Programmable base editing of zebrafish genome using a modified CRISPR-Cas9 system.

Precise genetic modifications in model animals are essential for biomedical research. Here, we report a programmable "base editing" system to induce precise base conversion with high efficiency in zebrafish. Using cytidine deaminase fused to Cas9 nickase, up to 28% of site-specific single-base mutations are achieved in multiple gene loci. In addition, an engineered Cas9-VQR variant with 5'-NGA PAM specificities is used to induce base conversion in zebrafish. This shows that Cas9 variants can be used to expand the utility of this technology. Collectively, the targeted base editing system represents a strategy for precise and effective genome editing in zebrafish.The use of base editing enables precise genetic modifications in model animals. Here the authors show high efficient single-base editing in zebrafish using modified Cas9 and its VQR variant with an altered PAM specificity

CRISPR-Cas orthologues and variants: optimizing the repertoire, specificity and delivery of genome engineering tools

Robust and cost-effective genome editing in a diverse array of cells and model organisms is now possible thanks to the discovery of the RNA-guided endonucleases of the CRISPR-Cas system. The commonly used Cas9 of Streptococcus pyogenes shows high levels of activity but, depending on the application, has been associated with some shortcomings. Firstly, the enzyme has been shown to cause mutagenesis at genomic sequences resembling the target sequence. Secondly, the stringent requirement for a specific motif adjacent to the selected target site can limit the target range of this enzyme. Lastly, the physical size of Cas9 challenges the efficient delivery of genomic engineering tools based on this enzyme as viral particles for potential therapeutic applications. Related and parallel strategies have been employed to address these issues. Taking advantage of the wealth of structural information that is becoming available for CRISPR-Cas effector proteins, Cas9 has been redesigned by mutagenizing key residues contributing to activity and target recognition. The protein has also been shortened and redesigned into component subunits in an attempt to facilitate its efficient delivery. Furthermore, the CRISPR-Cas toolbox has been expanded by exploring the properties of Cas9 orthologues and other related effector proteins from diverse bacterial species, some of which exhibit different target site specificities and reduced molecular size. It is hoped that the improvements in accuracy, target range and efficiency of delivery will facilitate the therapeutic application of these site-specific nucleases

Advances in Engineering the Fly Genome with the CRISPR-Cas System.

Drosophila has long been a premier model for the development and application of cutting-edge genetic approaches. The CRISPR-Cas system now adds the ability to manipulate the genome with ease and precision, providing a rich toolbox to interrogate relationships between genotype and phenotype, to delineate and visualize how the genome is organized, to illuminate and manipulate RNA, and to pioneer new gene drive technologies. Myriad transformative approaches have already originated from the CRISPR-Cas system, which will likely continue to spark the creation of tools with diverse applications. Here, we provide an overview of how CRISPR-Cas gene editing has revolutionized genetic analysis in Drosophila and highlight key areas for future advances

Nat Rev Genet

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.

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

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

학위논문 (박사)-- 서울대학교 대학원 : 자연과학대학 화학부, 2019. 2. 이연.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.인간과 여러 동식물들의 게놈 지도가 완성되면서 유전체 조절에 대한 관심이 올라가게 되었다. 유전학에서는 세포 수준에서 발현되는 유전자의 조절이 필수 불가결한 요소이다. 다양한 유전자의 기능이 밝혀지고, 질병과 관련된 유전자의 조절을 하는 데 많은 연구가 진행됨에 따라 유전자를 표적으로 할 수 있는 Zinc finger nuclease, TALE nuclease, CRISPR-Cas9과 같은 유전자 결합 단백질에 대한 연구도 활발히 진행되고 있다. CRISPR-Cas9은 다루기 쉽고, 효율도 좋은 강력한 단백질 시스템으로 자리잡고 있으나, 목표한 유전자가 아닌 비특이적인 결합으로 다른 유전자와 결합을 할 수 있다는 단점이 있다. 이를 해결하기 위해서 특이성이 올라간 Cas9을 개발하는 연구들이 진행되고 있다. 주된 연구 방식으로는 구조적 정보를 기반으로 RNA와 보다 정확하게 결합할 수 있도록 단백질의 정보를 바꿔주는 합리적인 디자인 방법과 임의의 돌연변이를 정보를 넣어주고 이를 대장균과 같은 생물체를 이용하여 유도진화를 통해서 목표로 하는 단백질을 얻어내는 방식이 있다. 이번 연구에서는, 대장균을 숙주로 유도 진화 방법을 이용하여 정확성이 향상된 Cas9 단백질을 만드는 일을 진행하였다. 한 번에 양성선택과 음성선택을 동시에 진행할 수 있는 Sniper 스크리닝을 고안하고 이를 이용해서 목표로 하는 단백질인 Sniper-Cas9을 선별해 낼 수 있었다. 선별한 후 기존에 개발된 여러가지 Cas9 단백질들과 비교를 통해서 찾아낸 Sniper-Cas9이 정확성이 향상되었고 다른 단백질들은 사용할 수 없는 여러가지 sgRNA와도 문제 없이 결합을 할 수 있다는 것을 확인하였다.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/Cas9‐mediated genome editing: from basic research to translational medicine

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