Exploiting SNP s for biallelic CRISPR mutations in the outcrossing woody perennial Populus reveals 4‐coumarate:CoA ligase specificity and redundancy

Abstract

CRISPR (clustered regularly interspaced short palindromic repeats), first discovered as an immune system of prokaryotes, has become a powerful tool for genome editing in eukaryotes (Gaj et al., 2013). Co-expression of the CRISPR-associated endonuclease (Cas9) with a chimeric guide-RNA (gRNA) targeting a GN19NGG motif results in a double-strand DNA break near NGG, the protospacer adjacent motif (PAM) (Jinek et al., 2012). Processing by the endogenous DNA repair machinery generates small indels that, when located within a coding sequence, can disrupt the reading frame and render the gene nonfunctional. The CRISPR/Cas9 system has been successfully applied in several herbaceous systems (Belhaj et al., 2013; Harrison et al., 2014). Here we report its application in the woody perennial Populus using the 4-coumarate:CoA ligase (4CL) gene family as a case study. We achieved 100% mutational efficiency for two 4CL genes targeted, with every transformant examined carrying biallelic modifications. The CRISPR/Cas9 system is highly sensitive to single nucleotide polymorphisms (SNPs), as cleavage for a third 4CL gene was abolished due to SNPs in the target sequence. For outcrossing species with a highly heterozygous genome, gRNA design must take into account the frequent occurrence of SNPs to achieve efficient genome editing. Two 4CL genes, 4CL1 and 4CL2, associated with lignin and flavonoid biosynthesis, respectively (Hu et al., 1998; Harding et al., 2002), were targeted for CRISPR/Cas9 editing. The Populus tremula × alba clone 717-1B4 (717) routinely used for transformation is divergent from the genome-sequenced Populus trichocarpa (Hamzeh & Dayanandan, 2004). Therefore, the 4CL1 and 4CL2 gRNAs designed from the reference genome were interrogated with in-house 717 RNA-Seq data to ensure the absence of SNPs which could limit Cas9 efficiency (Supporting Information Fig. S1). A third gRNA designed for 4CL5, a genome duplicate of 4CL1, was also included. The corresponding 717 sequence harbors one SNP in each allele near/within the PAM, both of which are expected to abolish targeting by the 4CL5-gRNA (Fig. S1). All three gRNA target sites are located within the first exon (Fig. S1a). For 717 transformation, the gRNA was expressed from the Medicago U6.6 promoter, along with a human codon-optimized Cas9 (Mali et al., 2013) under control of the CaMV 35S promoter in binary vector p201N-Cas9 (Jacobs et al., 2015) (Methods S1). Transformation with the Cas9-only vector serves as a control. Altogether, we generated over 30 independent transgenic lines per construct. Randomly selected 4CL1 and 4CL2 lines were subjected to amplicon-sequencing following the protocol of Jacobs et al. (2015). The data were then processed by AGEseq, a custom program we developed for analysis of genome editing by sequencing (L-J. Xue & C-J. Tsai, unpublished) (Methods S1), and biallelic mutations were confirmed in all cases (Fig. 1a). The indel patterns differed between alleles in most cases, with the vast majority of the mutations predicted to disrupt the open reading frame. Although 4CL1 and 4CL5 share a high level (89%) of nucleotide identity, no off-target cleavage of 4CL5 was detected in the 4CL1 mutants (Fig. S2a), supporting specificity of the 4CL1-gRNA. For plants transformed with the 4CL5-gRNA, no editing events were detected as predicted by the presence of SNPs within the corresponding 717 sequence (Fig. S2b). Five randomly selected Cas9 lines were also analyzed and no editing was detected in the three 4CL loci as shown for two representative lines (Figs 1a, S2). These results indicate that the CRISPR editing system is highly efficient and sequence-specific in poplar. Down-regulation of 4CL1 has been shown to reduce lignin accrual, alter monolignol composition and cause stem wood discoloration in several species, including Populus (Kajita et al., 1996; Hu et al., 1999; Voelker et al., 2010). Indeed, a reddish-brown wood color was observed in each of the 36 mutant 4CL1 lines we obtained (Fig. 1b), but with a uniformity that was unlike the patchy discoloration associated with uneven 4CL1 suppression in previous studies (Kajita et al., 1996; Voelker et al., 2010). Lignin content and syringyl-to-guaiacyl (S : G) monolignol ratio were reduced in the 4CL1 mutants (Fig. 1c,d). The lignin and wood discoloration phenotypes were remarkably steadfast among independent mutant lines with varying indel patterns, consistent with a null 4CL1. This high level of mutation efficiency and phenotypic reproducibility is unmatched in previous (antisense, sense co-suppression, or RNAi) studies where typically only a small number of the transformants showed significant changes (Kajita et al., 1996; Hu et al., 1999; Voelker et al., 2010). Despite the efficiency, lignin content was only reduced by c. 23%, suggesting functional redundancy of the 4CL family. The 4CL2 has been associated with flavonoid biosynthesis based on its expression in nonlignifying tissues (Hu et al.,