Plant Genome Editing Using FnCpf1 and LbCpf1 Nucleases at Redefined and Altered PAM Sites

Zhong, Zhiyong; Zhang, Yingxiao; You, Qi; Tang, Xu; Ren, Qiao; Liu, Shishi; Liu, Yang; Wang, Yan; Liu, Xiaopei; Liu, Binglin; Zhang, Tao; Zheng, Xuelian; Le, Ysa; Zhang, Yong; Qi, Yiping

doi:10.1016/j.molp.2018.03.008

Cited by 145 publications

(114 citation statements)

References 12 publications

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“…These results indicated that a TTTV PAM is beneficial for the efficiency of editing by FnCpf1. More recently, another group reported similar results in rice protoplasts (Zhong et al ).…”

supporting

confidence: 60%

Multiplex gene editing in rice with simplified CRISPR‐Cpf1 and CRISPR‐Cas9 systems

Wang

Mao

et al. 2018

JIPB

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“…These results indicated that a TTTV PAM is beneficial for the efficiency of editing by FnCpf1. More recently, another group reported similar results in rice protoplasts (Zhong et al ).…”

supporting

confidence: 60%

Multiplex gene editing in rice with simplified CRISPR‐Cpf1 and CRISPR‐Cas9 systems

Wang

Mao

et al. 2018

JIPB

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“…() also demonstrated temperature‐dependent performance of AsCas12a‐mediated editing in vertebrates. Consistent with these results, we achieved high editing efficiencies with LbCas12a and FnCas12a in rice when higher temperatures (28–30 °C) were applied (Tang et al ., ; Zhong et al ., ). Thus, it may be worth developing a sophisticated high temperature treatment regime for maize immature embryos during transformation to attempt to optimize Cas12a‐mediated genome editing as claimed by Cigan et al .…”

Section: Resultsmentioning

confidence: 97%

Activities and specificities of CRISPR/Cas9 and Cas12a nucleases for targeted mutagenesis in maize

Lee

Zhang

Kleinstiver

et al. 2018

Plant Biotechnology Journal

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203

151

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CRISPR/Cas9 and Cas12a (Cpf1) nucleases are two of the most powerful genome editing tools in plants. In this work, we compared their activities by targeting maize glossy2 gene coding region that has overlapping sequences recognized by both nucleases. We introduced constructs carrying SpCas9-guide RNA (gRNA) and LbCas12a-CRISPR RNA (crRNA) into maize inbred B104 embryos using Agrobacterium-mediated transformation. On-target mutation analysis showed that 90%-100% of the Cas9-edited T0 plants carried indel mutations and 63%-77% of them were homozygous or biallelic mutants. In contrast, 0%-60% of Cas12a-edited T0 plants had on-target mutations. We then conducted CIRCLE-seq analysis to identify genome-wide potential off-target sites for Cas9. A total of 18 and 67 potential off-targets were identified for the two gRNAs, respectively, with an average of five mismatches compared to the target sites. Sequencing analysis of a selected subset of the off-target sites revealed no detectable level of mutations in the T1 plants, which constitutively express Cas9 nuclease and gRNAs. In conclusion, our results suggest that the CRISPR/Cas9 system used in this study is highly efficient and specific for genome editing in maize, while CRISPR/Cas12a needs further optimization for improved editing efficiency.

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“…Instead of G-rich PAMs required by Cas9, Cas12a recognizes T-rich PAMs and thus further increases the number of potential target sites. Recent publications in yeast and rice have revealed that a PAM of 5 0 -TTTV-3 0 is also strongly preferred by FnCas12a [69,70]. In contrast to Cas9 which generates bluntended DSBs proximal to the PAM, Cas12a generates staggered DSBs distal from the PAM.…”

Section: Using Cas12a (Formerly Named Cpf1) In Plantsmentioning

confidence: 99%

Transforming plant biology and breeding with CRISPR/Cas9, Cas12 and Cas13

2018

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Currently, biology is revolutionized by ever growing applications of the CRISPR/Cas system. As discussed in this Review, new avenues have opened up for plant research and breeding by the use of the sequence-specific DNases Cas9 and Cas12 (formerly named Cpf1) and, more recently, the RNase Cas13 (formerly named C2c2). Although double strand break-induced gene editing based on error-prone nonhomologous end joining has been applied to obtain new traits, such as powdery mildew resistance in wheat or improved pathogen resistance and increased yield in tomato, improved technologies based on CRISPR/Cas for programmed change in plant genomes via homologous recombination have recently been developed. Cas9- and Cas12- mediated DNA binding is used to develop tools for many useful applications, such as transcriptional regulation or fluorescence-based imaging of specific chromosomal loci in plant genomes. Cas13 has recently been applied to degrade mRNAs and combat viral RNA replication. By the possibility to address multiple sequences with different guide RNAs and by the simultaneous use of different Cas proteins in a single cell, we should soon be able to achieve complex changes of plant metabolism in a controlled way.

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Plant Genome Editing Using FnCpf1 and LbCpf1 Nucleases at Redefined and Altered PAM Sites

Cited by 145 publications

References 12 publications

Multiplex gene editing in rice with simplified CRISPR‐Cpf1 and CRISPR‐Cas9 systems

Multiplex gene editing in rice with simplified CRISPR‐Cpf1 and CRISPR‐Cas9 systems

Activities and specificities of CRISPR/Cas9 and Cas12a nucleases for targeted mutagenesis in maize

Transforming plant biology and breeding with CRISPR/Cas9, Cas12 and Cas13

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