Cas12a-mediated gene targeting by sequential transformation strategy in Arabidopsis thaliana

Abstract

Gene targeting (GT) allows precise manipulation of genome sequences, such as knock-ins and sequence substitutions, but GT in seed plants remains a challenging task. Engineered sequence-specific nucleases (SSNs) are known to facilitate GT via homology-directed repair (HDR) in organisms. Here, we demonstrate that Cas12a and a temperature-tolerant Cas12a variant (ttCas12a) can efficiently establish precise and heritable GT at two loci in Arabidopsis thaliana (Arabidopsis) through a sequential transformation strategy. As a result, ttCas12a showed higher GT efficiency than unmodified Cas12a. In addition, the efficiency of transcriptional and translational enhancers for GT via sequential transformation strategy was also investigated. These enhancers and their combinations were expected to show an increase in GT efficiency in the sequential transformation strategy, similar to previous reports of all-in-one strategies, but only a maximum twofold increase was observed. These results indicate that the frequency of double strand breaks (DSBs) at the target site is one of the most important factors determining the efficiency of genetic GT in plants. On the other hand, a higher frequency of DSBs does not always lead to higher efficiency of GT, suggesting that some additional factors are required for GT via HDR. Therefore, the increase in DSB can no longer be expected to improve GT efficiency, and a new strategy needs to be established in the future. This research opens up a wide range of applications for precise and heritable GT technology in plants.

Keywords: Arabidopsis thaliana; Cas12a; Enhancer; Gene targeting; Genome engineering; Sequential transformation.

Fig. 1

Overview of sequential transformation strategies for Cas12a/ttCas12a-mediated GT establishment. A Generation of parental lines. Parental line construct contains a Cas12a/ttCas12a cassette driven by the DD45 promoter, a crRNA targeting the intergenic region driven by the AtU6 promoter, and a hygromycin selection marker gene cassette driven by the 35S promoter. Cas12a/ttCas12a expression constructs were transformed into Col-0 accession by Agrobacterium to generate parental lines. Screening of T1 transgenic parental lines with 50 mg/L hygromycin yielded approximately 35–40 individual lines. To evaluate the obtained parental line candidates, mutation frequencies in the target intergenic region, Cas12a/ttCas12a copy number, and hygromycin-resistant phenotype of the progenies were examined. B Sequential transformation strategy for GT establishment. Parental lines with greater hygromycin resistance, higher mutation frequency, and lower copy number of Cas12a/ttCas12a were used for sequential transformations. The donor constructs used for sequential transformation contain a crRNA cassette at the target locus driven by the AtU6 promoter, a repair donor template for the HDR, and a cassette of herbicide resistance marker gene bar driven by the 35S promoter. T1 transformants of the donor constructs were first screened with Basta spray and then genotyped to obtain GT-positive events

Fig. 2

Evaluation of Cas12a/ttCas12a parental lines in Arabidopsis. A Schematic diagram of parental line constructs. Cas12a/ttCas12 represents a parental construct containing without enhancer, dCas12a/dttCas12 represents a parental construct containing the dMac3 translational enhancer, UCas12a/UttCas12a represents a parental construct containing the AtUbq10 transcriptional enhancer, and UdCas12a/UdttCas12 represents a parental construct containing both enhancers. The blue rectangular block and black line represent the 5' UTR sequence transcriptional enhancer of AtUbq10 with the first intron. Magenta square indicates the dMac3 translational enhancer. B Proportion of mutation frequencies in individual T1 generations of the eight parental line constructs. Mutation frequency of intergenic target site was determined by T7EI digestion assay. C Statistical analysis of mutation frequency in parental lines. The numbers in parenthesis represent the number of individual samples analyzed. One-way ANOVA and Tukey test were applied to analyze standard differences (P < 0.05). D Hygromycin-resistant phenotypes in different generations. Homozygous transgenic lines of UdCas12a-#14–23 and UdttCas12a-#8–31 were selected in the T2 to T6 generation (Supplementary Figure S4). Seeds were germinated on 1/2 MS plates containing 50 mg/L hygromycin. Col-0 and CS69955 (previously reported Cas9 parental line) were used as controls

Fig. 3

Characterization of GFP-KI events at the EMB2410 locus. A Diagram of donor construct for sequential transformation. The donor template has a GFP-KI fragment with 1 Kbp long homology arms at the EMB2410 target locus. Black arrows indicate full-length primers used for genotyping, green arrows in GFP fragments indicate specific primers. B PCR genotyping of precise GFP-KI at the EMB2410 target locus in T1 transformants. C Correlation analysis of mutation frequency at the target intergenic region in parental lines and precise GFP-KI GT efficiency at the EMB2410 locus. D Heritable GFP-KI GT in UdttCas12a-#8–152 T2 generation. Col-0 was used as a control. M; DNA size marker

Fig. 4

Statistical analysis of DSB and GT efficiency at the EMB2410 locus. A, B, Mutation frequency of GT-negative GFP-KI at EMB2410 locus transgenic plants in Cas12a (A) and ttCas12a (B) parental lines. Mutation frequencies were determined by PCR with full-length primers, sequencing and TIDE. The numbers in parenthesis represent the number of individual samples analyzed. The lines show mean with SD of individual values. One-way ANOVA and Tukey test were applied to analyze standard differences (P < 0.05). C, D, Correlation analysis of mutation frequency at EMB2410 locus and Cas12a/ttCas12a copy number in Cas12a (C) and ttCas12a (D) parental lines. The Cas12a/ttCas12a copy number was determined by q-PCR in T1 parental line plants and calculated by the 2^−∆∆CT method. Actin7 was used as an internal reference. E, F, Correlation analysis of mutation frequency and GFP-KI GT efficiency at the EMB2410 locus in Cas12a (E) and ttCas12a (F) parental lines. Squares indicate precise GT, triangles indicate total HDR. G, H, Correlation analysis of precise and total HDR efficiency in Cas12a (G) and ttCas12a (H) parental lines

Fig. 5

Characterization of amino acid substitution at the ALS locus. A Overview of efficient screening for amino acid substitution GT events at the ALS locus. T1 transformants were screened with Basta spray, followed by imazethapyr spray to screen for amino acid substitution GT events and further genotyping. B Detail of ALS substitution. The donor template harbors S653I and G654E substitutions, which also overlap the PvuI restriction enzyme site and are flanked by 2 Kbp homology arms. The black arrows represent the full-length primer for ALS genotyping. Red arrow indicates the target site of crRNA for the ALS locus. Bold font in blue box represents bases replacement and amino acid substitution. Red letters indicate PAM. C Phenotypes of Basta and imazethapyr spray screening. The image on the left shows the primary screening with Basta spray on T1 generation transformants. On the right is the second screening by imazethapyr spraying. D PCR genotyping of precise ALS S653I, G654E in T1 transformants. PCR-based genotyping was performed on the imazethapyr-resistant plants. Full-length primers were used for PCR followed by PvuI enzyme digestion. Three parental lines, ttCas12a-#5, UttCas12a-#19, and UdttCas12a-#8, were used for ALS base substitution GT by sequential transformation strategy. E Inheritance of the precise S653I and G654E substitution GT events at the ALS locus. Col-0 was used as a control. M; DNA size marker