Evaluating the cleavage efficacy of CRISPR-Cas9 sgRNAs targeting ineffective regions of Arabidopsis thaliana genome

PeerJ

article

Afsheen Malik¹  Alvina Gul¹  Faiza Munir¹  Rabia Amir¹  Hadi Alipour²  Mustafeez Mujtaba Babar³  Syeda Marriam Bakhtiar⁴  Rehan Zafar Paracha⁵  Zoya Khalid⁶  Muhammad Qasim Hayat¹ 

[1] Department of Plant Biotechnology, Atta-ur-Rahman School of Applied Biosciences, National University of Sciences and Technology;Department of Plant Production and Genetics, Faculty of Agriculture and Natural Resources, Urmia University;Shifa College of Pharmaceutical Sciences, Shifa Tameer-e-Millat University;Department of Bioinformatics and Biosciences, Capital University of Science and Technology;Research Center for Modeling and Simulation, National University of Sciences and Technology;Computational Biology Research Lab, Department of Computer Science, National University of Computer and Emerging Sciences-FAST

关键词: CRISPR-Cas9;    Cleavage efficacy;    Non-coding region;    sgRNA design tool;    Ineffective region;    Genome editing;    Arabidopsis thaliana;   

DOI  :  10.7717/peerj.11409

学科分类：社会科学、人文和艺术（综合）

来源: Inra

【 摘 要 】

The CRISPR-Cas9 system has recently evolved as a powerful mutagenic tool for targeted genome editing. The impeccable functioning of the system depends on the optimal design of single guide RNAs (sgRNAs) that mainly involves sgRNA specificity and on-target cleavage efficacy. Several research groups have designed algorithms and models, trained on mammalian genomes, for predicting sgRNAs cleavage efficacy. These models are also implemented in most plant sgRNA design tools due to the lack of on-target cleavage efficacy studies in plants. However, one of the major drawbacks is that almost all of these models are biased for considering only coding regions of the DNA while excluding ineffective regions, which are of immense importance in functional genomics studies especially for plants, thus making prediction less reliable. In the present study, we evaluate the on-target cleavage efficacy of experimentally validated sgRNAs designed against diverse ineffective regions of Arabidopsis thaliana genome using various statistical tests. We show that nucleotide preference in protospacer adjacent motif (PAM) proximal region, GC content in the PAM proximal seed region, intact RAR and 3rd stem loop structures, and free accessibility of nucleotides in seed and tracrRNA regions of sgRNAs are important determinants associated with their high on-target cleavage efficacy. Thus, our study describes the features important for plant sgRNAs high on-target cleavage efficacy against ineffective genomic regions previously shown to give rise to ineffective sgRNAs. Moreover, it suggests the need of developing an elaborative plant-specific sgRNA design model considering the entire genomic landscape including ineffective regions for enabling highly efficient genome editing without wasting time and experimental resources.

【 授权许可】

CC BY