An Effective Strategy for Reliably Isolating Heritable and Cas9-Free Arabidopsis Mutants Generated by CRISPR/Cas9-Mediated Genome Editing

Abstract

Mutations generated by CRISPR/Cas9 in Arabidopsis (Arabidopsis thaliana) are often somatic and are rarely heritable. Isolation of mutations in Cas9-free Arabidopsis plants can ensure the stable transmission of the identified mutations to next generations, but the process is laborious and inefficient. Here, we present a simple visual screen for Cas9-free T2 seeds, allowing us to quickly obtain Cas9-free Arabidopsis mutants in the T2 generation. To demonstrate this in principle, we targeted two sites in the AUXIN-BINDING PROTEIN1 (ABP1) gene, whose function as a membrane-associated auxin receptor has been challenged recently. We obtained many T1 plants with detectable mutations near the target sites, but only a small fraction of T1 plants yielded Cas9-free abp1 mutations in the T2 generation. Moreover, the mutations did not segregate in Mendelian fashion in the T2 generation. However, mutations identified in the Cas9-free T2 plants were stably transmitted to the T3 generation following Mendelian genetics. To further simplify the screening procedure, we simultaneously targeted two sites in ABP1 to generate large deletions, which can be easily identified by PCR. We successfully generated two abp1 alleles that contained 1,141- and 711-bp deletions in the ABP1 gene. All of the Cas9-free abp1 alleles we generated were stable and heritable. The method described here allows for effectively isolating Cas9-free heritable CRISPR mutants in Arabidopsis.

Figure 1.

Design of a CRISPR/Cas9 vector to facilitate a visual screen for Cas9-free Arabidopsis seeds in the T2 generation. A, Schematic representation of the new CRISPR/Cas9 vector that contains a Cas9 expression cassette driven by the cauliflower mosaic virus (CaMV) 35S promoter and a U6 promoter-controlled gRNA production unit. More importantly, it also expresses mCherry from the strong promoter At2S3 in seeds. NLS, Nuclear localization signal. B, Visual screen for T2 seeds that no longer harbor the CRISPR/Cas9 construct. The Cas9-free seeds do not produce the red fluorescence.

Figure 2.

Generation of abp1 mutants using the mCherry-containing CRISPR/Cas9 editing vector. A, Schematic representation of the ABP1 gene and the sequences of the selected target sites for editing ABP1. PAM sites (NGG or CCN) are highlighted in dark blue. CRP2 and CRP3 target opposite strands of the ABP1 genomic DNA. The restriction enzyme sites used for genotyping and screening for mutations are underlined. BsaJI recognizes CCNNGG, while TaqI cuts TCGA. Note that Cas9 usually cuts 3 bp upstream of the PAM site. Therefore, screening with BsaJI is not optimal. B, Restriction digestion screen of T1 plants transformed with CRP2/CRISPR vector using the enzyme BsaJI. Plants with mutations generate PCR bands resistant to BsaJI digestion (arrow). Among the 15 samples shown, four potentially have been edited at the ABP1 locus (3, 5, 11, and 14). C, Restriction digestion of PCR products from T1 plants that have disrupted the TaqI site at the CRP3 target site. Note that sample 75 has very little wild-type (WT) DNA. The arrow points to a TaqI-resistant PCR band. D, Three abp1 mutants with deletions/insertions at the CRP2 target site. abp1-c4d has a 4-bp deletion, and abp1-c3d has a 3-bp deletion; abp1-c8i has a very complex mutation. E, Two editing events at the CRP3 site that resulted in two stable Cas9-free abp1 alleles. One has a 12-bp deletion and the other deletes 42 bp near the target site. Note that the 42-bp deletion is not shown in full.

Figure 3.

CRISPR/Cas9-mediated deletions of a large DNA fragment between two gRNA target sites in Arabidopsis. A, We produced CRISPR plasmids that target three sites of the ABP1 gene. We combined the RGR and CRP2 modules to delete the first three exons. We also combined the RGR and CRP3 modules in another plasmid. RGR is controlled by the UBQ10 promoter. Green boxes refer to ABP1 exons. Vertical arrows point to gRNA target sites. ABP1-U409 and ABP1-CRP2-GT2 are the primer pair used in the PCR screening. The RGR sequence and design are shown in Supplemental Figure S2. B, PCR amplification using ABP1-U409 and ABP1-CRP2-GT2 primers and the genomic DNA from Cas9-free T2 plants generated from a single T1 plant transformed with the RGR-CRP2 dual gRNA vector. About half of the plants contained a deletion. Note that this primer pair preferentially amplifies the small fragment and cannot differentiate homozygous from heterozygous plants. C, Schematic representation of the abp1-c2 mutation, which is a deletion of 1,141 bp including the first three exons and 304 bp of the ABP1 promoter. The dashed line represents the deleted region. D, Identification of a second abp1 allele that has a large deletion. Only two plants (105 and 115) out of 96 Cas9-free T2 plants from a single RGR-CRP2 T1 plant contained a deletion (arrows). E, Further sequencing analysis shows that the deletion is 711 bp, which is the exact expected size generated by gRNAs targeting RGR and CRP2 sites.

Figure 4.

Reliably isolating stable and heritable targeted mutants using CRISPR/Cas9 genome-editing technology in Arabidopsis. A, Flow chart for isolating CRISPR alleles of Arabidopsis mutants. The key is to use the visual screen to quickly identify Cas9-free T2 seeds. Mutations in Cas9-free T2 plants are stably transmitted to next generations following Mendelian genetics (Supplemental Table S1). B, Schematic representation of the mosaic nature of mutations generated by CRISPR/Cas9 in T1 plants. If a founder cell for a flower is mutated, the seeds generated from that particular flower will contain heritable mutations (blue or purple). However, seeds in the majority of the siliques do not contain heritable mutations. Red refers to seeds with the mCherry-CRISPR/Cas9 construct.