Plant genome editing with TALEN and CRISPR

Abstract

Genome editing promises giant leaps forward in advancing biotechnology, agriculture, and basic research. The process relies on the use of sequence specific nucleases (SSNs) to make DNA double stranded breaks at user defined genomic loci, which are subsequently repaired by two main DNA repair pathways: non-homologous end joining (NHEJ) and homology directed repair (HDR). NHEJ can result in frameshift mutations that often create genetic knockouts. These knockout lines are useful for functional and reverse genetic studies but also have applications in agriculture. HDR has a variety of applications as it can be used for gene replacement, gene stacking, and for creating various fusion proteins. In recent years, transcription activator-like effector nucleases and clustered regularly interspaced palindromic repeats (CRISPR) and CRISPR associated protein 9 or CRISPR from Prevotella and Francisella 1 have emerged as the preferred SSNs for research purposes. Here, we review their applications in plant research, discuss current limitations, and predict future research directions in plant genome editing.

Keywords: CRISPR; Cas9; Cpf1; HDR; NHEJ; Plant genome editing; TALEN.

Fig. 1

Major DNA repair pathways in plants. Non-homologous end joining (NHEJ) and homology directed repair (HDR) are two main repair pathways. Classical NHEJ may lead to insertions or deletions, while microhomology based alternative NHEJ always results in deletions. Homology directed repair is less efficient, but can result in precise integration of a donor DNA template into the genome

Fig. 2

TALEN and CRISPR-Cas9. a A TALEN is composed of two monomers with each containing a TALE DNA binding domain and a FokI nuclease domain. Fok1 dimerizes to create a double-strand break. b CRISPR-Cas9 is a two-component system composed of Cas9 and a gRNA. Once Cas9 finds a PAM site, if the gRNA binds to the DNA, a double break occurs three base pairs upstream the PAM

Fig. 3

NHEJ based genome editing applications. a NHEJ repair of an SSN induced break can create a premature stop codon. A stop codon is indicated by a red octagon. GOI is an acronym for gene of interest. b Non-protein coding genes such as microRNA and long non-coding RNA can be rendered non-functional through targeted mutations by SSNs. c Regulatory elements involved in the activation or repression of genes can be disrupted by SSNs. d Pieces of chromosomes that may involve regulatory networks or related genes can be deleted by SSNs

Fig. 4

Paired Cas9 nickase and FokI-dCas9 systems. Alternative Cas9 proteins can decrease off-target effects. a Two nickases are required to make a double-strand break, increasing the gRNA requirement and length of target sequence. b A catalytically dead Cas9 is paired to a Fok1 nuclease, also resulting in an increased length of target sequence for enhanced targeting specificity

Fig. 5

HDR based genome editing applications. a Gene replacement is applicable for basic research and agriculture. b HDR can add a tag to a protein for easy purification and study. c Fluorescent proteins such as green fluorescent protein (GFP) can be fused to a gene of interest for in vivo study. d Gene stacking is useful for placing genes physically close together on a chromosome. This is accomplished by creating a target site for HDR at the end of each gene, which allows for modular addition of genes

Fig. 6

TALE and CRISPR-Cas9 based transcriptome modulation systems. a The activator VP64 is fused to TALE for gene activation. b The repressor SRDX is fused to TALE for gene repression. c The activator VP64 is fused to dCas9 for gene activation. d The repressor SRDX is fused to dCas99 for gene repression