Article
Biotechnology & Applied Microbiology
Junhong Choi, Wei Chen, Chase C. Suiter, Choli Lee, Florence M. Chardon, Wei Yang, Anh Leith, Riza M. Daza, Beth Martin, Jay Shendure
Summary: The PRIME-Del method induces deletions with high precision using a pair of prime editing sgRNAs, outperforming CRISPR-Cas9 and sgRNA pairs in programming deletions up to 10 kb. This method can be broadly useful for precise, flexible programming of genomic deletions and potentially other rearrangements.
NATURE BIOTECHNOLOGY
(2022)
Article
Biotechnology & Applied Microbiology
Tingting Jiang, Xiao-Ou Zhang, Zhiping Weng, Wen Xue
Summary: The study optimized prime editing tools for creating precise genomic deletions and direct replacement of a genomic fragment with a desired sequence without the need for an exogenous DNA template. By conjugating Cas9 nuclease to reverse transcriptase and using two PE guide RNAs targeting complementary DNA strands, the PEDAR method achieved precise and specific deletion and repair of target sequences.
NATURE BIOTECHNOLOGY
(2022)
Article
Biotechnology & Applied Microbiology
Qiupeng Lin, Shuai Jin, Yuan Zong, Hong Yu, Zixu Zhu, Guanwen Liu, Liquan Kou, Yanpeng Wang, Jin-Long Qiu, Jiayang Li, Caixia Gao
Summary: Designing prime binding sites with a certain melting temperature and using two pegRNAs can substantially enhance the efficiency of prime editing.
NATURE BIOTECHNOLOGY
(2021)
Article
Biotechnology & Applied Microbiology
Steven Erwood, Teija M. Bily, Jason Lequyer, Joyce Yan, Nitya Gulati, Reid A. Brewer, Liangchi Zhou, Laurence Pelletier, Evgueni A. Ivakine, Ronald D. Cohn
Summary: This study demonstrates the use of the CRISPR prime editing method for high-throughput variant classification and functional characterization. The results show that the method is efficient and accurate, and can be applied to other genes with appropriate cellular assays.
NATURE BIOTECHNOLOGY
(2022)
Article
Multidisciplinary Sciences
Sebastien Levesque, Diana Mayorga, Jean-Philippe Fiset, Claudia Goupil, Alexis Duringer, Andreanne Loiselle, Eva Bouchard, Daniel Agudelo, Yannick Doyon
Summary: Prime editing allows precise genetic modifications without the need for donor DNA templates, but its efficiency remains a challenge. In this study, a robust co-selection strategy was designed by co-editing the Na+/K+ ATPase, enabling easy genetic modifications and revealing certain patterns in the editing process. This lays the foundation for creating cellular models and developing cell-type specific co-selection strategies.
NATURE COMMUNICATIONS
(2022)
Article
Multidisciplinary Sciences
Justin A. Bosch, Gabriel Birchak, Norbert Perrimon
Summary: The study successfully applied prime editing in Drosophila to introduce premature stop codons and achieve efficient germline transmission, demonstrating its potential for studying gene function in Drosophila.
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
(2021)
Article
Biochemistry & Molecular Biology
Miaojin Zhou, Shuqing Tang, Nannan Duan, Mi Xie, Zhuo Li, Mai Feng, Lingqian Wu, Zhiqing Hu, Desheng Liang
Summary: This study demonstrates the therapeutic potential of targeted-deletion of ISS-N1 via prime editing for restoring FL-SMN expression in SMA patient-specific induced pluripotent stem cells (SMA-iPSCs).
INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES
(2022)
Article
Biochemical Research Methods
Shuai Jin, Qiupeng Lin, Qiang Gao, Caixia Gao
Summary: Prime editors (PEs) have been used to accelerate crop improvement and breeding in plants by installing desired base edits. However, the editing efficiency of PEs in plants is generally low. In this protocol, methods such as optimizing prime editing guide RNA (pegRNA), using dual-pegRNAs, and engineering PEs have been shown to enhance editing efficiency. A design platform, PlantPegDesigner, based on rice prime editing experimental data, has also been developed. This protocol provides detailed steps for designing and optimizing pegRNAs using PlantPegDesigner, constructing engineered plant PE vectors, evaluating editing efficiencies, and comparing the effectiveness and byproducts of PEs.
Article
Biochemical Research Methods
Kylie Standage-Beier, Stefan J. Tekel, David A. Brafman, Xiao Wang
Summary: This study introduces a software tool called PINE-CONE which enables high-throughput automated design of pegRNAs and prime editing strategies. By translating edit coordinates and sequences into required designs, PINE-CONE accelerates the application of PE technology in synthetic biology and biomedical research.
ACS SYNTHETIC BIOLOGY
(2021)
Article
Biology
Thomas Thumberger, Tinatini Tavhelidse-Suck, Jose Arturo Gutierrez-Triana, Alex Cornean, Rebekka Medert, Bettina Welz, Marc Freichel, Joachim Wittbrodt
Summary: Precise and targeted genome editing using CRISPR/Cas9 is crucial for basic research and translational applications. Researchers have developed a method called "hei-tag" that enhances the efficiency of Cas9 and base editors by adding a myc-tag linked to an optimized NLS. This method improves editing efficiency and is effective in various model and non-model systems.
Review
Genetics & Heredity
Juliane Weller, Ananth Pallaseni, Jonas Koeppel, Leopold Parts
Summary: The first fruits of the CRISPR-Cas revolution are entering the clinic, offering solutions to previously incurable genetic diseases. Control over generated mutations is crucial for the success of gene editing therapies. This review presents the current understanding and prediction of CRISPR-Cas outcomes in mammalian cells, providing insights for efficient experimental design.
Article
Biotechnology & Applied Microbiology
Shisheng Huang, Zhenwu Zhang, Wanyu Tao, Yao Liu, Xiangyang Li, Xiaolong Wang, Javad Harati, Peng-Yuan Wang, Xingxu Huang, Chao -Po Lin
Summary: In this study, we used an RNA polymerase II promoter instead of an RNA polymerase III promoter to produce Csy4-processed intronic prime editing guide RNAs (pegRNAs), and achieved efficient genetic editing through other optimizations. We also found that simultaneous suppression of both DNA mismatch repair and DNA damage response can achieve efficient and accurate editing in human embryonic stem cells.
Article
Biology
Hanqin Li, Oriol Busquets, Yogendra Verma, Khaja Mohieddin Syed, Nitzan Kutnowski, Gabriella R. Pangilinan, Luke A. Gilbert, Helen S. Bateup, Donald C. Rio, Dirk Hockemeyer, Frank Soldner
Summary: Recent development of prime editing technology has the potential to simplify the generation of human pluripotent stem cell-based disease models. It is more efficient and precise than conventional gene editing methods, particularly in generating disease-associated mutations. By optimizing the delivery modalities, editing efficiency can be further improved.
Article
Biotechnology & Applied Microbiology
Jonas Koeppel, Juliane Weller, Elin Madli Peets, Ananth Pallaseni, Ivan Kuzmin, Uku Raudvere, Hedi Peterson, Fabio Giuseppe Liberante, Leopold Parts
Summary: Prime editing efficiency is influenced by insertion sequence features, secondary structure, and DNA repair context. A library of 3,604 sequences of various lengths is designed to measure the insertion frequency into four genomic sites in human cell lines, revealing that length, nucleotide composition, and secondary structure affect insertion rates. Additionally, the 3' flap nucleases TREX1 and TREX2 suppress the insertion of longer sequences. By combining sequence and repair features, a machine learning model can predict the relative frequency of insertions into a site with an R value of 0.70. User-friendly software based on accurate predictions is demonstrated to aid in choosing high-efficiency codon variants for common fusion tags, along with an empirical catalog of insertion rates for many useful sequences.
NATURE BIOTECHNOLOGY
(2023)
Article
Cell Biology
Yuko Nitahara-Kasahara, Shuji Mizumoto, Yukiko U. Inoue, Shota Saka, Guillermo Posadas-Herrera, Aki Nakamura-Takahashi, Yuki Takahashi, Ayana Hashimoto, Kohei Konishi, Shinji Miyata, Chiaki Masuda, Emi Matsumoto, Yasunobu Maruoka, Takahiro Yoshizawa, Toshiki Tanase, Takayoshi Inoue, Shuhei Yamada, Yoshihiro Nomura, Shin'ichi Takeda, Atsushi Watanabe, Tomoki Kosho, Takashi Okada
Summary: Researchers generated mouse models for musculocontractural Ehlers-Danlos syndrome (mcEDS) through CRISPR/Cas9 genome editing, revealing pathophysiological features such as growth impairment, skin fragility, and muscle-related phenotypes caused by depletion of dermatan sulfate (DS). This study provides insights into the pathophysiology of mcEDS and may contribute to the development of novel treatment strategies.
DISEASE MODELS & MECHANISMS
(2021)
Article
Biochemical Research Methods
Daesik Kim, Beum-Chang Kang, Jin-Soo Kim
Summary: Digenome-seq is a cell-free method for identifying genome-wide off-target sites of programmable nucleases and deaminases, which is more sensitive and comprehensive than cell-based methods and does not involve DNA end enrichment through PCR amplification. The process takes about several weeks, including purification and preparation of RNPs, digestion of genomic DNA, and bioinformatic analysis after WGS.
Article
Gastroenterology & Hepatology
Yi Rang Na, Daun Jung, Michelle Stakenborg, Hyeri Jang, Gyo Jeong Gu, Mi Reu Jeong, Soo Youn Suh, Hak Jae Kim, Yoon Hey Kwon, Tae Sik Sung, Seung Bum Ryoo, Kyu Joo Park, Jong Pil Im, Ji Yong Park, Yun Sang Lee, Heonjong Han, Boyoun Park, Sungwook Lee, Daesik Kim, Ho Su Lee, Isabelle Cleynen, Gianluca Matteoli, Seung Hyeok Seok
Summary: Dysfunctional resolution of intestinal inflammation and altered mucosal healing are critical in the pathogenesis of inflammatory bowel disease (IBD). In this study, we identified a subset of PTGER4-expressing intestinal macrophages with mucosal healing properties in humans and mice. Mechanistically, increased mucosal levels of PGE(2) trigger the secretion of CXCL1 in PTGER4(+) macrophages via MAPKs, promoting epithelial cell differentiation and proliferation in colitis. Targeting macrophages with liposomes loaded with an MAPK agonist could restore defective epithelial regeneration and promote mucosal healing, offering potential therapeutic targets for patients with IBD.
Article
Biotechnology & Applied Microbiology
Jiyeon Kweon, Jung-Ki Yoon, An-Hee Jang, Ha Rim Shin, Ji-Eun See, Gayoung Jang, Jong-Il Kim, Yongsub Kim
Summary: The engineered prime editors leverage various PAM-flexible Cas9 variants to broaden the range of target sites and achieve high editing activity, successfully generating multiple types of mutations in cells. Additionally, they successfully introduce mutations such as BRAF V600E that cannot be induced by conventional prime editors, expanding the applicability of CRISPR-based prime editing technologies in biological research.
Article
Biochemistry & Molecular Biology
Ha Rim Shin, Ji-Eun See, Jiyeon Kweon, Heon Seok Kim, Gi-Jun Sung, Sojung Park, An-Hee Jang, Gayoung Jang, Kyung-Chul Choi, Inki Kim, Jin-Soo Kim, Yongsub Kim
Summary: CRISPR-based base editors are widely used for nucleotide substitutions without causing DNA breaks. Efforts are being made to improve the efficiency of both cytosine and adenine base editors. A study has identified histone deacetylase inhibitors, particularly romidepsin, as a potential option to enhance base editing efficiency.
NUCLEIC ACIDS RESEARCH
(2021)
Article
Multidisciplinary Sciences
Ji-Eun See, Ha Rim Shin, Gayoung Jang, Jiyeon Kweon, Yongsub Kim
Summary: This study presents a new method for functional analysis of BRCA1 variants using CRISPR-mediated cytosine base editor, overcoming issues related to overexpression and post-transcriptional regulation. The researchers identified ambiguous BRCA1 variants and demonstrated the effectiveness of CRISPR-mediated base editors in reclassifying variants of uncertain significance.
JOVE-JOURNAL OF VISUALIZED EXPERIMENTS
(2021)
Article
Biotechnology & Applied Microbiology
Do Yon Kim, Jeong Mi Lee, Su Bin Moon, Hyun Jung Chin, Seyeon Park, Youjung Lim, Daesik Kim, Taeyoung Koo, Jeong-Heon Ko, Yong-Sam Kim
Summary: The research redesigned the guide RNA of Un1Cas12f1, enabling its miniature CRISPR system to achieve efficient and specific genome editing in human cells.
NATURE BIOTECHNOLOGY
(2022)
Article
Engineering, Biomedical
Hyewon Jang, Dong Hyun Jo, Chang Sik Cho, Jeong Hong Shin, Jung Hwa Seo, Goosang Yu, Ramu Gopalappa, Daesik Kim, Sung-Rae Cho, Jeong Hun Kim, Hyongbum Henry Kim
Summary: Prime editing has shown the potential to precisely correct disease-causing mutations and ameliorate disease phenotypes in mouse models of genetic diseases. By identifying optimal guide RNAs, it can precisely target genes without detectable off-target edits. However, further validation in more animal models is necessary.
NATURE BIOMEDICAL ENGINEERING
(2022)
Article
Multidisciplinary Sciences
Yeon-Sook Choi, Myung Ji Kim, Eun A. Choi, Sinae Kim, Eun Ji Lee, Min Ji Park, Mi-Ju Kim, Yeon Wook Kim, Hee-Sung Ahn, Jae Yun Jung, Gayoung Jang, Yongsub Kim, Hyori Kim, Kyunggon Kim, Jin Young Kim, Seung-Mo Hong, Song Cheol Kim, Suhwan Chang
Summary: Gal-3BP is identified as a highly secreted protein in PDAC and its overexpression is closely related to cell proliferation, migration, and invasion. It is found that Gal-3BP enhances galectin-3-mediated signaling, leading to increased PDAC metastasis. Blocking Gal-3BP with antibodies can effectively suppress PDAC metastasis.
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
(2022)
Article
Oncology
Jae Sik Kim, Joo Ho Lee, Sang-Rok Jeon, Yongsub Kim, Seung Hyuck Jeon, Hong-Gyun Wu
Summary: This study used a CRISPR-based knockout screen to determine the mechanism of EGF-induced apoptosis and identified 266 genes that may be responsible for EGF-induced apoptosis. The experimental validation showed that DUSP1 may be a critical component of EGF-induced apoptosis and a potential target for EGFR-overexpressing cancers.
CANCER RESEARCH AND TREATMENT
(2023)
Article
Medicine, Research & Experimental
Eunyoung Choi, Hye-Yeon Hwang, Eunji Kwon, Daesik Kim, Taeyoung Koo
Summary: RNA-guided CRISPR-Cas12a endonucleases derived from Lachnospiraceae bacterium demonstrate a broad PAM preference and efficient recognition of certain non-canonical PAMs. Mutations in LbABE8e improve its base editing activity, allowing for effective gene editing at sites with non-canonical PAMs. The engineered LbCas12a and LbABE8e variants show potential for targeted genome engineering and therapeutic editing in cancer cells.
MOLECULAR THERAPY-NUCLEIC ACIDS
(2022)
Article
Biochemistry & Molecular Biology
Eun-Young Kim, Ji-Eun Kim, Young-Eun Kim, Bongkun Choi, Dong Hyun Sohn, Si-On Park, Yeon-Ho Chung, Yongsub Kim, William H. Robinson, Yong-Gil Kim, Eun-Ju Chang
Summary: The study found that parkin dysfunction associated with the progression of parkinsonism contributes to low bone mineral density. The decreased parkin in monocytes is linked to increased bone-resorbing activity of osteoclasts. Parkin-deficient mice exhibit an osteoporotic phenotype with increased bone-resorbing capacity and susceptibility to inflammatory arthritis.
CELL AND BIOSCIENCE
(2023)
Article
Medicine, Research & Experimental
Gayoung Jang, Ha Rim Shin, Hyo-Sang Do, Jiyeon Kweon, Soojin Hwang, Soyoung Kim, Sun Hee Heo, Yongsub Kim, Beom Hee Lee
Summary: This study utilized CRISPR-mediated base editors (BEs) and prime editors (PEs) to generate LNS-associated disease models and correct the disease models, suggesting a potential therapeutic strategy for this rare genetic disease.
MOLECULAR THERAPY-NUCLEIC ACIDS
(2023)
Article
Biochemistry & Molecular Biology
Jiyeon Kweon, An-Hee Jang, Eunji Kwon, Ungi Kim, Ha Rim Shin, Jieun See, Gayoung Jang, Chaeyeon Lee, Taeyoung Koo, Seokjoong Kim, Yongsub Kim
Summary: This article introduces the use of various CRISPR-Cas9 orthologs in genome engineering. One of the smallest Cas9 orthologs, cjCas9, derived from Campylobacter jejuni, has been developed into base editors capable of inducing endogenous base substitutions with high efficiency and specificity. The engineered cjCas9 and gRNA scaffolds can further improve the base editing efficiency of cjABE8e. The findings in this study expand the potential of using base editors for therapeutic approaches.
EXPERIMENTAL AND MOLECULAR MEDICINE
(2023)