Application of the CRISPR-Cas system for efficient genome engineering in plants.

Dear Editor, Recently, engineered endonucleases, such as Zinc-Finger Nucleases (ZFNs) (Carroll, 2011), Transcription Activator-Like Effector Nucleases (TALENs) (Mahfouz et al., 2011; Li et al., 2012), and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated (Cas) systems (Cong et al., 2013) have been successfully used for gene editing in a variety of species. These systems generate double-strand breaks (DSBs) at target loci to drive site-specific DNA sequence modifications. The modifications include sequence insertion and deletion and other mutations in the host genomes via the error-prone non-homologous end joining (NHEJ) pathway or sequence correction or replacement through the error-free homologous recombination (HR) pathway (Symington and Gautier, 2011). Here, we show that the CRISPR–Cas system can be applied to generate targeted gene mutations and gene corrections in plants, and the system can also be readily engineered to achieve deletion of large DNA fragments and for multiplex gene editing in plants. Both ZFNs and TALENs have tandem repeats in their DNA-binding domains that can be engineered to recognize specific DNA sequences; the resulting chimeric nucleases can thus be guided to the desired target sequences in the genome to generate DSBs. For each target site, a new ZFN or TALEN chimeric protein needs to be engineered to recognize the target. This has been a major hurdle in the wide use of these two gene-editing systems because engineering a new protein is no trivial task. In comparison, the newly developed CRISPR–Cas system uses a short single guide RNA (sgRNA) to direct the Cas9 endonuclease to complementary target DNA (Gaj et al., 2013), so only a new sgRNA is needed for a new target site. This system thus greatly simplifies the gene-editing process and widens target-site selection. An additional requirement for the Cas9 nuclease activity is the presence of the protospacer-associated motif (PAM) NGG downstream of the target site. This requirement is an important consideration in target-site selection (Jinek et al., 2012). Several expression vectors were constructed for CRISPR– Cas-based gene editing in Arabidopsis and rice, in which the designed sgRNAs and optimized SpCas9 (Cong et al., 2013) were driven by AtU6 or OsU6 and AtUBQ, OsUBQ, or CaMV 35S promoters, respectively (Supplemental Figure 1). A transient expression system was developed in Arabidopsis protoplasts according to an earlier study (Zhang et al., 2013) to assess the activity of the CRISPR–Cas construct psgR–Cas9–At. The yellow fluorescent protein (YFP)-based reporter contained two partially overlapped YFP fragments that were interrupted by a multiple recognition site (MRS). Three different nucleases, I-SceI, gdTALEN, and CRISPR–Cas, were designed to target the MRS region (Supplemental Figure 2). When the MRS sequence is recognized and cleaved, a functional copy of the YFP gene could be restored through the HR pathway in the cells, so that the efficiency of these endonucleases can be estimated by counting the number of cells emitting yellow fluorescent light using flow cytometry. In an optimized experiment, 11.0% sgR–MRS/YFFP co-transfected protoplasts showed fluorescence—a frequency lower than the 18.8% for gdTALEN but comparable to the 12.5% for I-SceI (Supplemental Figure 2). These results suggested that the CRISPR–Cas system was functional in generating DSBs and triggering gene correction in plant cells. To test the importance of the PAM sequence for target recognition in plants, an improper sgRNA (sgR-MRS*) with a shifted PAM sequence (from GGG to GGA) was used to target the MRS in the YFFP reporter (Supplemental Table 1 and Supplemental Figure 2). The proportion of YFP florescent cells in the sgR-MRS*-targeted protoplasts was 5.4%, compared to the 11% for the sgR-MRS target with a correct PAM. Thus, the altered PAM sequence greatly reduced but did not abolish the activity of CRISPR–Cas9, suggesting that, although PAM is important, it is not absolutely required for the function of CRISPR–Cas in plant cells. The sgRNA and Cas9 expression cassettes were cloned into a binary vector that contains a nonfunctional GUUS reporter (Figure 1A and Supplemental Figure 1) for Agrobacteriummediated transformation. Among 44 T1 transgenic Arabi­ dopsis plants tested, five showed a GUS signal in their cotyledons (Figure 1B). We did not see any plant organ with a uniform GUS signal, and observed GUS expression in one guard cell but not in the other one in the same stoma (Figure 1B), indicating that the CRISPR–Cas induced cleavage and HR repair events happened in individual cells. Using a SURVEYOR assay, we found that 35 of the 44 plants, including three of the five GUS-positive lines, had mutations at the target site (Figure 1C and Supplemental Table 2). These