Abstract
CRISPR/Cas9 has become one of the most promising techniques for genome editing in plants and works very well in poplars with an Agrobacterium-mediated transformation system. We selected twelve genes, including SOC1, FUL, and their paralogous genes, four NFP-like genes and TOZ19 for three different research topics. The gRNAs were designed for editing, and, together with a constitutively expressed Cas9 nuclease, transferred either into the poplar hybrid Populus × canescens or into P. tremula. The regenerated lines showed different types of editing and revealed several homozygous editing events which are of special interest in perennial species because of limited back-cross ability. Through a time series, we could show that despite the constitutive expression of the Cas9 nuclease, no secondary editing of the target region occurred. Thus, constitutive Cas9 expression does not seem to pose any risk to additional editing events. Based on various criteria, we obtained evidence for a relationship between the structure of gRNA and the efficiency of gene editing. In particular, the GC content, purine residues in the gRNA end, and the free accessibility of the seed region seemed to be highly important for genome editing in poplars. Based on our findings on nine different poplar genes, efficient gRNAs can be designed for future efficient editing applications in poplars.
Highlights
Since the first publications regarding application of CRISPR/Cas9 (CRISPR—Clustered Regularly Interspaced Short Palindromic Repeats/Cas9—CRISPR Associated 9) for genome editing in a plant species [1], this technique has become one of the most promising methods in molecular engineering
This target region is recognized by a complementary guide RNA which forms a complex with the endonuclease
For knockdown of SOC1, gRNA for SOC1 (gRNA1) was designed that was fully complementary to the genomic sequence in poplar clone poplar clone INRA 717-1B4 (P1)
Summary
Since the first publications regarding application of CRISPR/Cas (CRISPR—Clustered Regularly Interspaced Short Palindromic Repeats/Cas9—CRISPR Associated 9) for genome editing in a plant species [1], this technique has become one of the most promising methods in molecular engineering. The CRISPR/Cas method is based on the Streptococcus pyogenes endonuclease Cas that causes a DNA double strand break at a specific region (reviewed in Bortesi et al [5]) This target region is recognized by a complementary guide RNA (gRNA) which forms a complex with the endonuclease. Following transient or stable transgenic gRNA transfer, the CRISPR/Cas method has been adapted to many plant [8,9], animal [10,11,12], and fungal species [13]. Relevant for the application of CRISPR/Cas approaches is that even one-nucleotide mutations cause a frameshift that may lead to a knockout of the targeted gene. Transformation line N 485 based on the transgenic clone N 473-9 was intended to add the T-DNA with the gRNAs to edit AGL8.1 and AGL8.2. From six putative double transgenic regenerates, four lines turned out to contain the T-DNA
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