식에 관여한다. PAM 은 Cas9 단백질의 DNA 결합에 관여하는 필수적인 염기서열이며, 실험 설계 단계에서 sgRNA와 함께 고려되어야 한다. Cas9 단백질은 DNA에 double-strand break를 발생시켜 DNA 보수 기작을 유도한다. 목표 유전자는 DNA 보수 기작을 거치는 동안 돌연변이가 발생하거나 유전자가 편집된다. 식물은 인간의 주식, 치료제, 바이오 연료원 등을 제공하며 삶의 유지와 질적 향상에 밀접한 역할을 한다. 유전체에 임의 돌연변 이를 유발하는 기존의 화학적 물리적 돌연변이 유발 방법과 달리 CRISPR/Cas9 system은 식물 유전체 특정 위치에 돌연변이를 유발시킬 수있다. CRISPR/Cas9 system은 실제적으로 이미 개발된 Agrobacterium-mediated transformation과 particle bombardment 기술 등 식물 형질전환 기법을 통해 식물체에 적용 가능하다. 유전체 크기가 크고 반복 서열의 비중이 높은 일부 작물에서는 목표 유전자 특이성 확보가 어렵고 원하지 않는 유전자가 돌연변이 되는 off-target 발생 가능성이 높으나, 애기장대, 담배, 벼 등 모델 식물과 일부 작물에서 실제 적용 사례가 보고 되어 있다. CRISPR/Cas9 system은 실험이 단순하고 효율적이며 한 번에 여러 유전자에 적용 가능하고 비용이 저렴한 큰 장점을 가지고 있으며 식물을 포함한 동물, 심지어 인간에게도 적용 가능하다. CRISPR/Cas9 system을 활용한 유전체 편집 기술은 인간의 삶의 질 향상에 핵심적인 역할을 담당할 것으로 예측된다.

CRISPR/Cas-Based Nanobiosensor Using Plasmonic Nanomaterials to Detect Disease Biomarkers

Jin-Ha Choi,Jinho Yoon,Meizi Chen,Minkyu Shin,Li Ling Goldston,이기범,최정우 한국바이오칩학회 2025 BioChip Journal Vol.19 No.2

The development of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein (Cas) technology (CRISPR/Cas) as a gene-editing tool has the potential to revolutionize nucleic acid analysis. Recently, CRISPR/Cas systems have demonstrated considerable promise in the development of biosensors for the detection of essential disease biomarkers because they exhibit nonspecific collateral cleavage properties upon target sequence recognition. How-ever, the CRISPR/Cas-based biosensors developed thus far have limitations, such as complicated steps, low sensitivity, low selectivity, and low signal-to-noise ratios. These limitations can be overcome by incorporating the unique characteristics of plasmonic nanomaterials into CRISPR/Cas systems to enhance the signal and improve the sensitivity of these biosensors. From this perspective, current interdisciplinary studies on CRISPR/Cas-based nanobiosensors comprising plasmonic nanoma-terials can contribute to the development of highly sensitive CRISPR/Cas-based nanobiosensors. These nanobiosensors can detect attractive disease biomarkers, such as viral nucleic acids, small molecules, and proteins. This review article provides a thorough overview of nanobiosensors that incorporate CRISPR/Cas systems combined with plasmonic nanomaterials to enhance biosensing performance. We believe this review will inspire novel approaches and further innovation in the fields of molecular diagnostics and biomedicine aimed at using CRISPR/Cas systems and plasmonic nanomaterials for more per-sonalized and effective medical treatments.

Recent trends in CRISPR‑Cas system: genome, epigenome, and transcriptome editing and CRISPR delivery systems

배태근,허준석,김도경,허준호 한국유전학회 2019 Genes & Genomics Vol.41 No.8

Background The CRISPR-Cas systems have emerged as a robust genome editing tool useful in various fields of research. With the discovery and development of the orthologous CRISPR-Cas systems, their genome editing efficiency have improved. Objective In this review, we aim to present the recent developments and applications of the CRISPR-Cas systems. Methods First, we introduce how the advancements of CRISPR technology enabled genome editing to single base precision. Then, we discuss the CRISPR based methods for targeted transcriptional regulation, epigenome editing, and RNA editing. Finally, we review the CRISPR delivery systems highlighting recent attempts to integrate nanotechnology to develop novel CRISPR delivery modalities. Conclusion Here, we review the recent trends in CRISPR-based biotechnologies, encompassing genome editing, epigenome regulation and direct RNA targeting and provide an overview of methods employed for CRISPR delivery with an emphasis on the most recent nanotechnology-based delivery strategies. We anticipate that the development of CRISPR based technology will continue to explore novel methods.

현장에서 가축질병을 진단하기 위한 CRISPR/Cas 시스템의 활용

이원희(Wonhee Lee),이윤석(Yoonseok Lee) 한국생명과학회 2020 생명과학회지 Vol.30 No.3

최근, 국내에서 발생하는 대가축의 질병은 바이러스 혹은 세균 등과 같은 병원체가 사료 섭취, 가축 간의 신체접촉, 호흡 등 다양한 경로를 통해 전파되어 발병되는 전염성 질병이다. 전염성 질병은 가축의 건강을 위협하고 생산성을 감소시키기 때문에 현장에서 조기 진단하여 개체 격리와 같은 통제 관리가 필수적이다. 기존 사용되고 있는 진단 키트들은 현장에서 사용하기에 용이하지 않으며 극소량의 감도에서 진단이 제한적인 단점을 가지고 있다. 그러므로, 현장에서 극소량의 감도와 진단의 편이성을 고려하여 DNA와 RNA 수준에서 진단할 수 있는 CRISPR/Cas 시스템은 최적의 시스템이라 할 수 있다. 본 연구논문에서는 대가축의 전염성 질병들을 현장에서 조기 진단함에 있어 CRISPR/Cas 시스템의 활용전략에 대해 소개하고자 한다. 최근 발견된 CRISPR/Cas 효소들은 2개의 클래스와 6가지 하위유형으로 분류되었다. 이 중에서 클래스 2에 포함되는 Cas 효소들은 대표적으로 제 2형에 Cas9, 제 5형에 Cas12a와 Cas12b, 제 6형에 Cas13a와 Cas13b가 있다. 현재까지 개발된 CRISPR/Cas 시스템들은 간단한 시각 신호를 통해 표적에 대한 정량 및 다중 감지가 가능하고 특히, 극소량 수준의 초고감도에서도 표적만을 진단할 수 있으며 단시간 이내에 진단 결과를 얻을 수 있다. 하지만 초고감도 DNA 혹은 RNA를 진단하기 위해 최적의 신호 증폭 방법과 결합되어야 하고 표적 DNA 혹은 RNA를 진단에 적합하도록 DNA를 RNA로, RNA를 DNA로 전변해야 하는 단점이 있다. 따라서, 현장에서 대가축의 전염성 질병을 조기에 진단할 수 있는 CRISPR/Cas 바이오센서를 개발하는데 있어 가축의 전염 매개체로부터 추출되는 병원체 유형(DNA 혹은 RNA)을 고려하여 최적의 Cas 효소를 선정하여야 하고 이에 따른 적절한 신호 증폭 방법이 결합되어야 한다. 따라서, CRISPR/Cas 시스템은 유전자 편집 방법을 사용하는 빠르고 효율적인 진단 도구이며 이 시스템은 소의 전염병을 조기에 진단하고 감염 확산방지에 도움될 수 있을 것으로 판단 되어진다. Recently, cattle epidemic diseases are caused by a pathogen such as a virus or bacterium. Such diseases can spread through various pathways, such as feed intake, respiration, and contact between livestock. Diagnosis based on the ELISA (Enzyme-linked immunosorbent assay) and PCR (Polymerase chain reaction) methods has limitations because these traditional diagnostic methods are time consuming assays that require multiple steps and dedicated equipment. In this review, we propose the use of the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) Cas system based on DNA and RNA levels for early point-of-care diagnosis in cattle. In the CRISPR/Cas system, Cas effectors are classified into two classes and six subtypes. The Cas effectors included in class 2 are typically Cas9 in type II, Cas12 in type V (Cas12a and Cas12b) and Cas13 in type VI (Cas13a and Cas13b). The CRISPR/Cas system uses reporter molecules that are attached to the ssDNA strands. When the Cas enzyme cuts the ssDNA, these reporters either fluoresce or change color, indicating the presence of a specific disease marker. There are several steps in the development of a CRISPR/Cas system. The first is to select the Cas enzyme depending on DNA or RNA from pathogens (viruses or bacteria). Based on that, the next step is to integrate the optimal amplification, transducing method, and signal reporter. The CRISPR/Cas system is a powerful diagnostic tool using a gene-editing method, which is faster, better, and cheaper than traditional methods. This system could be used for early diagnosis of epidemic cattle diseases and help to control their spread.

Genetics of CRISPR arrays in Salmonella Typhimurium 14028 associated with foreign DNA decay

김정남 한국유전학회 2018 Genes & Genomics Vol.40 No.8

Clustered regularly interspaced short palindromic repeats (CRISPRs) are a genetic locus of prokaryotes and contain highly conserved direct repeats, spacers, and CRISPR-associated genes. Spacers in CRISPRs are known as adaptive immune markers and reveal what types of phage or foreign DNA have been introduced in the past. The primary objective of this study was to analyze spacer sequences in CRISPR arrays of 15 Salmonella enterica subspecies and to determine if Salmonella CRISPRs are indeed involved in resistance to foreign DNAs. Using a bioinformatics algorithm, the CRISPR arrays of 15 subspecies of S. enterica were predicted. The transformation efficiencies of the wild-type and mutant strains lacking a space were determined using the plasmid harboring the same sequences with the space. Analysis of the CRISPR arrays indicated that S. Typhimurium encoded three possible CRISPR regions in the genome. Notably, 48 or 55 spacers were predicted in the genomes of S. Typhimurium 14028 and LT2 strains, respectively, and 39 were precisely identical. To confirm this prediction, the predicted CRISPR regions of S. Typhimurium 14028 were sequenced using the specific primers. Interestingly, a homology search of individual spacers found that the 2nd spacer of CRISPR 2 was nearly identical to a partial genome region of phage FSL SP-016. The mutant strain showed two to threefold increased transformation efficiency compared to that of the wild-type strain. These results demonstrate that the spacer sequence is dependent on genetic relations, especially for adaptive immunity against phage or foreign DNAs.