CRISPR-P 2.0: An Improved CRISPR-Cas9 Tool for Genome Editing in Plants

Líu, Hao; Ding, Yuduan; Zhou, Yanqing; Jin, Wei; Xie, Kabin; Chen, Lingling

doi:10.1016/j.molp.2017.01.003

Cited by 541 publications

(369 citation statements)

References 14 publications

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“…The crRNA expression cassette under the control of the PpU6 promoter was generated by digesting the pU6‐sgRNA plasmid (Collonnier et al ., ) with Nco I and Xba I. The candidate crRNA target sequences were designed using crispor software (http://crispor.tefor.net/) (Haeussler et al ., ) and crispr‐p 2.0 (http://crispr.hzau.edu.cn/CRISPR2/) (Liu et al ., ).…”

Section: Methodsmentioning

confidence: 97%

A CRISPR/LbCas12a‐based method for highly efficient multiplex gene editing in Physcomitrella patens

Liu

et al. 2019

The Plant Journal

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Summary Due to their high efficiency, specificity, and flexibility, programmable nucleases, such as those of the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a (Cpf1) system, have greatly expanded the applicability of editing the genomes of various organisms. Genes from different gene families or genes with redundant functions in the same gene family can be examined by assembling multiple CRISPR RNAs (crRNAs) in a single vector. However, the activity and efficiency of CRISPR/Cas12a in the non‐vascular plant Physcomitrella patens are largely unknown. Here, we demonstrate that LbCas12a together with its mature crRNA can target multiple loci simultaneously in P. patens with high efficiency via co‐delivery of LbCas12a and a crRNA expression cassette in vivo. The mutation frequencies induced by CRISPR/LbCas12a at a single locus ranged from 26.5 to 100%, with diverse deletions being the most common type of mutation. Our method expands the repertoire of genome editing tools available for P. patens and facilitates the creation of loss‐of‐function mutants of multiple genes from different gene families.

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Section: Methodsmentioning

confidence: 97%

A CRISPR/LbCas12a‐based method for highly efficient multiplex gene editing in Physcomitrella patens

Liu

et al. 2019

The Plant Journal

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“…CRISPR‐P 2.0 supports the designing of guide sequence for Cpf1 and other many CRISPR/Cas systems . It provides a comprehensive analysis of guide sequences, such as the GC content, secondary structure of sgRNA, sites for restriction endonucleases …”

Section: Tools To Design Target‐sgrna For Gementioning

confidence: 99%

CRISPR/Cas Systems in Genome Editing: Methodologies and Tools for sgRNA Design, Off‐Target Evaluation, and Strategies to Mitigate Off‐Target Effects

et al. 2020

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Life sciences have been revolutionized by genome editing (GE) tools, including zinc finger nucleases, transcription activator‐Like effector nucleases, and CRISPR (clustered regulatory interspaced short palindromic repeats)/Cas (CRISPR‐associated) systems, which make the targeted modification of genomic DNA of all organisms possible. CRISPR/Cas systems are being widely used because of their accuracy, efficiency, and cost‐effectiveness. Various classes of CRISPR/Cas systems have been developed, but their extensive use may be hindered by off‐target effects. Efforts are being made to reduce the off‐target effects of CRISPR/Cas9 by generating various CRISPR/Cas systems with high fidelity and accuracy. Several approaches have been applied to detect and evaluate the off‐target effects. Here, the current GE tools, the off‐target effects generated by GE technology, types of off‐target effects, mechanisms of off‐target effects, major concerns, and outcomes of off‐target effects in plants and animals are summarized. The methods to detect off‐target effects, tools for single‐guide RNA (sgRNA) design, evaluation and prediction of off‐target effects, and strategies to increase the on‐target efficiency and mitigate the off‐target impact on intended genome‐editing outcomes are summarized.

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“…To detect all the potential off-target mutations, six sgRNA and their PAM sequences were aligned with the TM-1 reference genome using CRISPR-P and Cas-OFFinder software (Bae et al, 2014;Liu et al, 2017). With ≤5 mismatches in the sgRNA and PAM sequences, there were 3296 (PAM: NGG), 410 (PAM: NAG) and 707 (PAM: NGA) potential off-target sites identified (Table S6, Figure S8 and Appendix S1).…”

Section: Low Frequency Off-target Mutations Detected In Cas9-edited Pmentioning

confidence: 99%

Whole genome sequencing reveals rare off‐target mutations and considerable inherent genetic or/and somaclonal variations in CRISPR/Cas9‐edited cotton plants

Manghwar

Liang

et al. 2018

Plant Biotechnology Journal

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Summary The CRISPR /Cas9 system has been extensively applied for crop improvement. However, our understanding of Cas9 specificity is very limited in Cas9‐edited plants. To identify on‐ and off‐target mutation in an edited crop, we described whole genome sequencing ( WGS ) of 14 Cas9‐edited cotton plants targeted to three genes, and three negative (Ne) control and three wild‐type ( WT ) plants. In total, 4188–6404 unique single‐nucleotide polymorphisms ( SNP s) and 312–745 insertions/deletions (indels) were detected in 14 Cas9‐edited plants compared to WT , negative and cotton reference genome sequences. Since the majority of these variations lack a protospacer‐adjacent motif ( PAM ), we demonstrated that the most variations following Cas9‐edited are due either to somaclonal variation or/and pre‐existing/inherent variation from maternal plants, but not off‐target effects. Of a total of 4413 potential off‐target sites (allowing ≤5 mismatches within the 20‐bp sg RNA and 3‐bp PAM sequences), the WGS data revealed that only four are bona fide off‐target indel mutations, validated by Sanger sequencing. Moreover, inherent genetic variation of WT can generate novel off‐target sites and destroy PAM s, which suggested great care should be taken to design sg RNA for the minimizing of off‐target effect. These findings suggested that CRISPR /Cas9 system is highly specific for cotton plants.

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CRISPR-P 2.0: An Improved CRISPR-Cas9 Tool for Genome Editing in Plants

Cited by 541 publications

References 14 publications

A CRISPR/LbCas12a‐based method for highly efficient multiplex gene editing in Physcomitrella patens

A CRISPR/LbCas12a‐based method for highly efficient multiplex gene editing in Physcomitrella patens

CRISPR/Cas Systems in Genome Editing: Methodologies and Tools for sgRNA Design, Off‐Target Evaluation, and Strategies to Mitigate Off‐Target Effects

Whole genome sequencing reveals rare off‐target mutations and considerable inherent genetic or/and somaclonal variations in CRISPR/Cas9‐edited cotton plants

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