Large chromosomal deletions and heritable small genetic changes induced by CRISPR/Cas9 in rice

Abstract

The Cas9/sgRNA of the CRISPR/Cas system has emerged as a robust technology for targeted gene editing in various organisms, including plants, where Cas9/sgRNA-mediated small deletions/insertions at single cleavage sites have been reported in transient and stable transformations, although genetic transmission of edits has been reported only in Arabidopsis and rice. Large chromosomal excision between two remote nuclease-targeted loci has been reported only in a few non-plant species. Here we report in rice Cas9/sgRNA-induced large chromosomal segment deletions, the inheritance of genome edits in multiple generations and construction of a set of facile vectors for high-efficiency, multiplex gene targeting. Four sugar efflux transporter genes were modified in rice at high efficiency; the most efficient system yielding 87-100% editing in T0 transgenic plants, all with di-allelic edits. Furthermore, genetic crosses segregating Cas9/sgRNA transgenes away from edited genes yielded several genome-edited but transgene-free rice plants. We also demonstrated proof-of-efficiency of Cas9/sgRNAs in producing large chromosomal deletions (115-245 kb) involving three different clusters of genes in rice protoplasts and verification of deletions of two clusters in regenerated T0 generation plants. Together, these data demonstrate the power of our Cas9/sgRNA platform for targeted gene/genome editing in rice and other crops, enabling both basic research and agricultural applications.

Figure 1.

Constructs for Cas9 and guide RNA expression in rice cells and plants. (A) Binary vector pUbi-Cas9 and intermediate vectors pENTR-dgRNA and pENTR-sgRNA. Cas9 in pUbi-Cas9 is transcribed under control of the maize ubiquitin 1 promoter and NOS terminator. The dual crRNA and tracrRNA genes and single guide RNAs [sgRNA(+85) and sgRNA(+48)] are driven by the rice U6.1 and U6.2 gene promoters. pUbi-Cas9 can accept guide RNA expression cassettes from the intermediate vectors with flanking attL1 and attL2 sites via their cognate attR1 and attR2 sites using the Gateway recombination system. (B) Sequences of guide RNAs used in this study. The dual guide RNAs consist of two separate RNAs, tracrRNA and crRNA containing complementary base pairing regions. The crRNA contains two cloning sites (not shown) for insertion of a 30-bp target DNA. sgRNA(+85) and sgRNA(+48) contain 85-bp and 48-bp RNA tails, respectively.

Figure 2.

sgRNA(+85)/SWEET13-1 not sgRNA(+48)/SWEET13-2 in the cassette of two sgRNAs produces high-efficiency di-allelic mutations in rice SWEET13 in T0 transgenic lines. (A) The gel picture of electrophoresis on the T7E1 treated rice SWEET13 amplicons from the Cas9/sgRNA(+85)-SWEET13-1 T0 transgenic plants. The PCR products amplified from the sgRNA-targeted SWEET13 locus were treated with T7EI and subjected to 1% agarose gel electrophoresis. The arrow indicates the expected T7E1 cleavage product, suggesting possible mutations. M, 1-kb ladder DNA marker; WT, parental Kitaake control. (B) Sequences of SWEET13 at the sgRNA-targeted site (bold and underlined in the wild-type sequence) in each T0 transgenic lines as in (A). The nucleotide changes (dashes for deletion and lower letter for insertion) are also indicated to the right side of each sequence suffixed with a letter, if needed, to distinguish different alleles.

Figure 3.

sgRNA(+85)/SWEET1a instead of sgRNA(+48)/SWEET16 in the cassette of two sgRNAs induces high-efficiency di-allelic mutations of SWEET1a in the primary (T0) transgenic rice plants. (A) Analyses of T0 transgenic lines for site-specific mutations of SWEET1a and for the presence of transgenes Cas9 and sgRNA. The first panel shows the agarose gel image of the T7 endonuclease I (T7EI) treated amplicon of SWEET1a with the expected ∼200-bp cleavage products indicated by the arrow. M, 1-kb ladder DNA marker; lanes 1–10 each denote one representative plant from each independent line; WT, parental Kitaake. Lines 1, 6 and 8 (light black) are T7EI negative. The lower two panels show the PCR results of Cas9 and sgRNA transgenes in the 10 independent transgenic lines. (B) Sequencing confirmation of SWEET1a mutations in seven of the T7EI-positive T0 lines shown in (A). All seven lines contain di-allelic mutations (dash for deletion, lower letters and ‘++’ for insertions) at the sgRNA-targeted sites (bold and underlined). The number of altered nucleotides is shown at the right side of each sequence coupled with a lower-case letter, if needed, indicating the uniqueness of different alleles.

Figure 4.

Two sgRNA(+85) versions produce mutations at two loci in T0 transgenic plants. (A) Detection of mutations and transgenes in five T0 transgenic lines. The upper two panels show the PCR products of SWEET13 and SWEET1b at the relevant sites that were treated with T7EI and separated in 1% agarose gels. The arrow points to the DNA band of the T7EI cleavage products of similar size. The lower three panels show the PCR products of Cas9 and two sgRNAs in the five transgenic lines using specific respective primers. (B) Sequencing confirmation of the site-specific mutations (di-allelic) in the T0 transgenic lines. Dashes denote nucleotide deletions and lower-case letters denote nucleotide insertions. The number of altered nucleotides is shown at the right side of each sequence coupled with a letter, if needed, indicating the uniqueness of different alleles.

Figure 5.

Inheritance of the Cas9/sgRNA modified SWEET13 and removal of transgenes in T1 progeny. (A) Each of the di-allelic mutations (4- and 11-bp deletions) in one representative T0 line (#7) was transmitted to its progeny (T1 plant 7–1 and -3). The sgRNA-targeted site is denoted in the wild-type sequence as bold letters and the PAM sequence underlined. Nucleotide deletions are denoted as dashed lines. (B) PCR-detected presence and absence of individual transgenes (Cas9, hptII and sgRNA) from different sources as indicated above each lane of the agarose gel picture. SWEET13 was used as a control for sample quality.

Figure 6.

Large chromosomal deletion induced by Cas9/sgRNA in rice. (A) Schematic of a cluster of five diterpenoid genes within an ∼170-kb region on rice chromosome 4, indicating sgRNA-targeted sites in colors and PAM sequences underlined. (B) Agarose gel electrophoresis image of the PCR products amplified from the genomic DNA of protoplast pools transfected with sgRNA constructs as indicated by a number corresponding to the sgRNA construct at the right side of picture. (C) Sequences of large DNA segment deletions induced by sgRNAs. The wild-type cluster is depicted as DNA sequence (including sgRNA-targeted sites in distinct colors) and lines (for the intervening sequences). The sequences of individual representative clones (Δ1–7) derived from PCR products shown in (B) are also depicted, with the deleted sequences indicated by dashes between the sgRNA-targeted sites.

Figure 7.

Large segment deletions induced by Cas9/sgRNAs in rice chromosome 2. (A) Schematic illustration of 10 diterpenoid genes spanning about 245 kb on rice chromosome 2, indicating the sgRNA-targeted sites in colors and PAM sequences underlined. (B) Agarose gel electrophoresis of the PCR products amplified from the genomic DNA of protoplasts transfected with sgRNA constructs as indicated by the number that corresponds the sgRNA constructs at the right side of picture. (C) Sequences of large DNA segment deletions induced by sgRNAs. The wild-type cluster is depicted as DNA sequence (sgRNA-targeted sites in distinct colors) and lines (sequences not shown). The sequences of individual representative clones (Δ1 to Δ7) derived from PCR products shown in (B) are presented with the deleted sequences as dashed lines linking the junction sequences between the sgRNA-targeted sites.

Figure 8.

Deletion of 245 kb induced by Cas9/sgRNA activity in rice plants. (A) Schematic illustration of the 245-kb region with two sgRNA-targeted sites as denoted in colors and PAM sequences underlined and bold. (B) Callus lines containing the ∼245-kb deletions identified with a PCR approach and confirmed with sequencing of amplicons. The agarose gel image indicates the four callus lines (number in red) are positive for large fragment deletions. The sequences beneath the image are the junction sequences with the deletions (dashed lines) or extra nucleotides (lower case letters). (C) DNA sequence changes in the representative plants generated from three callus lines (#16, 17 and 21) with the Cas9/sgRNA induced large deletions (dashed lines) in one chromosome and small nucleotide changes (dashed lines for deletions and lower case letters for insertions) in the homologous chromosome.