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

Pu, Xiaojun; Liu, Lina; Li, Ping; Huo, Heqiang; Dong, Xueyi; Xie, Kabin; Yang, Hong; Liu, Li

doi:10.1111/tpj.14478

Cited by 19 publications

(13 citation statements)

References 60 publications

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“…The Gransden wild-type strain of P. patens used for all experiments are provided by Prof. Mitsuyasu Hasebe. Protonemata and gametophores were cultured on solid BCDAT medium at 25 • C with a 16-h light/8-h dark cycle (60 to 80 µmol photons m −2 s −1 ), as previously described [67].…”

Section: Plant Materials and Growth Conditionsmentioning

confidence: 99%

Genome-Wide Analysis of the MYB Transcription Factor Superfamily in Physcomitrella patens

Yang

Liu

et al. 2020

IJMS

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MYB transcription factors (TFs) are one of the largest TF families in plants to regulate numerous biological processes. However, our knowledge of the MYB family in Physcomitrella patens is limited. We identified 116 MYB genes in the P. patens genome, which were classified into the R2R3-MYB, R1R2R3-MYB, 4R-MYB, and MYB-related subfamilies. Most R2R3 genes contain 3 exons and 2 introns, whereas R1R2R3 MYB genes contain 10 exons and 9 introns. N3R-MYB (novel 3RMYB) and NR-MYBs (novel RMYBs) with complicated gene structures appear to be novel MYB proteins. In addition, we found that the diversity of the MYB domain was mainly contributed by domain shuffling and gene duplication. RNA-seq analysis suggested that MYBs exhibited differential expression to heat and might play important roles in heat stress responses, whereas CCA1-like MYB genes might confer greater flexibility to the circadian clock. Some R2R3-MYB and CCA1-like MYB genes are preferentially expressed in the archegonium and during the transition from the chloronema to caulonema stage, suggesting their roles in development. Compared with that of algae, the numbers of MYBs have significantly increased, thus our study lays the foundation for further exploring the potential roles of MYBs in the transition from aquatic to terrestrial environments.

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Section: Plant Materials and Growth Conditionsmentioning

confidence: 99%

Genome-Wide Analysis of the MYB Transcription Factor Superfamily in Physcomitrella patens

Yang

Liu

et al. 2020

IJMS

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“…While mutant forms of Cas9 have been created to alter or improve its function, Cas12a enzymes from different prokaryotic species, typically Acidaminococcus sp. BV3L6 (AsCas12a) and Lachnospiraceae bacterium ND2006 (LbCas12a), have been used to maximize genome editing in living cells (Jacobsen et al 2019;Pu et al 2019). Cas12a proteins from different species exhibit markedly different cleavage properties, most notably LbCas12a functions better at lower temperatures which is ideal for delivery into ectothermic organisms such as zebrafish or plants (Kim et al 2017;Tang et al 2017;Malzahn et al 2019).…”

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confidence: 99%

“…CRISPR reagents can be delivered into plants by Agrobacterium mediated T-DNA transfer (Char et al 2017;Lee et al 2019), biolistic plasmid delivery (Svitashev et al 2016;Gil-Humanes et al 2017;Hamada et al 2018) or biolistic delivery of ribonucleoprotein (RNP) complexes (Svitashev et al 2016;Liang et al 2018Liang et al , 2019. Using purified Cas9 or Cas12a proteins and chemically synthesized guide RNAs eliminates the possibility of continuous expression and ensures that these reagents are present transiently and thus minimizing the opportunity for off-target editing to occur (Svitashev et al 2016).…”

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confidence: 99%

“…The crRNAs have a similar GC content, 50% for crRNA1, 45% for crRNA2, and 47.6% for crRNA3. Base repeats can in some cases influence the secondary structure of crRNA as well as crRNA-DNA binding ability which can cause detrimental effects on total editing (Svitashev et al 2016). Towards this end, crRNA1 and crRNA3 have four repeats in total with a maximum repeat of three bases (GGG) followed by three repeats of two bases (AA, TT and CC), whereas crRNA2 has three repeats in total (TT, TT and GG), with all of them being two base repeats.…”

mentioning

confidence: 99%

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Comparison of CRISPR-Cas9/Cas12a Ribonucleoprotein Complexes for Genome Editing Efficiency in the Rice Phytoene Desaturase (OsPDS) Gene

Banakar

Schubert

Collingwood

et al. 2020

Rice

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Background: Delivery of CRISPR reagents into cells as ribonucleoprotein (RNP) complexes enables transient editing, and avoids CRISPR reagent integration in the genomes. Another technical advantage is that RNP delivery can bypass the need of cloning and vector construction steps. In this work we compared efficacies and types of edits for three Cas9 (WT Cas9 nuclease, HiFi Cas9 nuclease, Cas9 D10A nickase) and two Cas12a nucleases (AsCas12a and LbCas12a), using the rice phytoene desaturase (PDS) gene as a target site. Findings: Delivery of two Cas9 nucleases (WT Cas9, and HiFi Cas9) and one Cas12a nuclease (LbCas12a) resulted in targeted mutagenesis of the PDS gene. LbCas12a had a higher editing efficiency than that of WT Cas9 and HiFi Cas9. Editing by Cas9 enzymes resulted in indels (1-2 bp) or larger deletions between 20-bp to 30-bp, which included the loss of the PAM site; whereas LbCas12a editing resulted in deletions ranging between 2 bp to 20 bp without the loss of the PAM site. Conclusions: In this work, when a single target site of the rice gene OsPDS was evaluated, the LbCas12a RNP complex achieved a higher targeted mutagenesis frequency than the AsCas12a or Cas9 RNPs.

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“…The Clustered Regulatory Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated (Cas) system has brought a remarkable development in genome engineering efficiency in different organisms during the past few years (Doudna and Charpentier, 2014;Albadri et al, 2017;Ermert et al, 2019;Pu et al, 2019;Song et al, 2019). Briefly, the CRISPR-cas system employs a guide RNA (gRNA) and an endonuclease, mostly a single nuclease Cas9 (Makarova et al, 2011;Chylinski et al, 2014), as the two working elements for a site directed DNA cutting.…”

Section: Crispr-casmentioning

confidence: 99%

The Gibberellin Producer Fusarium fujikuroi: Methods and Technologies in the Current Toolkit

Cen

Lin

Wang

et al. 2020

Front. Bioeng. Biotechnol.

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In recent years, there has been a noticeable increase in research interests on the Fusarium species, which includes prevalent plant pathogens and human pathogens, common microbial food contaminants and industrial microbes. Taken the advantage of gibberellin synthesis, Fusarium fujikuroi succeed in being a prevalent plant pathogen. At the meanwhile, F. fujikuroi was utilized for industrial production of gibberellins, a group of extensively applied phytohormone. F. fujikuroi has been known for its outstanding performance in gibberellin production for almost 100 years. Research activities relate to this species has lasted for a very long period. The slow development in biological investigation of F. fujikuroi is largely due to the lack of efficient research technologies and molecular tools. During the past decade, technologies to analyze the molecular basis of host-pathogen interactions and metabolic regulations have been developed rapidly, especially on the aspects of genetic manipulation. At the meanwhile, the industrial fermentation technologies kept sustained development. In this article, we reviewed the currently available research tools/methods for F. fujikuroi research, focusing on the topics about genetic engineering and gibberellin production.

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

Cited by 19 publications

References 60 publications

Genome-Wide Analysis of the MYB Transcription Factor Superfamily in Physcomitrella patens

Genome-Wide Analysis of the MYB Transcription Factor Superfamily in Physcomitrella patens

Comparison of CRISPR-Cas9/Cas12a Ribonucleoprotein Complexes for Genome Editing Efficiency in the Rice Phytoene Desaturase (OsPDS) Gene

The Gibberellin Producer Fusarium fujikuroi: Methods and Technologies in the Current Toolkit

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