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An overview of designing and selection of sgRNAs for precise genome editing by the CRISPR-Cas9 system in plants

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Abstract

A large number of computational tools have been documented in recent years for identification of target-specific valid single-guide (sg) RNAs (18–20 nucleotide long sequence) that is an important component for the efficient utilization of the CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats—CRISPR-associated Protein) system. Despite optimization of Cas9, other major concerns are on-target efficiency and off-target activity that depend upon the sequence(s) of target-specific sgRNA(s). However, a very little attention has been paid for identification of the best-hit sgRNA for precise targeting as well as minimizing the off-target effects. The aim of this present work is to offer comparative insight into existing CRISPR software tools with their unique features (including targeted genome) and utilities. These available web tools were found to be designed based upon only a few limited mathematical models. Among all these available web tools, three (Benchling, Desktop and CRISPR-P) have been curated as exclusively available for plant genome-editing purpose. These three software tools have been comprehensively described and analyzed with single same target enquiry from two randomly selected genes (IDM2 and IDM3 from Arabidopsis thaliana). Interestingly, all these selected tools generated different results (sgRNAs) even for the same query. In fact, the sequence of sgRNA is considered an important parameter to determine the efficiency and specificity of sgRNAs for precise genome editing. Thus, there is an urgent requirement to pay attention for a validated sgRNA-designing tool for precise DNA editing in plants. In conclusion, this work will encourage building up a consensus for developing a universal valid sgRNA designing for different organisms including plants.

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Abbreviations

Cas:

CRISPR-associated protein/gene

CRISPR:

Clustered regularly interspaced short palindromic repeats

sgRNA:

Single-guide RNA

crRNA:

CRISPR RNA

PAM:

Protospacer adjacent motif

CFD:

Cutting frequency determination

ZFN:

Zinc finger nuclease

CLD:

CRISPR library designer

TALEN:

Transcription activator-like effector nuclease

tracRNA:

Trans-activating CRISPR RNA

DESKGEN:

Desktop genetics

NGS:

Next-generation sequencing

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Acknowledgements

The authors express deepest gratitude to Vice Chancellor of Central University of Punjab, India for providing financial support during course of this work. “UGC-BSR start up grant” sanctioned to Vinay Kumar, who sponsors this research. The authors thank anonymous reviewers and editors for critical reading the manuscript and suggesting substantial improvements.

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Authors and Affiliations

  1. Department of Plant Sciences, School for Basic and Applied Sciences, Central University of Punjab, Bathinda, 161001, India

    Ajay Prakash Uniyal, Komal Mansotra & Vinay Kumar

  2. Center of Innovative and Applied Biotechnology, Mohali, 140306, India

    Sudesh Kumar Yadav

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  1. Ajay Prakash Uniyal
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  2. Komal Mansotra
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  4. Vinay Kumar
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VK conceived and designed the present research. VK and KM conducted the experiments. KM and VK analyzed the data. APU, SKY and VK wrote the manuscript. All the authors read and approved the manuscript.

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Correspondence to Vinay Kumar.

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Uniyal, A.P., Mansotra, K., Yadav, S.K. et al. An overview of designing and selection of sgRNAs for precise genome editing by the CRISPR-Cas9 system in plants. 3 Biotech 9, 223 (2019). https://doi.org/10.1007/s13205-019-1760-2

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