A Multipurpose Toolkit to Enable Advanced Genome Engineering in Plants

Abstract

We report a comprehensive toolkit that enables targeted, specific modification of monocot and dicot genomes using a variety of genome engineering approaches. Our reagents, based on transcription activator-like effector nucleases (TALENs) and the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system, are systematized for fast, modular cloning and accommodate diverse regulatory sequences to drive reagent expression. Vectors are optimized to create either single or multiple gene knockouts and large chromosomal deletions. Moreover, integration of geminivirus-based vectors enables precise gene editing through homologous recombination. Regulation of transcription is also possible. A Web-based tool streamlines vector selection and construction. One advantage of our platform is the use of the Csy-type (CRISPR system yersinia) ribonuclease 4 (Csy4) and tRNA processing enzymes to simultaneously express multiple guide RNAs (gRNAs). For example, we demonstrate targeted deletions in up to six genes by expressing 12 gRNAs from a single transcript. Csy4 and tRNA expression systems are almost twice as effective in inducing mutations as gRNAs expressed from individual RNA polymerase III promoters. Mutagenesis can be further enhanced 2.5-fold by incorporating the Trex2 exonuclease. Finally, we demonstrate that Cas9 nickases induce gene targeting at frequencies comparable to native Cas9 when they are delivered on geminivirus replicons. The reagents have been successfully validated in tomato (Solanum lycopersicum), tobacco (Nicotiana tabacum), Medicago truncatula, wheat (Triticum aestivum), and barley (Hordeum vulgare).

Figure 1.

Two Sets of Vectors for Direct Cloning or Modular Assembly of Genome Engineering Reagents. (A) Direct cloning vectors were designed to speed up the cloning process. Specificity determining elements (gRNAs or TAL repeats) are cloned directly into the transformation backbone (e.g., T-DNA). (B) The modular assembly vectors enable combination of different functional elements. Specificity determining elements (gRNAs, TAL repeats, and donor templates for gene targeting) are first cloned into intermediate module vectors. Custom-selected modules are then assembled together into the transformation backbone (e.g., T-DNA).

Figure 2.

TALEN-Mediated Mutagenesis in M. truncatula Using Direct Cloning Vectors. (A) Maps of two M. truncatula genes targeted for mutagenesis using a single TALEN pair. PCR primers used for screening in (B) are shown as green arrowheads. The sequence of the TALEN target site is shown with TALEN binding sites underlined. (B) HaeIII PCR digestion screening of eight T1 progeny of plant WPT52-4. The amplified locus is indicated on the right. bar and ACTIN are controls that amplify the transgene and a native gene, respectively. HM340 (D), wild-type product digested with HaeIII; HM340 (UD), undigested wild-type product. (C) DNA sequences of undigested amplicons from plant WPT52-4-4. The reference sequence of the unmodified locus is shown on the top. TALEN binding sites are underlined. HaeIII restriction site used for the screening is in red.

Figure 3.

Comparison of Different Systems for Expressing Multiple gRNAs. (A) Structure of six constructs for expressing gRNA9 and gRNA10, which both target the tomato ARF8A gene. CmYLCV, Cestrum yellow leaf curling virus promoter; Csy4, 20-bp Csy4 hairpin; tRNA, 77-bp pre-tRNA^Gly gene; rb, 15-bp ribozyme cleavage site; ribozyme, 58-bp synthetic ribozyme; AtU6, Arabidopsis U6 promoter; At7SL, Arabidopsis 7SL promoter. (B) Possible editing outcomes due to expression of both gRNAs. Sequences targeted by gRNA9 and gRNA10 are shown on the top. Cleavage sites are indicated by arrows. (C) Overall mutation frequencies as determined by deep sequencing. Error bars represent se of three replicates (pools of protoplasts). Statistical significance was determined by Tukey’s test. *P < 0.05 and ***P < 0.001.

Figure 4.

Evaluation of Eight gRNAs in Various Polycistronic Expression Systems and the Effect of Position in the Array on gRNA Activity. (A) Structure of seven constructs for expressing gRNAs 49 to 56 targeting four tomato genes for deletion. (B) Detection of deletions between gRNA sites using qPCR. Each gene is targeted for deletion with two gRNAs designed to create a 3-kb deletion to prevent amplification of the nondeleted, wild-type template. Deletion frequency was quantified by qPCR using primers shown as red and blue arrowheads. (C) Deletion frequencies in tomato protoplasts as measured by qPCR. Positions of respective gRNAs in the array are specified for each construct. Error bars represent se of two replicates.

Figure 5.

Multiplexed Mutagenesis Using the Csy4 System in Tomato Protoplasts. (A) Maps of six tomato genes targeted for simultaneous deletion using 12 gRNAs. gRNA sites are shown as black arrows. Arrowheads represent primers used to detect the deletions. Lengths of PCR products from the wild-type locus are shown on the right; lengths of the deletion products are in parentheses. (B) Deletions of expected length were detected in each of the six genes by PCR. Blue and black arrowheads mark the unmodified and deletion products, respectively. Wild-type DNA from nontransformed cells was used as template. (C) DNA sequences of representative deletion products. The sequence of the unmodified locus for each of the six genes is shown on the top. gRNA target sites are underlined and PAM sequences are in red.

Figure 6.

Multiplexed Mutagenesis Using the Csy4 System in Wheat Protoplasts. (A) Maps of three wheat genes targeted for simultaneous mutagenesis using six gRNAs. gRNA sites are shown as black arrows. Arrowheads represent primers used to detect the deletions. Lengths of PCR products from the wild-type locus are shown on the right; lengths of the deletion products are in parentheses. (B) Deletions of expected length were detected in each of the six genes by PCR. Blue and black arrowheads mark the unmodified and deletion products, respectively. Wild-type DNA from nontransformed cells was used as template. (C) DNA sequences of representative deletion products. The sequence of the unmodified locus for each of the three genes is shown on the top. gRNA target sites are underlined and PAM sequences are in red.

Figure 7.

Both CmYLCV and PvUbi1 Promoters Are Functional in Barley Protoplasts and Induce Targeted Deletions Using the Csy4 gRNA Expression System. (A) Map of the barley MLO gene targeted for deletion using two gRNAs. gRNA sites are shown as black arrows. Arrowheads represent primers used to detect the deletions. Length of the PCR product from the wild-type locus is shown on the right; length of the deletion product is in parentheses. (B) Deletions between the gRNA38 and gRNA39 sites were detected in the barley MLO gene when either the CmYLCV or PvUbi1 promoters were used to express the gRNAs. Blue and black arrowheads mark the unmodified and deletion products, respectively. Two replicates (pools of protoplasts) of the experiment are shown. (C) DNA sequences of representative deletion products. The sequence of the unmodified locus for each of the three genes is shown on the top. gRNA target sites are underlined and PAM sequences are in red.

Figure 8.

Targeted Deletion of 58 kb in M. truncatula. (A) Map of the region on chromosome 2 targeted for deletion. Cyan rectangles represent individual genes. gRNA sites are shown as black arrows. Primers for detection of deletions are shown below as green arrows. (B) PCR detection of large deletions in callus tissue, T0, T1, and T2 plants. Primers used to detect deletions of different sizes are indicated on the right of each gel picture. Primer pairs F1a/R1a, F1b/R1b, F1c/R1c, F1d/R1d, and F1e/R1e amplify the nondeleted wild-type sequence. Primers bar F2/R2 amplify the bar transgene. NT, no template control. (C) DNA sequences of the 58-kb deletion events amplified with primers F3/R5 from callus tissue and T0 plants transformed with the Csy4 vector. The reference sequence of the unmodified locus is shown on the top. gRNA target sites are underlined and PAM sequences are highlighted in red. (D) Phenotypes of T1 plants heterozygous and homozygous for the 58-kb deletion. Note the dwarf stature of the mutants. Images have been adjusted to minimize soil noise by decreasing the brightness of the white and neutral color channels. All images have been adjusted equally.

Figure 9.

Trex2 Enhances Mutagenesis in Tomato and Barley Protoplasts. (A) T7 endonuclease I assay results for two sites in tomato and one site in barley. Two replicates (pools of protoplasts) and an average fold increase in mutagenesis frequency are shown for the tomato sites. The average fold increase in the two experiments is shown at the top. (B) DNA sequences of NHEJ-derived mutations found in bacterial clones of PCR products that encompass the gRNA target site in the tomato ANT1 gene. Mutations are shown in 15 of 92 bacterial clones derived from tomato cells transformed with Cas9 and gRNA only and 26 of 88 bacterial clones derived from cells transformed with Cas9, gRNA, and Trex2. Sequences of three clones containing insertions at the gRNA site found in the sample lacking Trex2 are not shown. The reference sequence is shown on the top. The 20-bp gRNA target site is underlined and the PAM sequence is highlighted in red. (C) DNA sequences of NHEJ-derived mutations found in bacterial clones of PCR products that encompass the gRNA target site in the barley MLO gene. Mutations are shown in 18 bacterial clones derived from barley cells transformed with Cas9 and gRNA only and 19 bacterial clones derived from cells transformed with Cas9, sgRNA, and Trex2. The samples were enriched for mutations by digesting the PCR amplicons with NcoI before cloning. The reference sequence is shown above; the 20-bp gRNA target site is underlined and the PAM is highlighted in red. The sequence of the NcoI recognition site is in italics. The 1-bp insertion is highlighted in green.

Figure 10.

Gene Targeting with Cas9 Nickases and GVRs in Tobacco. (A) Illustration of the gene targeting approach in tobacco. gRNAs gNt_F2 and gNt_R2 target the defective gus:nptII transgene stably inserted in the tobacco genome. The missing coding sequence is restored through homology directed repair using the donor template, resulting in a functional GUS gene. (B) Effect of single and double nicks on gene targeting in tobacco. The Cas9 genes and gRNAs were delivered to tobacco leaves on a BeYDV replicon by agroinfiltration. The leaves were stained in an X-Gluc solution 5 d later, and blue (GUS positive) spots were quantified by image analysis. Data were normalized to the Cas9 nuclease sample. Error bars represent the se of 5 independent experiments. AtCas9, Cas9 nuclease; AtD10A, Cas9 D10A nickase; AtH840A, Cas9 H840A nickase; (-gRNA), no gRNA control. (C) DNA sequences of the left and right recombination junctions from leaves infiltrated with the D10A paired nickase construct. Multiple bacterial clones were sequenced and were identical. Junctions at both ends of each homology arms are shown.