A CRISPR–Cpf1 system for efficient genome editing and transcriptional repression in plants

Abstract

Clustered regularly interspaced short palindromic repeats (CRISPR)-Cpf1 has emerged as an effective genome editing tool in animals. Here we compare the activity of Cpf1 from Acidaminococcus sp. BV3L6 (As) and Lachnospiraceae bacterium ND2006 (Lb) in plants, using a dual RNA polymerase II promoter expression system. LbCpf1 generated biallelic mutations at nearly 100% efficiency at four independent sites in rice T0 transgenic plants. Moreover, we repurposed AsCpf1 and LbCpf1 for efficient transcriptional repression in Arabidopsis, and demonstrated a more than tenfold reduction in miR159b transcription. Our data suggest promising applications of CRISPR-Cpf1 for editing plant genomes and modulating the plant transcriptome.

Towards mastering CRISPR-induced gene knock-in in plants: Survey of key features and focus on the model Physcomitrella patens

“…Cpf1 from Acidaminococcus sp. (AsCpf1) and Lachnospiraceae bacterium (LbCpf1) have been shown to be functional in rice, soybean and tobacco [34,[56][57][58]. In these plants, LbCpf1 mutagenesis efficiency, which seems to be correlated with the target sequence [56], appeared always higher than those of AsCpf1, reaching sometimes 100% in rice [58].…”

CRISPR-based tools for plant genome engineering

“…Spacer sequences were inserted into the crRNA that guides Cpf1 to the target by annealing a phosphorylated oligonucleotide dimer into a Type IIS cloning site before assembly into the final plasmid (Figure 2A). In contrast, Tang et al [39] flanked the crRNA with self-cleaving ribozymes enabling them to drive expression in rice from a strong constitutive promoter ( Figure 2B). Notably, Tang et al [39] reported 100% mutation efficiency, with very few plants being genetic chimeras.…”

“…In contrast, Tang et al [39] flanked the crRNA with self-cleaving ribozymes enabling them to drive expression in rice from a strong constitutive promoter ( Figure 2B). Notably, Tang et al [39] reported 100% mutation efficiency, with very few plants being genetic chimeras.…”

“…Viral replicons based on Bean Yellow Dwarf Virus (BeYDV), Tomato Leaf Curl Virus (ToLCV) and Wheat Dwarf Virus (WDV) have been used to increase the number of copies of the repair template in many plant species, successfully increasing the frequency of targeted DNA insertion up to 10-fold (A) Tang et al [39] used Multisite Gateway cloning (recombination sites are annotated as attL and attR) to assemble a Cpf1 expression cassette (UbiCpf1) and a CRISPR RNA (crRNA) cassette into a binary backbone containing a plant selectable marker cassette (S.M.). The crRNA was flanked by the Hammerhead (HH) and Hepatitis Delta Virus (HDV) ribozyme sequences to self-process after transcription from a constitutive RNA polymerase II promoter (Ubi).…”

Self-cleaving ribozymes enable the production of guide RNAs from unlimited choices of promoters for CRISPR/Cas9 mediated genome editing

“…One of the major findings is that the choice of promoters for expressing Cas9 has a dramatic impact on the efficiency and heritability of gene editing in Arabidopsis . Another effort has been to isolate and characterize additional nucleases such as Cpf1 that have characteristically different PAM requirements and that generate DSBs with cohesive ends (Tang et al, 2017). …”