621 research outputs found

    Nat Rev Genet

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    CRISPR-Cas9 RNA-guided nucleases are a transformative technology for biology, genetics and medicine owing to the simplicity with which they can be programmed to cleave specific DNA target sites in living cells and organisms. However, to translate these powerful molecular tools into safe, effective clinical applications, it is of crucial importance to carefully define and improve their genome-wide specificities. Here, we outline our state-of-the-art understanding of target DNA recognition and cleavage by CRISPR-Cas9 nucleases, methods to determine and improve their specificities, and key considerations for how to evaluate and reduce off-target effects for research and therapeutic applications.DP1 GM105378/GM/NIGMS NIH HHS/United StatesDP1GM105378/DP/NCCDPHP CDC HHS/United States2020-05-15T00:00:00Z27087594PMC72255727696vault:3548

    Expanding CRISPR/Cas9 Genome Editing Capacity in Zebrafish Using SaCas9.

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    The type II CRISPR/Cas9 system has been used widely for genome editing in zebrafish. However, the requirement for the 5'-NGG-3' protospacer-adjacent motif (PAM) of Cas9 from Streptococcus pyogenes (SpCas9) limits its targeting sequences. Here, we report that a Cas9 ortholog from Staphylococcus aureus (SaCas9), and its KKH variant, successfully induced targeted mutagenesis with high frequency in zebrafish. Confirming previous findings, the SpCas9 variant, VQR, can also induce targeted mutations in zebrafish. Bioinformatics analysis of these new Cas targets suggests that the number of available target sites in the zebrafish genome can be greatly expanded. Collectively, the expanded target repertoire of Cas9 in zebrafish should further facilitate the utility of this organism for genetic studies of vertebrate biology

    Sharpening the Molecular Scissors: Advances in Gene-Editing Technology

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    The ability to precisely modify human genes has been made possible by the development of tools such as meganucleases, zinc finger nucleases, TALENs, and CRISPR/Cas. These now make it possible to generate targeted deletions, insertions, gene knock outs, and point variants; to modulate g

    Directed evolution of CRISPR-Cas9 to increase its specificity

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    ํ•™์œ„๋…ผ๋ฌธ (๋ฐ•์‚ฌ)-- ์„œ์šธ๋Œ€ํ•™๊ต ๋Œ€ํ•™์› : ์ž์—ฐ๊ณผํ•™๋Œ€ํ•™ ํ™”ํ•™๋ถ€, 2019. 2. ์ด์—ฐ.The use of CRISPR-Cas9 as a therapeutic reagent is hampered by its off-target effects. The determination of the Cas9 crystal structure (Nishimasu, H., et al., 2014) enabled scientists to rationally design mutant Cas9 proteins (enhanced Specificity Cas9 (eSpCas9) and Cas9-High Fidelity (Cas9-HF)) with higher specificities than wild-type Cas9 (WT-Cas9). Their design was based on the hypothesis that weakening non-specific interactions between a Cas9-RNA complex and its substrate DNA would reduce both on-target and off-target activities alike. Since on-target activity is generally much higher than off-target activity, these mutant Cas9 variants would show higher specificities than WT while retaining on-target activities. However, it has been reported that both eSpCas9 and Cas9-HF were poorly active at some target sites (Kim, S. et al., 2017Kulcsar, P.I., et al., 2017Zhang, D., et al., 2017) calling for alternative approaches to improve Cas9 specificity. More recently, three additional Cas9 variants, termed evoCas9, HypaCas9 and xCas9-3.7 with improved specificity and activity were developed, reflecting unmet needs in this field. Although rationally designed S. pyogenes Cas9 (SpCas9) variants that display higher specificities than the wild-type SpCas9 protein are available, these attenuated Cas9 variants are often poorly efficient in human cells. Here, we develop a directed evolution approach in E. coli to obtain Sniper-Cas9, which shows high specificities without sacrificing on-target activities in human cells. Unlike other engineered Cas9 variants, Sniper-Cas9 shows WT-level on-target activities with extended or truncated sgRNAs with further reduced off-target activities and works well in a preassembled ribonucleoprotein (RNP) format to allow DNA-free genome editing.์ธ๊ฐ„๊ณผ ์—ฌ๋Ÿฌ ๋™์‹๋ฌผ๋“ค์˜ ๊ฒŒ๋†ˆ ์ง€๋„๊ฐ€ ์™„์„ฑ๋˜๋ฉด์„œ ์œ ์ „์ฒด ์กฐ์ ˆ์— ๋Œ€ํ•œ ๊ด€์‹ฌ์ด ์˜ฌ๋ผ๊ฐ€๊ฒŒ ๋˜์—ˆ๋‹ค. ์œ ์ „ํ•™์—์„œ๋Š” ์„ธํฌ ์ˆ˜์ค€์—์„œ ๋ฐœํ˜„๋˜๋Š” ์œ ์ „์ž์˜ ์กฐ์ ˆ์ด ํ•„์ˆ˜ ๋ถˆ๊ฐ€๊ฒฐํ•œ ์š”์†Œ์ด๋‹ค. ๋‹ค์–‘ํ•œ ์œ ์ „์ž์˜ ๊ธฐ๋Šฅ์ด ๋ฐํ˜€์ง€๊ณ , ์งˆ๋ณ‘๊ณผ ๊ด€๋ จ๋œ ์œ ์ „์ž์˜ ์กฐ์ ˆ์„ ํ•˜๋Š” ๋ฐ ๋งŽ์€ ์—ฐ๊ตฌ๊ฐ€ ์ง„ํ–‰๋จ์— ๋”ฐ๋ผ ์œ ์ „์ž๋ฅผ ํ‘œ์ ์œผ๋กœ ํ•  ์ˆ˜ ์žˆ๋Š” Zinc finger nuclease, TALE nuclease, CRISPR-Cas9๊ณผ ๊ฐ™์€ ์œ ์ „์ž ๊ฒฐํ•ฉ ๋‹จ๋ฐฑ์งˆ์— ๋Œ€ํ•œ ์—ฐ๊ตฌ๋„ ํ™œ๋ฐœํžˆ ์ง„ํ–‰๋˜๊ณ  ์žˆ๋‹ค. CRISPR-Cas9์€ ๋‹ค๋ฃจ๊ธฐ ์‰ฝ๊ณ , ํšจ์œจ๋„ ์ข‹์€ ๊ฐ•๋ ฅํ•œ ๋‹จ๋ฐฑ์งˆ ์‹œ์Šคํ…œ์œผ๋กœ ์ž๋ฆฌ์žก๊ณ  ์žˆ์œผ๋‚˜, ๋ชฉํ‘œํ•œ ์œ ์ „์ž๊ฐ€ ์•„๋‹Œ ๋น„ํŠน์ด์ ์ธ ๊ฒฐํ•ฉ์œผ๋กœ ๋‹ค๋ฅธ ์œ ์ „์ž์™€ ๊ฒฐํ•ฉ์„ ํ•  ์ˆ˜ ์žˆ๋‹ค๋Š” ๋‹จ์ ์ด ์žˆ๋‹ค. ์ด๋ฅผ ํ•ด๊ฒฐํ•˜๊ธฐ ์œ„ํ•ด์„œ ํŠน์ด์„ฑ์ด ์˜ฌ๋ผ๊ฐ„ Cas9์„ ๊ฐœ๋ฐœํ•˜๋Š” ์—ฐ๊ตฌ๋“ค์ด ์ง„ํ–‰๋˜๊ณ  ์žˆ๋‹ค. ์ฃผ๋œ ์—ฐ๊ตฌ ๋ฐฉ์‹์œผ๋กœ๋Š” ๊ตฌ์กฐ์  ์ •๋ณด๋ฅผ ๊ธฐ๋ฐ˜์œผ๋กœ RNA์™€ ๋ณด๋‹ค ์ •ํ™•ํ•˜๊ฒŒ ๊ฒฐํ•ฉํ•  ์ˆ˜ ์žˆ๋„๋ก ๋‹จ๋ฐฑ์งˆ์˜ ์ •๋ณด๋ฅผ ๋ฐ”๊ฟ”์ฃผ๋Š” ํ•ฉ๋ฆฌ์ ์ธ ๋””์ž์ธ ๋ฐฉ๋ฒ•๊ณผ ์ž„์˜์˜ ๋Œ์—ฐ๋ณ€์ด๋ฅผ ์ •๋ณด๋ฅผ ๋„ฃ์–ด์ฃผ๊ณ  ์ด๋ฅผ ๋Œ€์žฅ๊ท ๊ณผ ๊ฐ™์€ ์ƒ๋ฌผ์ฒด๋ฅผ ์ด์šฉํ•˜์—ฌ ์œ ๋„์ง„ํ™”๋ฅผ ํ†ตํ•ด์„œ ๋ชฉํ‘œ๋กœ ํ•˜๋Š” ๋‹จ๋ฐฑ์งˆ์„ ์–ป์–ด๋‚ด๋Š” ๋ฐฉ์‹์ด ์žˆ๋‹ค. ์ด๋ฒˆ ์—ฐ๊ตฌ์—์„œ๋Š”, ๋Œ€์žฅ๊ท ์„ ์ˆ™์ฃผ๋กœ ์œ ๋„ ์ง„ํ™” ๋ฐฉ๋ฒ•์„ ์ด์šฉํ•˜์—ฌ ์ •ํ™•์„ฑ์ด ํ–ฅ์ƒ๋œ Cas9 ๋‹จ๋ฐฑ์งˆ์„ ๋งŒ๋“œ๋Š” ์ผ์„ ์ง„ํ–‰ํ•˜์˜€๋‹ค. ํ•œ ๋ฒˆ์— ์–‘์„ฑ์„ ํƒ๊ณผ ์Œ์„ฑ์„ ํƒ์„ ๋™์‹œ์— ์ง„ํ–‰ํ•  ์ˆ˜ ์žˆ๋Š” Sniper ์Šคํฌ๋ฆฌ๋‹์„ ๊ณ ์•ˆํ•˜๊ณ  ์ด๋ฅผ ์ด์šฉํ•ด์„œ ๋ชฉํ‘œ๋กœ ํ•˜๋Š” ๋‹จ๋ฐฑ์งˆ์ธ Sniper-Cas9์„ ์„ ๋ณ„ํ•ด ๋‚ผ ์ˆ˜ ์žˆ์—ˆ๋‹ค. ์„ ๋ณ„ํ•œ ํ›„ ๊ธฐ์กด์— ๊ฐœ๋ฐœ๋œ ์—ฌ๋Ÿฌ๊ฐ€์ง€ Cas9 ๋‹จ๋ฐฑ์งˆ๋“ค๊ณผ ๋น„๊ต๋ฅผ ํ†ตํ•ด์„œ ์ฐพ์•„๋‚ธ Sniper-Cas9์ด ์ •ํ™•์„ฑ์ด ํ–ฅ์ƒ๋˜์—ˆ๊ณ  ๋‹ค๋ฅธ ๋‹จ๋ฐฑ์งˆ๋“ค์€ ์‚ฌ์šฉํ•  ์ˆ˜ ์—†๋Š” ์—ฌ๋Ÿฌ๊ฐ€์ง€ sgRNA์™€๋„ ๋ฌธ์ œ ์—†์ด ๊ฒฐํ•ฉ์„ ํ•  ์ˆ˜ ์žˆ๋‹ค๋Š” ๊ฒƒ์„ ํ™•์ธํ•˜์˜€๋‹ค.Table of Contents Abstract i Table of Contents iii List of Figures v Abstract in Koreans 106 Introduction 1 Materials and Methods 4 1. Plasmid construction 4 2. EMX1 genome insertion 4 3. Library construction 5 4. Positive and negative screening for evolving SpCas9 6 5. Plasmids encoding Cas9 variants and sgRNA 7 6. Cell culture and transfection conditions 8 7. Recombinant Cas9 protein production 8 8. Preparation of guide RNAs for RNP production 9 9. RNP delivery 10 10. Whole-genome and digenome sequencing 10 11. Targeted deep sequencing 11 12. iPS cell genome editing 11 13. T-cell genome editing 12 Results 15 1. Simultaneous positive and negative selection using E. coli 15 2. Construction of the Cas9 library and Sniper screen 21 3. Clone selection and characterization 36 4. Comparison of Sniper-Cas9 with other engineered Cas9 variants 45 5. On-target and off-target activities of Sniper-Cas9 RNP 53 6. Comparison of Sniper-Cas9 with xCas9-3.7 58 7. Unbiased genome-wide off-target analysis of Sniper-Cas9 66 8. On-target and off-target activities of Sniper-Cas9 BE3 80 9. Sniper-Cas9 RNP as potential therapeutic modality 83 Discussion 94 References 101Docto

    CRISPR/Cas9โ€mediated genome editing: from basic research to translational medicine

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    The recent development of the CRISPR/Cas9 system as an efficient and accessible programmable genome-editing tool has revolutionized basic science research. CRISPR/Cas9 system-based technologies have armed researchers with new powerful tools to unveil the impact of genetics on disease development by enabling the creation of precise cellular and animal models of human diseases. The therapeutic potential of these technologies is tremendous, particularly in gene therapy, in which a patient-specific mutation is genetically corrected in order to treat human diseases that are untreatable with conventional therapies. However, the translation of CRISPR/Cas9 into the clinics will be challenging, since we still need to improve the efficiency, specificity and delivery of this technology. In this review, we focus on several in vitro, in vivo and ex vivo applications of the CRISPR/Cas9 system in human disease-focused research, explore the potential of this technology in translational medicine and discuss some of the major challenges for its future use in patients.Portuguese Foundation for Science and Technology: UID/BIM/04773/2013 1334 Spanish Ministry of Science, Innovation and Universities RTI2018-094629-B-I00 Portuguese Foundation for Science and Technology SFRH/BPD/100434/2014 European Union (EU) 748585 LPCC-NRS/Terry Fox grantsinfo:eu-repo/semantics/publishedVersio
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