Review of CRISPR/Cas9 sgRNA Design Tools

Cui, Yingbo; Xu, Jiaming; Cheng, Minxia; Liao, Xiangke; Peng, Shaoliang

doi:10.1007/s12539-018-0298-z

Cited by 202 publications

(120 citation statements)

References 112 publications

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“…Unintended binding, modification, and cleavage of nucleic acids, so called the off-target effect, is a major challenge to the CRISPR/Cas9 system (3). To overcome this problem, many sgRNA designing tools have been developed to increase specificity of sgRNAs at the silicon level (23,24). A number of methods have also been employed to avoid off-target cutting or identify undesired mutations, such as using single-based editors correct base-pairing to avoid inducing a dsDNA break, or replace Cas9 with Cas13a to target RNA editing (3).…”

Section: Discussionmentioning

confidence: 99%

Optimizing CRISPR/Cas9 technology for precise correction of the Fgfr3-G374R mutation in achondroplasia in mice

Kai

Zhang

et al. 2019

Journal of Biological Chemistry

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Edited by Xiao-Fan Wang CRISPR/Cas9 is a powerful technology widely used for genome editing, with the potential to be used for correcting a wide variety of deleterious disease-causing mutations. However, the technique tends to generate more indels (insertions and deletions) than precise modifications at the target sites, which might not resolve the mutation and could instead exacerbate the initial genetic disruption. We sought to develop an improved protocol for CRISPR/Cas9 that would correct mutations without unintended consequences. As a case study, we focused on achondroplasia, a common genetic form of dwarfism defined by missense mutation in the Fgfr3 gene that results in glycine to arginine substitution at position 374 in mice in fibroblast growth factor receptor 3 (Fgfr3-G374R), which corresponds to G380R in humans. First, we designed a GFP reporter system that can evaluate the cutting efficiency and specificity of single guide RNAs (sgRNAs). Using the sgRNA selected based on our GFP reporter system, we conducted targeted therapy of achondroplasia in mice. We found that we achieved higher frequency of precise correction of the Fgfr3-G374R mutation using Cas9 protein rather than Cas9 mRNA. We further demonstrated that targeting oligos of 100 and 200 nucleotides precisely corrected the mutation at equal efficiency. We showed that our strategy completely suppressed phenotypes of achondroplasia and whole genome sequencing detected no off-target effects. These data indicate that improved protocols can enable the precise CRISPR/Cas9-mediated correction of individual mutations with high fidelity.CRISPR/Cas9 is a RNA-guided genome editing tool developed from a microbial adaptive immune defense system (1-3). Because of its high efficiency and easy-to-use nature, the CRISPR/Cas9 system is revolutionizing many research fields, Figure 4. Observation of gene corrected and mutant mice. A, whole mount skeleton staining of Fgfr3 G1120A/ϩ dwarf (left) and corrected mice (right) at 1 month of age. B, X-ray images and its statistical analysis (C) showing the length of femur of dwarf and corrected mice. D, comparison of body weight between dwarf mice, corrected mice, and knockout mice. Correction of the Fgfr3-G374R related achondroplasia

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Section: Discussionmentioning

confidence: 99%

Optimizing CRISPR/Cas9 technology for precise correction of the Fgfr3-G374R mutation in achondroplasia in mice

Kai

Zhang

et al. 2019

Journal of Biological Chemistry

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“…However, parallel and equally justifiable efforts to increase the accuracy of CRISPR systems involve changing the structure of the sgRNA. The rules of sgRNA design are constantly evolving to improve specificity, with multiple groups contributing to this effort [195,[198][199][200]. In addition to sequence, the chemical composition of the sgRNA can also affect specificity.…”

Section: Hyperaccurate Crispr Systemsmentioning

confidence: 99%

Gene editing and CRISPR in the clinic: current and future perspectives

et al. 2020

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Genome editing technologies, particularly those based on zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and CRISPR (clustered regularly interspaced short palindromic repeat DNA sequences)/Cas9 are rapidly progressing into clinical trials. Most clinical use of CRISPR to date has focused on ex vivo gene editing of cells followed by their re-introduction back into the patient. The ex vivo editing approach is highly effective for many disease states, including cancers and sickle cell disease, but ideally genome editing would also be applied to diseases which require cell modification in vivo. However, in vivo use of CRISPR technologies can be confounded by problems such as off-target editing, inefficient or off-target delivery, and stimulation of counterproductive immune responses. Current research addressing these issues may provide new opportunities for use of CRISPR in the clinical space. In this review, we examine the current status and scientific basis of clinical trials featuring ZFNs, TALENs, and CRISPR-based genome editing, the known limitations of CRISPR use in humans, and the rapidly developing CRISPR engineering space that should lay the groundwork for further translation to clinical application.

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“…These tools differ in the type of models used, selected features, genomes etc. The tool sgRNA Designer [6] followed the rules proposed by root laboratory [7]. Their training dataset contained genes from human and mouse cells.…”

Section: Previous Workmentioning

confidence: 99%

CRISPRpred(SEQ): a sequence based tool for sgRNA on target activity prediction [(almost) beating Deep Learning pipelines by traditional machine learning]

Rafid¹,

Toufikuzzaman²,

Rahman³

et al. 2019

Preprint

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An accurate and fast genome editing tool can be used to treat genetic diseases, modify crops genetically etc. However, a tool that has low accuracy can be risky to use, as incorrect genome editing may have severe consequences. Although many tools have been developed in the past, there are still room for further improvement. In this paper, we present CRISPRpred(SEQ), a sequence based tool for sgRNA on target activity prediction that leverages only traditional machine learning techniques. We compare the results of CRISPRpred(SEQ) with that of DeepCRISPR, the current state-of-the-art, which uses a deep learning pipeline. In spite of using only traditional machine learning methods, we are able to beat DeepCRISPR for the three out of four cell lines in the benchmark dataset convincingly (2.174%, 6.905% and 8.119% improvement for the three cell lines), which is quite outstanding. *

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Review of CRISPR/Cas9 sgRNA Design Tools

Cited by 202 publications

References 112 publications

Optimizing CRISPR/Cas9 technology for precise correction of the Fgfr3-G374R mutation in achondroplasia in mice

Optimizing CRISPR/Cas9 technology for precise correction of the Fgfr3-G374R mutation in achondroplasia in mice

Gene editing and CRISPR in the clinic: current and future perspectives

CRISPRpred(SEQ): a sequence based tool for sgRNA on target activity prediction [(almost) beating Deep Learning pipelines by traditional machine learning]

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