CRISPR/Cas9 genome targeting systems have been applied to a variety of species. However, most CRISPR/Cas9 systems reported for plants can only modify one or a few target sites. Here, we report a robust CRISPR/Cas9 vector system, utilizing a plant codon optimized Cas9 gene, for convenient and high-efficiency multiplex genome editing in monocot and dicot plants. We designed PCR-based procedures to rapidly generate multiple sgRNA expression cassettes, which can be assembled into the binary CRISPR/Cas9 vectors in one round of cloning by Golden Gate ligation or Gibson Assembly. With this system, we edited 46 target sites in rice with an average 85.4% rate of mutation, mostly in biallelic and homozygous status. We reasoned that about 16% of the homozygous mutations in rice were generated through the non-homologous end-joining mechanism followed by homologous recombination-based repair. We also obtained uniform biallelic, heterozygous, homozygous, and chimeric mutations in Arabidopsis T1 plants. The targeted mutations in both rice and Arabidopsis were heritable. We provide examples of loss-of-function gene mutations in T0 rice and T1 Arabidopsis plants by simultaneous targeting of multiple (up to eight) members of a gene family, multiple genes in a biosynthetic pathway, or multiple sites in a single gene. This system has provided a versatile toolbox for studying functions of multiple genes and gene families in plants for basic research and genetic improvement.

The clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein9 (Cas9) genome editing system (CRISPR/Cas9) is adapted from the prokaryotic type II adaptive immunity system. The CRISPR/Cas9 tool surpasses other programmable nucleases, such as ZFNs and TALENs, for its simplicity and high efficiency. Various plant-specific CRISPR/Cas9 vector systems have been established for adaption of this technology to many plant species. In this review, we present an overview of current advances on applications of this technology in plants, emphasizing general considerations for establishment of CRISPR/Cas9 vector platforms, strategies for multiplex editing, methods for analyzing the induced mutations, factors affecting editing efficiency and specificity, and features of the induced mutations and applications of the CRISPR/Cas9 system in plants. In addition, we provide a perspective on the challenges of CRISPR/Cas9 technology and its significance for basic plant research and crop genetic improvement.

No abstract

No abstract

The recent development of sequence-specific nuclease systems, i.e., TALENs and CRISPR/Cas9, has made genomic targeting easier in many organisms including plants (Li et al., 2012;Cong et al., 2013;Joung and Sander, 2013;Li, et al., 2013;Shan et al., 2013;Liang et al., 2014;Zhang et al., 2014). Mutations induced by CRISPR/Cas9 usually occur around the cleavage sites at three bases upstream of the protospacer-adjacent motif (PAM), producing insertion and deletion of nucleotides. For diploid organisms, such targeted mutations may happen in one or both homologous chromosomes. Previous reports showed that CRISPR/ Cas9-based genomic editing in some plants mainly produced complicated mosaic (chimeric) mutations in the somatic cells of the first generation transgenic plants Mao et al., 2013), and the presence of targeted mutations could be detected by a combination of Cas9 protein and in vitro produced single guide RNAs (sgRNAs) (Gao and Zhao, 2014). However, genomic targeting (Zhang et al., 2014) and our results (Ma X et al., unpublished results) in rice T 0 plants show that the majority of targeted mutations are in uniform allelic statuses, mostly biallelic (two distinct variations), homozygous (two identical mutations), and heterozygous (wild-type/single mutation), and many targeted mutations in Arabidopsis T 1 plants using our CRISPR/Cas9 system are simple heterozygous and biallelic mutations. Most of the targeted mutations are 1-bp insertions and small segment deletions; nucleotide substitutions and insertions of 2 bp or more are very rare. Direct sequencing of PCR products containing such heterozygous and biallelic mutations results in superimposed sequencing chromatograms. A commercial sequence analysis software package, CodonCode Aligner (http:// www.codoncode.com/), has been developed to decode heterozygous DNA sequences by splitting the overlapping sequencing traces into pseudo-alleles. However, this decoding program is very sensitive to the quality of sequencing chromatograms and often outputs false results. For example, we tested decoding of 21 sequencing chromatograms with heterozygous and biallelic mutations from T 0 rice plants using CodonCode Aligner but 16 cases produced false results; four cases of the decoding are shown in Supplemental Figure 1. Therefore, PCR products containing non-homozygous mutations need to be cloned and multiple clones for each targeted site are sequenced, which is tedious, inefficient, and expensive. Here, we present a simple and highly reliable method for rapid decoding of such superimposed sequencing chromatograms from direct sequencing of PCR products with heterozygous and biallelic mutations.Typically, starting from the mutation sites, heterozygous and biallelic mutations ( Figure 1A) produce double traces (overlapping peaks) in the sequencing chromatograms ( Figure 1B). To decode such a sequencing chromatogram, a short degenerate sequence

The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-mediated genome targeting system has been applied to a variety of organisms, including plants. Compared to other genome-targeting technologies such as zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), the CRISPR/Cas9 system is easier to use and has much higher editing efficiency. In addition, multiple "single guide RNAs" (sgRNAs) with different target sequences can be designed to direct the Cas9 protein to multiple genomic sites for simultaneous multiplex editing. Here, we present a procedure for highly efficient multiplex genome targeting in monocot and dicot plants using a versatile and robust CRISPR/Cas9 vector system, emphasizing the construction of binary constructs with multiple sgRNA expression cassettes in one round of cloning using Golden Gate ligation. We also describe the genotyping of targeted mutations in transgenic plants by direct Sanger sequencing followed by decoding of superimposed sequencing chromatograms containing biallelic or heterozygous mutations using the Web-based tool DSDecode. © 2016 by John Wiley & Sons, Inc.

No abstract

No abstract