Targeted mutagenesis in rice using CRISPR-Cas system

Abstract

Genome editing of model organisms is essential for gene function analysis and is thus critical for human health and agricultural production. The current technologies used for genome editing include ZFN (zinc-finger nuclease), meganucleases, TALEN (Transcription activator-like effector nucleases), etc . 1 . These technologies can generate double stranded breaks (DSBs) to either disrupt gene function through generation of premature stop codons by non-homologous end joining (NHEJ) pathway, or to facilitate gene targeting through homologous recombination (HR) with an incoming template. Recently, a new technology for genome editing, CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas (CRISPR-associated) systems, has been developed 2 . CRISPR/Cas systems are adaptive defense systems in prokaryotic organisms to fight against alien nucleic acids 3 . The spacer sequences acquired from foreign DNA are positioned between host repeats, and transcribed together as CRISPR RNA (crRNA). In the type II CRISPR system, a single nuclease Cas9, guided by a dual-crRNA:tracrRNA, is sufficient to cleave cognate DNA homologous to the spacer 2 . Efficient cleavage also requires the presence of protospacer adjacent motif (PAM) 5′-NGG-3′ following the spacer sequence. The dual-crRNA:tracrRNA has been further streamlined to a single RNA chimera, called sgRNA (single guide RNA) 2 . Compared with protein-guided technologies, CRISPR/Cas system is much easier to implement, as only short guide RNAs need to be customized to target the genes of interest. Up to now, the CRISPR/Cas system has been successfully applied to efficient genome editing in many eukaryotic organisms including human 1 , mice 4 , zebra fish 5 , fly 6 , worm 7 , and yeast 8 . However, the application of CRISPR/Cas system in plants has not been reported. Rice ( Oryza sativa L.) is a major staple crop in the grass family (Poaceae), feeding half of the world's population. Rice is also used as a model monocot plant for biological studies because it has a relatively small genome compared to other cereal crops and is easy to be manipulated genetically. We demonstrate in this study that the CRISPR/Cas technology can achieve efficient targeted mutagenesis in transgenic rice. Our work paves the way for large-scale genome editing in rice, which is important for quality improvement and yield increase of rice. To accommodate the CRISPR/Cas system to Agrobacterium -mediated plant transformation, we designed Gateway ™ binary T-DNA vectors for co-expression of CAS9 and guide RNA (either sgRNA or dual-crRNA:tracrRNA, see Figure 1A ). Gene-specific spacer sequence was cloned into entry vectors for expression of guide RNA ( Supplementary information, Figure S1 and Data S1 ), which was then cloned into destination vectors containing the CAS9 expression cassette. To reconstitute Cas9 ribonucleoprotein complex in the nucleus, Cas9 was attached with a nuclear localization signal, and guide RNAs were driven by pol III type promoter of U3 snRNA. CAS9 coding sequence was codon-optimized for expression in rice ( Supplementary information, Figure S2 ), and was driven by the maize Ubiquitin ( Ubi ) promoter. To facilitate Cas9 binding and R-loop formation, we chose the sgRNA with the secondary structure containing a dangling spacer, an extended hairpin region and a long 3′ end ( Figure 1B ). Figure 1 Targeted mutagenesis in rice using CRISPR-Cas system. (A) Schematic diagram of the vectors used in this study. CAS9 is cloned into the destination vector. Guide RNAs are cloned into entry vectors. Modular design of vectors permits multiplex genome editing. (B) Schematic showing the folding of sgRNA and co-folding of crRNA and tracrRNA. Spacer sequences for targeting DGU.US or CAO1 are also shown. The long hairpin and long tail are used to maintain structural stability. (C) Schematic representation of the DGU.US system to detect the generation of DSB. DSB generated by Cas9 between the direct repeats (U) can be repaired through SSA, thus restoring the GUS function. PAM is underlined. (D) GUS staining after co-transformation of DGU.US with (left) or without the CRISPR construct (right). Scale bar, 0.5 mm. (E) Leaf blade of wild type (WT) and transformants with cao1 phenotype. (F) Targeted mutagenesis of CAO1 gene. The position of target site is shown on the gene structure. The spacer sequence is shaded in gray, and the PAM is underlined. Sequencing results of mutant alleles are aligned to the reference genome sequence. The indels are shown in red letters or dashes. The sequence of the 92-bp insertion in CAO1 gene is 5′-TCG TTC CCT TTC ATC AAG TGG GTA AGG TAG GCG AAC CAC TTA AGC TTT GCC CCC CAC CTC CTT TGG GGA TCC CTA CTT ACC CGA GCT TCC GG-3′. (G) Chlorophyll analysis in WT and transgenic plants. (H) Targeted mutagenesis of LAZY1 gene. The position of target site is shown on the gene structure. The spacer sequence is shaded in gray, and the PAM is underlined. Sequencing results of mutant