A CRISPR/Cas9 toolkit for multiplex genome editing in plants
© Xing et al.; licensee BioMed Central Ltd. 2014
Received: 9 October 2014
Accepted: 6 November 2014
Published: 29 November 2014
To accelerate the application of the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/ CRISPR-associated protein 9) system to a variety of plant species, a toolkit with additional plant selectable markers, more gRNA modules, and easier methods for the assembly of one or more gRNA expression cassettes is required.
We developed a CRISPR/Cas9 binary vector set based on the pGreen or pCAMBIA backbone, as well as a gRNA (guide RNA) module vector set, as a toolkit for multiplex genome editing in plants. This toolkit requires no restriction enzymes besides BsaI to generate final constructs harboring maize-codon optimized Cas9 and one or more gRNAs with high efficiency in as little as one cloning step. The toolkit was validated using maize protoplasts, transgenic maize lines, and transgenic Arabidopsis lines and was shown to exhibit high efficiency and specificity. More importantly, using this toolkit, targeted mutations of three Arabidopsis genes were detected in transgenic seedlings of the T1 generation. Moreover, the multiple-gene mutations could be inherited by the next generation.
We developed a toolkit that facilitates transient or stable expression of the CRISPR/Cas9 system in a variety of plant species, which will facilitate plant research, as it enables high efficiency generation of mutants bearing multiple gene mutations.
Approaches for precise, efficient gene targeting or genome editing are highly important for functional genomic analysis of plants and for the production of genetically engineering crops. For the majority of researchers, transfer DNA (T-DNA) and transposon insertional mutagenesis remain the main sources of mutants of genes of interest in model plants such as the dicot Arabidopsis thaliana and the monocot rice (Oryza sativa) ,. There is an increasing demand for plants bearing mutations in multiple genes in order to dissect the functions of gene family members with redundant functions and to analyze epistatic relationships in genetic pathways. However, the current method for generating plants carrying multiple mutated genes requires time-consuming and labor-intensive genetic crossing of single-mutant plants. Moreover, T-DNA insertional mutants cannot be obtained for every gene of interest. Therefore, new technologies that are affordable, efficient, and user-friendly are needed for plant genome targeting.
Double-strand breaks (DSBs) at specific genomic sites can introduce a mutation at the DNA break site via the error-prone non-homologous end-joining (NHEJ) pathway. DSBs can also result in homologous recombination (HR) between chromosomal DNA and foreign donor DNA through the HR pathway . Based on DSBs at target loci, sequence-specific nucleases, including homing meganucleases, zinc finger nucleases, and transcription activator-like effector (TALE) nucleases have emerged as powerful technologies for targeted genome editing in eukaryotic organisms .
Recently, another DSB-based breakthrough technology for genome editing, the CRISPR/Cas system, was developed ,. This system is based on the bacterial and archaeal clustered regularly interspaced short palindromic repeats (CRISPR) adaptive immune system for purging invading viral and plasmid DNA, which relies on the endonuclease activity of CRISPR-associated (Cas) proteins, with sequence specificity directed by CRISPR RNAs (crRNAs) -. The CRISPR/Cas system, which is employed in a variety of organisms, is derived from the Streptococcus pyogenes type II CRISPR system and consists of three genes, including one encoding Cas9 nuclease and two noncoding RNA genes: trans-activating crRNA (tracrRNA) and precursor crRNA (pre-crRNA). The programmable pre-crRNA, which contains nuclease guide sequences (spacers) interspaced by identical direct repeats, is processed to mature crRNA in combination with tracrRNA. The two RNA genes can be replaced by one RNA gene using an engineered single guide RNA (gRNA) containing a designed hairpin that mimics the crRNA–tracrRNA complex. The binding specificity of Cas9 with the target DNA is determined by both gRNA–DNA base pairing and a protospacer-adjacent motif (PAM, sequence: NGG) immediately downstream of the target region. Both nuclease domains of Cas9 (HNH and RuvC-like) cleave one strand of double-stranded DNA at the same site (three-nucleotide [nt] distance from the PAM), resulting in a DSB -. The CRISPR/Cas system has been harnessed to achieve efficient genome editing in a variety of organisms, including bacteria, yeast, plants, and animals, as well as human cell lines -. More importantly, using this RNA-guided endonuclease technology, multiple gene mutations and their germline transmission have been achieved -.
In vertebrates such as zebrafish, mice, rats, and monkeys, coinjection of gRNA and Cas9-encoding mRNA transcribed in vitro into single-cell-stage embryos can efficiently generate animals with multiple biallelic mutations that can be transmitted to the next generation with high efficiency ,-. However, this method is not feasible in plants, where transgenic lines stably expressing the CRISPR/Cas9 system are required for the generation of plants with one or more gene mutations. Agrobacterium-mediated transformation is a routine method used to generate transgenic plants, and a few binary vectors have been developed to deliver the CRISPR/Cas9 system into plant genomes via this method ,,,,-. Nevertheless, to accelerate the application of this system to a variety of plant species under normal or complex conditions (such as targeted mutation of genes in the background of T-DNA insertional mutants), a toolkit with additional plant selectable markers, more gRNA modules, and easier methods for assembling one or more gRNA expression cassettes is frequently required, especially for targeted mutation of multiple genes. We report the development of such a toolkit for multiplex genome editing in plants.
CRISPR/Cas9 binary vector set and gRNA module vector set for multiplex genome editing in plants
Validation of the CRISPR/Cas9 toolkit in maize protoplasts
To validate the toolkit and to compare the mutation efficiency of different Cas9 or Pol III promoters used to drive the gRNAs, we generated two sets of test vectors targeting the same maize genomic DNA site (ZmHKT1). One set comprises pBUN201-ZT1, pBUN301-ZT1, and pBUN401-ZT1, which harbor different Cas9 sequences, including hCas9-NLS-3 × FLAG in pBUN201-ZT1, 3 × FLAG-NLS-hCas9-NLS in pBUN301-ZT1 and 3 × FLAG-NLS-zCas9-NLS in pBUN401-ZT1. The hCas9 and zCas9 sequences are human-codon and Zea mays-codon optimized Cas9, respectively. Another set comprises pBUN401-ZT1, pBUN411-ZT1, and pBUN421-ZT1. These vectors differ based on the Pol III promoters used to drive the gRNA: AtU6-26p in pBUN401-ZT1, OsU3p in pBUN411-ZT1 and TaU3p in pBUN421-ZT1.
To verify mutation events, the PCR products were cloned, and the resulting colonies were screened by colony PCR and XcmI digestion of the colony PCR products. DNA from clones whose colony PCR products were resistant to XcmI digestion was sequenced (Figure 3D). Interestingly, we obtained eight insertional mutations, including one derived from hCas9 from the vector and seven from the ubiquitin promoter, which were presumably derived from the degraded vector rather than the maize genome (Figure 3E). These results suggest that the efficiency of targeted integration is relatively high when donor genes are provided.
Validation of the CRISPR/Cas9 toolkit in transgenic maize
Validation of the CRISPR/Cas9 toolkit in Arabidopsisfor the generation of mutants with multiple gene mutations
Germline transmission of T1 mutations to segregated nontransgenic T2 plants
Nontransgenic T2 plants
Mutation analysis of nontransgenic T2 triple mutant plants
NT T2 triple mutant lines
Dissecting the functions of gene family members with redundant functions and analyzing epistatic relationships in genetic pathways frequently require plant mutants bearing mutations in multiple genes. The recently developed CRISPR/Cas9 system provides an excellent method for genome editing ,,. However, to produce multiple gene mutations in plants, resources and methods for the assembly of multiple gRNA expression cassettes are frequently required. In this report, we describe methods used to generate gRNA modules and to assemble multiple gRNA expression cassettes using premade gRNA modules. These resources, comprising binary vectors and gRNA module vectors, are able to meet most of the requirements for use in a variety of plants under normal or complex conditions. These methods also allow researchers to customize their own gRNA modules and to assemble multiple gRNA expression cassettes for multiplex genome editing. Using this kit, we found that CRISPR/Cas9 could be used to knock out multiple plant genes simultaneously, and the efficiencies of multiple-gene mutations, in accordance with the “Bucket effect” theory in economics, depended on the lowest mutation efficiencies of the targeted genes.
Binary vectors are required for the use of CRISPR/Cas9 in plants. To fuse a 20-bp target sequence to the 5′-end of the gRNA scaffold, it is best to use type IIs restriction enzymes. Although a few type IIs restriction enzymes, such as AraI, BbsI/BpiI, BsaI/Eco31I, BsmBI/Esp3I, BspMI/BfuAI/BveI, and BtgZI are commercially available, few such enzymes can be used to linearize commonly used binary vectors, such as pCAMBIA series and pPZP series vectors ,, due to the presence of one or more sites in the backbones of these vectors. For example, not including the T-DNA region, the pCAMBIA backbone contains one BsaI, two BbsI, two BsmBI, two BspMI, and four BtgZI sites. Although no AarI site can be found in the pCAMBIA backbone, there is an AarI site in the Bar selectable marker gene of the T-DNA region of pCAMBIA3300. Fortunately, despite the presence of a BsaI site in the pVS1 replication region, which is required for plasmid propagation in Agrobacterium, there are no BsaI sites in commonly used elements, such as promoters including the double CaMV 35S promoter and the Ubi1 promoter, or in selectable markers including Kan, Hyg and Bar. Moreover, BsaI is the least expensive of the commonly used type IIs restriction enzymes. For example, the price per activity unit of BsaI/Eco31I is only approximately 1/50 that of AarI (Thermo Fisher Scientific and New England Biolabs). To utilize BsaI to assemble gRNA expression cassettes into pCAMBIA binary vectors, we disrupted the BsaI site of the pVS1 region. Thus, for the binary vector set we developed, no restriction enzyme but BsaI is required for the assembly of one or more gRNAs.
This toolkit provides the easiest method for generating plant CRISPR/Cas9 binary vectors. When constructing binary vectors carrying one or two gRNAs, only two 23-nt synthetic oligos (annealed to an insert) or a PCR fragment, respectively, are required, along with any of the binary vectors described in this report, to set up Golden Gate reactions. When constructing binary vectors carrying multiple gRNAs, two or more PCR fragments are required. Based on either the Golden Gate cloning method  or Gibson Assembly , two or more PCR fragments could easily be assembled into multiple gRNA expression cassettes onto any of the BsaI-linearized binary vectors in only one cloning step. Two strategies can be used to assemble more than four gRNA expression cassettes, i.e., generating more gRNA modules with additional validated Pol III promoters, and inserting (for the second time) gRNA expression cassettes harboring the spectinomycin-resistance gene into binary vectors that already contain four gRNAs followed by the assembly of additional gRNAs into the BsaI-linearized vectors. Thus, the binary vector set combined with the gRNA module vector set comprise an efficient, inexpensive, time-saving, user-friendly, multifaceted, extensible toolkit for the generation of CRISPR/Cas9 binary vectors carrying one or more gRNAs for targeted mutations of multiple genes.
We developed a CRISPR/Cas9-based binary vector set and a gRNA module vector set as a toolkit for multiplex genome editing in plants. We validated the kit using maize protoplasts, maize transgenic lines, and Arabidopsis transgenic lines and found that it exhibited high efficiency and specificity. The binary vector set combined with the gRNA module vector set comprise an efficient, inexpensive, time-saving, user-friendly, multifaceted, extensible toolkit for the generation of CRISPR/Cas9 binary vectors carrying one or more gRNAs for targeted mutations of multiple plant genes. This toolkit, which facilitates transient or stable expression of CRISPR/Cas9 in a variety of plant systems, can be applied to a variety of plants and is especially useful for high-efficiency generation of mutants bearing multiple gene mutations.
Golden gate method to construct a vector expressing one or two gRNAs
For assembly of one gRNA, equal volumes of 100 μmol/L oligos 1 and 2 were mixed, incubated at 65°C for 5 minutes, and cooled slowly to room temperature, resulting in a double-stranded insert with 4-nt 5′ overhangs at both ends. For assembly of two gRNAs, the two target sites were incorporated into PCR forward and reverse primers, respectively. The PCR fragment was amplified from pCBC-DT1T2 for dicot targets or pCBC-MT1T2 for monocot targets with two long primers or four shorter primers, among which two forward or two reverse primers were partially overlapping. The insert or the purified PCR fragment (T1T2-PCR), together with any of the binary vectors described in this report, were used to set up restriction-ligation reactions, as described elsewhere , using BsaI and T4 Ligase (New England Biolabs). The reaction was incubated in a thermocycler for 5 hours at 37°C, 5 min at 50°C and 10 min at 80°C. Detailed information including gRNA module sequences, PCR primers, colony PCR primers, and sequencing primers can be found in Additional file 3: Methods S2.
Golden gate cloning or Gibson assembly method to generate a vector expressing three or four gRNAs
Two methods were used to assemble more than three gRNAs: Golden Gate Cloning  and Gibson Assembly . For Golden Gate Cloning, two (T1-PCR and T2T3-PCR2) or three (T1-PCR, T2-PCR and T3T4-PCR2) PCR fragments were purified and mixed with any of the CRISPR/Cas9 binary vectors to set up Golden Gate reactions as described above. For Gibson Assembly, two (T1T2-PCR and T2T3-PCR) or three (T1T2-PCR, T2T3-PCR and T3T4-PCR) PCR fragments were purified and mixed with Gibson Assembly Master Mix (New England Biolabs) to set up reactions according to the manufacturer’s protocol. The fragment of desired size was gel purified and used as a PCR template for the second round of PCR amplification. The products from the second round of PCR were purified and mixed with any of the binary vectors described in this report to set up the Golden Gate reaction as described above. Detailed information including gRNA module sequences, PCR primers, colony PCR primers, and sequencing primers can be found in Additional file 4: Methods S3 and Additional file 5: Methods S4. The Fusion PCR method was also used to assemble more than three gRNAs; however, the efficiency of the second round of PCR was sometimes greatly reduced due to persistent non-specific amplifications.
Maize protoplast isolation and transfection
Seeds of B73 maize were immersed in sterile water overnight, sown in soil, and grown under a 16-h light/8-h dark cycle at 22°C in a growth room for 4–6 days. Tissues from the stems and sheaths of 20–30 seedlings were used for protoplast isolation according to a previously described method , with one modification, i.e., the protoplast pellets were collected by centrifugation at 100 × g for 3 min. PEG-mediated transfections were carried out as described . For each sample, 10–15 μg plasmid DNA was mixed with 200 μL protoplasts (approximately 2 × 105 cells). Freshly prepared PEG solution (200 μL) was added and the mixture was incubated at room temperature for 10–20 min in the dark. Subsequently, 800 μL W5 solution was added and mixed, and the protoplasts were pelleted by centrifugation at 100 × g for 3 min. The protoplasts were resuspended in 1.5 mL W5 solution and pelleted by centrifugation at 100 × g for 3 min. The protoplast were then resuspended in 800 μL W5 solution and cultured in the dark at 22°C for 14–16 h. Protoplast transfection was performed with three replicates per plasmid.
Verification of mutations of maize protoplasts
Three transfected protoplast samples from the same vector were pooled and the genomic DNA was extracted. The DNA fragment encompassing the CRISPR target site was amplified from genomic DNA by nested PCR with two pairs of gene-specific primers ZT-IDF0/-IDR0 and ZT-IDF/-IDR (Additional file 1: Table S1). For restriction enzyme digestion analysis of mutations, two restriction enzyme reactions for each PCR product were set up: in one reaction, the corresponding restriction enzyme was added; in the other reaction, the enzyme was replaced by water as a negative control. About 500 ng purified PCR products from each reaction was digested overnight in a 20-μL reaction. Together with the control, digested DNA was separated on a 2.0% ethidium bromide–stained agarose gel. For sequencing analysis of mutations, the purified PCR product was cloned into cloning vector pCBC and the resulting transformants were identified by colony PCR followed by restriction enzyme digestion analysis. Some of the restriction enzymes, such as XcmI and SphI, have activity in Taq PCR mixtures. At the end of the PCR, the enzymes were added to the PCR mixtures for overnight digestion, followed by agarose gel electrophoresis analysis. The digestion-resistant fragments were sequenced using a T7 primer.
Generation of transgenic maize and analysis of mutations
The CRISPR/Cas9 binary vector pBUE-2gRNA-ZH was transformed into Agrobacterium strain EHA105, and Agrobacterium-mediated method was used to transform immature embryos of B73 maize at China Agricultural University Transgenic Facility Center. The genomic DNA was extracted from 20 transgenic seedlings and the PCR fragment, primers and reactions were the same as those described above. For restriction enzyme digestion analysis, about 500 ng purified PCR products from each reaction was digested overnight with XcmI or SphI in a 20-μL reaction volume. For sequencing analysis, the PCR products from two representative transgenic seedlings were cloned into the cloning vector pCBC and positive clones were sequenced using the T7 primer.
Generation of transgenic Arabidopsisplants and analysis of mutations
The p2gR-TRI-A and p2gR-TRI-B vectors were transformed into Agrobacterium strain GV3101/pSoup using the freeze-thaw method, whereas pHSE-2gR-CHLI was transformed into Agrobacterium strain GV3101. Arabidopsis Col-0 wild-type plants were used for transformation via the floral dip method. The collected seeds were screened on MS plates containing 25 mg/L hygromycin. Genomic DNA was extracted from T1 transgenic plants grown in soil. Fragments surrounding the target sites were amplified by PCR using gene-specific primers TRY-IDF/R, CPC-IDF/R, and ETC2-IDF/R (Additional file 1: Table S1). The purified PCR product was cloned into cloning vector pCBC, and DNA from positive clones for each PCR fragment was sequenced using the T7 primer to identify mutations. To screen segregated nontransgenic T2 plants, genomic DNA was extracted from T2 plants grown in soil. With wild-type genomic DNA serving as a negative control and genomic DNA from T1 transgenic plants serving as a positive control, counterselection PCR was performed with three primer pairs, including Hyg-IDF/R and Hyg-IDF2/R2 for the hygromycin-resistance gene and zCas9-IDF/R for zCas9 (Additional file 1: Table S1). To analyze mutations of nontransgenic T2 plants, fragments surrounding the target sites of TRY, CPC or ETC2 were amplified by PCR using gene-specific primers TRY-IDF0/R0, CPC-IDF0/R0, and ETC2-IDF0/R0 (Additional file 1: Table S1). Purified PCR products were submitted for sequencing with primers (TRY/CPC/ETC2-seqF) located within the PCR fragments (Additional file 1: Table S1). Badly sequenced PCR products were then cloned into cloning vector pCBC and DNA from positive clones was sequenced using the T7 primer.
We thank Feng Zhang for the pX3300, Keith Joung for the pJDS246, Guo-Liang Wang for the pXSN and pXUN, Roger Hellens for the pSoup, M. Curtis for the pMDC99/100/123, Shu-Hua Yang for the help in maize protoplast transfection and the colleagues in CAU Transgenic Facility Center for the help in generation of transgenic maize. We would like to thank the native English speaking scientists of Elixigen Company for editing our manuscript. This work was supported by grants from the National Basic Research Program of China (2012CB114200), the National Science Foundation of China (31070329), and the National Transgenic Research Project (2011ZX08009).
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