Efficient Human Genome Editing Using SaCas9 Ribonucleoprotein Complexes

Wang, Yufei; Wang, Bang; Xie, Haihua; Ren, Qianwen; Liu, Xiexie; Li, Fanfan; Lv, Xiujuan; He, Xiubin; Cheng, Congsheng; Deng, Ran; Li, Jin; Zhao, Junzhao; Song, Zongming; Gu, Feng

doi:10.1002/biot.201800689

Cited by 20 publications

(17 citation statements)

References 39 publications

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“…It reported that genome editing was successful with SaCas9 in RNP form in human cell lines, including the correction of a pathogenic RS1 mutation for X-linked Juvenile Retinoschisis. Targeted deep sequencing did not reveal any off-target effects [ 99 ]. Paired Cas9 nickase is the mutated form of SpCas9 generated through mutation of the HNH or RuvC domain.…”

Section: Mitigation Of Off-target Effects: Improved Cas Variantsmentioning

confidence: 99%

Latest Developed Strategies to Minimize the Off-Target Effects in CRISPR-Cas-Mediated Genome Editing

Naeem

Majeed

Hoque

et al. 2020

Cells

280

179

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Gene editing that makes target gene modification in the genome by deletion or addition has revolutionized the era of biomedicine. Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 emerged as a substantial tool due to its simplicity in use, less cost and extraordinary efficiency than the conventional gene-editing tools, including zinc finger nucleases (ZFNs) and Transcription activator-like effector nucleases (TALENs). However, potential off-target activities are crucial shortcomings in the CRISPR system. Numerous types of approaches have been developed to reduce off-target effects. Here, we review several latest approaches to reduce the off-target effects, including biased or unbiased off-target detection, cytosine or adenine base editors, prime editing, dCas9, Cas9 paired nickase, ribonucleoprotein (RNP) delivery and truncated gRNAs. This review article provides extensive information to cautiously interpret off-target effects to assist the basic and clinical applications in biomedicine.

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Section: Mitigation Of Off-target Effects: Improved Cas Variantsmentioning

confidence: 99%

Latest Developed Strategies to Minimize the Off-Target Effects in CRISPR-Cas-Mediated Genome Editing

Naeem

Majeed

Hoque

et al. 2020

Cells

280

179

View full text Add to dashboard Cite

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“…Due to the different PAM requirements of these nucleases, it is hard to address this question under identical target site conditions. Although the purified Cas enzyme/ sgRNA or crRNA with the specific target with compatible PAM may partially address it, 15 plasmid-based genome editing would be more easy to be accessed. In this study, we developed a method to quantitatively determine the efficacy of HDR mediated by three distinct nucleases in human cells.…”

Section: Discussionmentioning

confidence: 99%

“…The next generation sequencing (NGS) protocol and data analysis were described previously (Supplementary Table S4). 15 The deep sequencing data are available at the NCBI Sequence Read Archive (SRA) under BioProject PRJNA644281 (SRA accession no., SRR12153401 to SRR12153396, sample accession nos., SAMN15456036 to SAMN15456041).…”

Section: Next Generation Sequencing and Data Analysismentioning

confidence: 99%

Rational Selection of CRISPR-Cas Triggering Homology-Directed Repair in Human Cells

Zhou

et al. 2021

Human Gene Therapy

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The CRISPR (clustered regularly interspaced short palindromic repeat)-Cas (CRISPR-associated) nucleases have been widely applied for genome engineering. Cas9 (Streptococcus pyogenes Cas9 [SpCas9] and Staphylococcus aureus Cas9 [SaCas9]) and Cpf1 (i.e., Francisella novicida U112 Cpf1 [FnCpf1], also named FnCas12a) were harnessed to perform gene editing in human cells. Precise genetic modification by homology-directed repair (HDR) is an attractive approach for in situ gene correction. However, so far, the comparative efficiencies of HDR mediated by different CRISPR orthologs remain unknown. To address this question, in this study, we developed a reporter system to investigate HDR efficiencies triggered by various CRISPR orthologs. We found that SpCas9 and SaCas9, the two most commonly used Cas9 enzymes, possessed a similar ability to induce HDR. Interestingly, with the increasing amount of coding plasmids or additional nuclear localization sequences, FnCpf1 could improve the HDR efficacy. Collectively, our study provides insights for the rational selection of appropriate tools for human genome manipulation.

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“…The purification method of FnCas12a and the synthesis of crRNA in vitro were performed as described by Wang et al (2019) . The coding sequence of FnCas12a is obtained from plasmid #69976 (Addgene), and the plasmid sequence for the expression of FnCas12a in E. coli is provided in Supplementary Material .…”

Section: Methodsmentioning

confidence: 99%

FnCas12a/crRNA-Mediated Genome Editing in Eimeria tenella

et al. 2021

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Eimeria species are intracellular parasites residing inside the intestinal epithelial cell, which cause poultry coccidiosis and result in significant financial losses in the poultry industry. Genome editing of Eimeria is of immense importance for the development of vaccines and drugs. CRISPR/Cas9 has been utilized for manipulating the genome of Eimeria tenella (E. tenella). Ectopic expression of Cas9, i.e., via plasmids, would introduce transgene, which substantially limits its application, especially for vaccine development. In this study, we initially optimized the condition of the transfection protocol. We demonstrated that with the optimized condition, the transfection of FnCas12a (also known as “FnCpf1”) protein and crRNA targeting EtHistone H4 triggered DNA double-strand breaks in vivo. We then used this strategy to knock-in a coding cassette for an enhanced yellow fluorescent protein (EYFP) and dihydrofolate reductase–thymidylate synthase gene (DHFR) as a selection marker to tag endogenous EtActin. The engineered E. tenella parasite possesses EYFP expression in its entire life cycle. Our results demonstrated that FnCas12a could trigger genome editing in E. tenella, which augments the applicability of the dissection of gene function and the development of anticoccidial drugs and vaccines for Eimeria species.

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Efficient Human Genome Editing Using SaCas9 Ribonucleoprotein Complexes

Cited by 20 publications

References 39 publications

Latest Developed Strategies to Minimize the Off-Target Effects in CRISPR-Cas-Mediated Genome Editing

Latest Developed Strategies to Minimize the Off-Target Effects in CRISPR-Cas-Mediated Genome Editing

Rational Selection of CRISPR-Cas Triggering Homology-Directed Repair in Human Cells

FnCas12a/crRNA-Mediated Genome Editing in Eimeria tenella

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