Efficient genome editing in plants using a CRISPR/Cas system

Abstract

In the past few years, the development of sequence-specific DNA nucleases has progressed rapidly and such nucleases have shown their power in generating efficient targeted mutagenesis and other genome editing applications. For zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), an engineered array of sequence-specific DNA binding domains are fused with the DNA nuclease Fok1 1 , 2 . These nucleases have been successful in genome modifications by generating double strand breaks (DSBs), which are then repaired through non-homologous end joining (NHEJ) or homologous recombination (HR) in different species, including mouse, tobacco and rice 3 , 4 , 5 . Recently, another breakthrough technology for genome editing, the CRISPR/Cas system, was developed. CRISPR (clustered regulatory interspaced short palindromic repeats) loci are variable short spacers separated by short repeats, which are transcribed into non-coding RNAs. The non-coding RNAs form a functional complex with CRISPR-associated (Cas) proteins and guide the complex to cleave complementary invading DNA 6 . After the initial development of a programmable CRISPR/Cas system, it has been rapidly applied to achieve efficient genome editing in human cell lines, zebrafish and mouse 7 , 8 , 9 , 10 . However, there is still no successful application in plants reported. We report here that the CRISPR/Cas system can be used to efficiently generate targeted gene mutations and corrections in plants. The Cas9 gene was driven by the CaMV 35S promoter and the chimeric single guide RNA (sgRNA) was driven by the AtU6-26 promoter in Arabidopsis or the OsU6-2 promoter in rice. We show that the engineered CRISPR/Cas was active in creating DSBs when transiently expressed in Arabidopsis protoplasts and stably expressed in transgenic Arabidopsis and rice plants. Our results demonstrate the feasibility of using engineered CRISPR/Cas as molecular scissors to create DSBs at specific sites of the plant genome to achieve targeted genome modifications in both dicot and monocot plants. We used the optimized coding sequence of hSpCas9 9 driven by the CaMV 35S promoter. For the non-coding RNA components of CRISPR, we expressed the sgRNA using native promoters for U6 RNAs in Arabidopsis ( Figure 1A and Supplementary information, Figure S1A ) or rice ( Supplementary information, Figure S1A ). The target site precedes an NGG, the requisite protospacer adjacent motif (PAM). To improve co-delivery, both the sgRNA and hSpCas9 were subcloned into one expression vector ( Figure 1A ). A split yellow fluorescent protein (YFP) reporter system, YF-FP, was used to test the functionality of the engineered CRISPR/Cas system in Arabidopsis protoplasts ( Figure 1B ). Co-transformation of the YF-FP reporter and the CRISPR/Cas construct led to the production of strong YFP signal with gene correction rate by HR at 18.8% ((4.76%–0.78%)/21.23%) ( Figure 1C ). The results suggest that the engineered CRISPR/Cas system is highly functional in generating DSBs on target DNA sequences in plant cells and that the DSBs can be repaired by HR to achieve gene correction. Figure 1 High-efficiency targeted gene editing in Arabidopsis and rice plants. (A) The vector for transient transformation of Arabidopsis protoplasts. The expression cassette of hSpCas9 is driven by the 35S promoter, whereas the sgRNA is driven by the AtU6-26 promoter. (B) The HR-based YF-FP reporter. The PAM sequence of the sgRNA target is colored in magenta and the sgRNA target in cyan. (C) The activity of CRISPR/Cas was measured using the YF-FP reporter in Arabidopsis protoplasts. YFP-positive cells were quantified by flow cytometry. (D) Summary of observed phenotypes and detected mutations in T1 and T0 transgenic plants of Arabidopsis and rice, respectively. (E and F) Representative T1 transgenic plants of the BRI1 sgRNA1 target. The left one is a T1 plant with normal vegetative growth, while the right one is a plant with a similar growth phenotype as bri1 mutants. The plants were screened on MS plates for 5 days and transplanted in soil for 1 week (E) or 3 weeks (F) before photographing. (G) One representative rice T0 transgenic plant of the YSA sgRNA1 target showing an expected albino leaf phenotype at the regeneration stage. Arrows indicate the albino leaves. (H) Genotyping of 12 T1 transgenic Arabidopsis plants of the BRI1 sgRNA1 target by RFLP analysis. The PCR products were digested with Eco RV. M, DNA marker. (I) Genotyping of 14 T0 transgenic rice plants of the YSA sgRNA1 target by RFLP analysis. The #13 sample corresponds to the plant in G . The PCR products were digested with Sfi I. M, DNA marker. Kas, wild-type rice Kasalath. (J and K) Representative sequences of mutant alleles identified from a single transgenic plant of the BRI1 sgRNA1 target (J) and T0 plant of the YSA sgRNA1 target (K) . For the YSA locus, the plant corresponds to the plant in G . The wild-type sequence is shown at the top with t