Exploiting SNPs 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 GN 19 NGG 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 9 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).

Whole-genome draft assembly of Populus tremula x P. alba clone INRA 717-1B4

“…A "substituted genome sequence of P. tremula x alba INRA 717-1B4" (sPta717) was created "by introducing genomic variants of INRA 717-1B4 into the P. trichocarpa reference genome" using 20x resequencing NGS (next generation sequencing) data (v1.1 of sPta717 at http://aspendb.uga.edu/index.php/ databases/spta-717-genome; Zhou et al, 2015). This resource has been applied for screening of 717 gRNAs for sequence variants to optimize gRNA design in CRISPR/Cas genome editing experiments (Zhou et al, 2015).…”

Genome engineering of woody plants: past, present and future

“…It was assumed that C. cellulolyticum might have less active NHEJ-based repair pathways. On the other hand, ''nickase type'' Cas9n had been shown to induce highly efficient ([95 %) HR-based gene targeting at the targeted locus [74]. This high gene targeting efficiency will open the door to high-throughput molecular genetics in wood decay bacteria.…”

“…Most recently, Zhou et al [74] demonstrated the successful gene knockout of 4-coumarate:CoA ligase, the 4CL1 gene in Populus tremula 9 alba clone 717-1B4. The 4CL1 gene is a key gene in lignin biosynthesis [17,75].…”

A novel method for high-frequency genome editing in rice, using the CRISPR/Cas9 system

“…최근 들 어 세균 획득 면역반응 system (CRISPR/Cas9(clustered Regularly Interspaced Short Palindromic Repeats/ CRISPR-associated protein 9)은 염기서열 특이적 핵산분해효소로서 작용할 수 있는 일 종의 맞춤형 유전자가위이다. CRISPR/Cas9 활용을 통해 특 정염기서열을 인지하고 그 부위의 이중나선을 절단함으로 써 유전체상 위치 특이적 돌연변이 유도, 유전자삽입, 대체 등을 포함한 다양한 염기서열 특이적 유전체 편집이 가능 하다 (Endo et al 2016;Wang et al 2016;Zhou et al 2015;Jiang et al 2013). 지금까지 많은 생물에서 CRISPR/Cas9를 이용하여 유전자 편집에 대한 보고는 되었으나 일반적인 연구실에서 적용 및 응용하기가 아직 부족한 실정이다.…”