Targeted Mutagenesis in Plant Cells through Transformation of Sequence-Specific Nuclease mRNA

<div><p>Plant genome engineering using sequence-specific nucleases (SSNs) promises to advance basic and applied plant research by enabling precise modification of endogenous genes. Whereas DNA is an effective means for delivering SSNs, DNA can integrate randomly into the plant genome, leading to unintentional gene inactivation. Further, prolonged expression of SSNs from DNA constructs can lead to the accumulation of off-target mutations. Here, we tested a new approach for SSN delivery to plant cells, namely transformation of messenger RNA (mRNA) encoding TAL effector nucleases (TALENs). mRNA delivery of a TALEN pair targeting the <i>Nicotiana benthamiana</i> ALS gene resulted in mutation frequencies of approximately 6% in comparison to DNA delivery, which resulted in mutation frequencies of 70.5%. mRNA delivery resulted in three-fold fewer insertions, and 76% were <10bp; in contrast, 88% of insertions generated through DNA delivery were >10bp. In an effort to increase mutation frequencies using mRNA, we fused several different 5’ and 3’ untranslated regions (UTRs) from <i>Arabidopsis thaliana</i> genes to the TALEN coding sequence. UTRs from an <i>A</i>. <i>thaliana</i> adenine nucleotide α hydrolases-like gene (At1G09740) enhanced mutation frequencies approximately two-fold, relative to a no-UTR control. These results indicate that mRNA can be used as a delivery vehicle for SSNs, and that manipulation of mRNA UTRs can influence efficiencies of genome editing.</p></div

Mutation size and frequency for mRNA and DNA delivery of SSNs.

<p>The bar graph illustrates the unique insertion profiles between samples transformed with mRNA and DNA reagents.</p

YFP expression in N. benthamiana protoplasts.

<p>Representative images of protoplasts ~24 hours after transformation. The top row shows protoplasts transformed with mRNA transcripts containing UTRs from the four genes tested. The lower row shows images for the controls, namely cells transformed with a DNA construct expressing YFP from a 35S promoter, YFP mRNA without UTRs and water. White arrowheads point to YFP expressing protoplasts.</p

TALEN activity in <i>N</i>. <i>benthamiana</i> protoplasts.

<p>A) Schematic of <i>ALS2</i>, indicating the TALEN target site (black triangle). The target site is 306 bp downstream of the stop codon. B) Bar graph depicting the frequency (%) of NHEJ-induced mutations created by the ALS2T1 TALEN with the four different UTRs as well as the no-UTR control. Data for both mRNA and DNA constructs are presented. The asterisk denotes a sample that is significantly different from the no-UTR control (p = 0.0065). Error bars denote standard error. C) Representative NHEJ-induced mutations. In the wild-type (WT) sequence, the TALEN binding site is indicated as underlined, bold text. Representative mutations have the number of deleted bases indicated at the right.</p

Schematic of mRNA expression constructs.

<p>A) The organization of a generic mRNA expression plasmid is shown with the T7 promoter, nuclease expression cassette and a poly-A sequence. B) Cassettes are illustrated that were used to express mRNA of the desired coding sequence (CDS). The sizes of the UTRs are given in bp. Note that the CDS is not drawn to scale.</p