Precise insertion and guided editing of higher plant genomes using Cpf1 CRISPR nucleases

Begemann, Matthew B.; Gray, Benjamin N.; January, Emma; Gordon, Gina C.; He, Yanni; Liu, Haijun; Wu, Xingrong; Brutnell, Thomas P.; Mockler, Todd C.; Oufattole, Mohammed

doi:10.1038/s41598-017-11760-6

Cited by 169 publications

(134 citation statements)

References 32 publications

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“…In addition, as cleavage occurs remote from the PAM and seed region, even after NHEJ-based small InDels are generated, further cleavage might still occur, which could still induce HR. In rice, using FnCas12a and LbCas12a targeted insertions via HR were accomplished and, at least for FnCas12a, with higher rates than SpCas9-based experiments [76]. Furthermore, when delivered as pre-assembled ribonucleoprotein complexes, LbCas12a was highly efficient at homology-directed DNA replacement in Chlamydomonas, which has been recalcitrant to efficient editing before [77].…”

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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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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“…CRISPR-Cas12a (formerly Cpf1), a class 2 type V-A CRISPR-Cas system, has also been applied for plant genome editing (Begemann et al, 2017;Endo et al, 2016a;Hu et al, 2017;Kim et al, 2017;Li et al, 2018a,b;Tang et al, 2017;Xu et al, 2016;Zhong et al, 2018). Unlike Cas9, Cas12a only requires CRISPR RNA (crRNA) without the need of trans-activating crRNA (tracrRNA) and it recognizes T-rich PAMs, resulting in staggered DNA double strand breaks (DSBs; Fagerlund et al, 2015;Zetsche et al, 2015).…”

Section: Introductionmentioning

confidence: 99%

Single transcript unit CRISPR 2.0 systems for robust Cas9 and Cas12a mediated plant genome editing

Tang

Ren

Yang

et al. 2019

Plant Biotechnology Journal

125

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Summary CRISPR ‐Cas9 and Cas12a are two powerful genome editing systems. Expression of CRISPR in plants is typically achieved with a mixed dual promoter system, in which Cas protein is expressed by a Pol II promoter and a guide RNA is expressed by a species‐specific Pol III promoter such as U6 or U3. To achieve coordinated expression and compact vector packaging, it is desirable to express both CRISPR components under a single Pol II promoter. Previously, we demonstrated a first‐generation single transcript unit ( STU )‐Cas9 system, STU ‐Cas9‐ RZ , which is based on hammerhead ribozyme for processing single guide RNA s (sg RNA s). In this study, we developed two new STU ‐Cas9 systems and one STU ‐Cas12a system for applications in plants, collectively called the STU CRISPR 2.0 systems. We demonstrated these systems for genome editing in rice with both transient expression and stable transgenesis. The two STU ‐Cas9 2.0 systems process the sg RNA s with Csy4 ribonuclease and endogenous tRNA processing system respectively. Both STU ‐Cas9‐Csy4 and STU ‐Cas9‐ tRNA systems showed more robust genome editing efficiencies than our first‐generation STU ‐Cas9‐ RZ system and the conventional mixed dual promoter system. We further applied the STU ‐Cas9‐ tRNA system to compare two C to T base editing systems based on rAPOBEC 1 and Pm CDA 1 cytidine deaminases. The results suggest STU ‐based Pm CDA 1 base editor system is highly efficient in rice. The STU ‐Cas12a system, based on Cas12a’ self‐processing of a CRISPR RNA (cr RNA ) array, was also developed and demonstrated for expression of a single cr RNA and four cr RNA s. Altogether, our STU CRISPR 2.0 systems further expanded the CRISPR toolbox for plant genome editing and other applications.

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“…Genome editing is a powerful tool for increasing plant yields, improving food quality, enhancing crop disease resistance and developing new cultivars to meet market needs (Begemann et al, 2017). At present, several strategies are being exploited to edit plant genomes, including CRISPR-Cas, meganucleases, TALENs and zinc finger nucleases (Mart ın-Pizarro and Pos e, 2018).…”

Section: Introductionmentioning

confidence: 99%

“…To date, Acidaminococcus sp. BV3L6 Cas12a (AsCas12a), Francisella novicida Cas12a (FnCas12a) and Lachnospiraceae bacterium ND2006 Cas12a (LbCas12a) have been used to edit the genomes of crop and model plants, including green alga, rice, soybeans and tobacco (Begemann et al, 2017;Endo et al, 2016;Ferenczi et al, 2017;Hu et al, 2017;Kim et al, 2017;Tang et al, 2017;Wang et al, 2017a,b,c;Xu et al, 2016;Yin et al, 2017), but not citrus. In addition, the performances of the three Cas12a homologs are different.…”

Section: Introductionmentioning

confidence: 99%

CRISPR‐LbCas12a‐mediated modification of citrus

Jia

Orbović

Wang

2019

Plant Biotechnology Journal

148

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Summary Recently, CRISPR‐Cas12a (Cpf1) from Prevotella and Francisella was engineered to modify plant genomes. In this report, we employed CRISPR‐LbCas12a (LbCpf1), which is derived from Lachnospiraceae bacterium ND2006, to edit a citrus genome for the first time. First, LbCas12a was used to modify the CsPDS gene successfully in Duncan grapefruit via Xcc‐facilitated agroinfiltration. Next, LbCas12a driven by either the 35S or Yao promoter was used to edit the PthA4 effector binding elements in the promoter (EBEPthA4‐CsLOBP) of CsLOB1. A single crRNA was selected to target a conserved region of both Type I and Type II CsLOBPs, since the protospacer adjacent motif of LbCas12a (TTTV) allows crRNA to act on the conserved region of these two types of CsLOBP. CsLOB1 is the canker susceptibility gene, and it is induced by the corresponding pathogenicity factor PthA4 in Xanthomonas citri by binding to EBEPthA4‐CsLOBP. A total of seven 35S‐LbCas12a‐transformed Duncan plants were generated, and they were designated as #D35s1 to #D35s7, and ten Yao‐LbCas12a‐transformed Duncan plants were created and designated as #Dyao1 to #Dyao10. LbCas12a‐directed EBEPthA4‐CsLOBP modifications were observed in three 35S‐LbCas12a‐transformed Duncan plants (#D35s1, #D35s4 and #D35s7). However, no LbCas12a‐mediated indels were observed in the Yao‐LbCas12a‐transformed plants. Notably, transgenic line #D35s4, which contains the highest mutation rate, alleviates XccΔpthA4:dCsLOB1.4 infection. Finally, no potential off‐targets were observed. Therefore, CRISPR‐LbCas12a can readily be used as a powerful tool for citrus genome editing.

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Precise insertion and guided editing of higher plant genomes using Cpf1 CRISPR nucleases

Cited by 169 publications

References 32 publications

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

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

Single transcript unit CRISPR 2.0 systems for robust Cas9 and Cas12a mediated plant genome editing

CRISPR‐LbCas12a‐mediated modification of citrus

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