Multiplex gene editing vector design based on the CRISPR/Cpf1 and CRISPR/Cas9 systems in maize
To evaluate the performance of the multiplex gene editing CRISPR/Cpf1 system in maize, we compared its efficiency against that of the CRISPR/Cas9 system using the same codon-optimization strategy for their better expression in maize. We cloned a maize RNA polymerase (Pol) III promoter as pZmU6 to drive crRNAs for the CRISPR/Cpf1 system or sgRNAs for the CRISPR/Cas9 system also for their better expression in maize, together with tZmU6 (Fig. 1a, Additional file 1: Fig. S1). In these expression cassettes, we designed and synthesized the sequence of the crRNA expression cassette for the CRISPR/Cpf1 system and the sgRNA expression cassette for the CRISPR/Cas9 system (Fig. 1b, Additional file 1: Fig. S1). Multiple crRNAs were separated by a simple short DR-based unit for the CRISPR/Cpf1 system, and multiple sgRNAs were separated by a tRNA-based unit for the CRISPR/Cas9 system (Fig. 1a).
The maize O2 gene is a gene involved in mutant kernels with the opaque phenotype. We used it as the target gene to examine the feasibility of the CRISPR/Cpf1 and CRISPR/Cas9 systems. We used CRISPR-GE (http://skl.scau.edu.cn/) to select target sites [32]. For a fair comparison, we selected target sites in the same region of the O2 gene locus as close as possible. The regions including three targets of the CRISPR/Cpf1 and CRISPR/Cas9 systems substantially overlapped (Fig. 1c). After altering series of effector factors, including guanine-cytosine content, region and potential off-target sites, as well as pairing with crRNA (sgRNA in the CRISPR/Cas9 system), we targeted three sites in the 2nd exon of the O2 gene with both the CRISPR/Cpf1 and CRISPR/Cas9 systems.
Acquisition of transgenic plants for the CRISPR/Cpf1 and CRISPR/Cas9 systems
For the CRISPR/Cpf1 and CRISPR/Cas9 systems, we obtained five transgenic lines by the Agrobacterium-mediated maize transformation method. We chose three lines that had a sufficient number of progenies. At the T0 generation, two regenerated transgenic plants per line were examined. We utilized specific primers for the bialaphos resistance (Bar) gene to verify all transgenic plants of the two systems (Additional file 1: Fig. S3, Table. S1).
The transcript expression levels of Cpf1 and Cas9 nucleases were analyzed by qRT-PCR (Fig. 2a, 2b, Additional file 1: Table. S1). We found that the expression levels of Cas9 in line 1 and line 3 were similar, while they were approximately sixty times higher in line 2 (Fig. 2a, Additional file 1: Fig. S4b). For Cpf1, the expression level in line 1 was the highest, followed by line 2 and line 3 (Fig. 2b, Additional file 1: Fig. S4c). Transcript expression verified the transgenic plants, and the expression levels contained differences between lines.
CRISPR/Cpf1 systems showed lower target gene editing efficiency than CRISPR/Cas9 in the T0 and T1 generations
To compare the editing efficiency between the CRISPR/Cpf1 and CRISPR/Cas9 systems, genomic DNA was isolated from leaf tissues of T0 plants. A 1481 bp region of O2 including all targets was amplified by PCR using primers ZmO2-exon2-F and R for sequencing and assembly (Additional file 1: Table. S1). The PCR products were either directly subjected to Sanger sequencing or cloned into pMD18-T and then subjected to colony sequencing. Sequencing results showed that all three lines of the CRISPR/Cas9 system contained editing results. Among the three lines, the 1st target was vulnerable to editing; for instance, it contained a 1 bp (T) deletion or insertion in different plants. The 2nd target contained a 1 bp (T) insertion and 21 bp deletions displayed on the 3rd target (Fig. 3a, Table 1). However, in the CRISPR/Cpf1 system, only the two seedlings in line 1 contained a 12 bp insertion between the 1st and 2nd targets (Fig. 3b, Table 2). Therefore, in maize, the CRISPR/Cas9 system is more effective than CRISPR/Cpf1 for multiplex gene editing in the T0 generation.
We then chose each of three lines from the CRISPR/Cpf1 and CRISPR/Cas9 systems for the T1 generation. The T1 progenies were produced by crossing the pollen of T0 lines to the W22 inbred line. At the T1 seedling stage, gene editing of the O2 gene was analyzed by PCR and sequencing analysis. Consistent with our expectation, all three lines from the CRISPR/Cas9 system contained editing results. The results showed that nineteen of thirty seedlings of line 3 contained editing results at the highest editing efficiency. The secondly high editing efficiency was in line 1; fifteen of thirty seedlings contained editing results. Eleven of all thirty seedlings of line 2 contained editing results (Additional file 1: Fig. S4b). Among the three lines, we verified that a number of mutations were inherited from the last generation, for instance, a 1 bp (T) deletion and insertion (Fig. 3a, Table 1). Furthermore, one plant newly contained a 1 bp (T) deletion on the 1st target and a 1 bp (T) insertion on the 2nd target as well as a 1 bp (G) insertion on the 3rd target (Fig. 3a, Table 1).
In terms of the CRISPR/Cpf1 system, four out of all thirty seedlings of line 1 contained editing results. As mentioned in the T0 generation, the same editing result, a 12 bp fragmental insertion, was observed, indicating that it was inherited (Fig. 3b, Table 2). Three out of all thirty seedlings of line 2 contained editing results, which indicated that there were no mutations in the T0 generation, and new mutations, such as a 3 bp deletion and a 2 bp insertion between the 2nd and 3rd targets, were generated in the T1 generation (Fig. 3b, Table 2). Line 3 continued to not contain editing result in the T1 generation (Additional file 1: Fig. S4c). Thus, the CRISPR/Cpf1 system still shows lower editing efficiency than the CRISPR/Cas9 system in the T1 generation. The results illustrated that the editing positions of the CRISPR/Cpf1 system mostly occurred between the two targets instead of within the common on-target region. Furthermore, the Cas9 nuclease has no relationship between editing efficiency and its transcript levels, while the editing efficiencies of each line in the CRISPR/Cpf1 system were directly and proportionally correlated with the level of Cpf1 nuclease transcript expression (Additional file 1: Fig. S4b, Fig. S4c).
Table 1
The DNA sequence of mutations displayed in the CRISPR/Cas9 system.
| Target sequence 1 (5’-3’) | In/Del | Symbol |
WT | GGAGATCCTCGGGCCCTTCTGGG | / | / |
T0 | GGAGATCCTCGGGCC———CTGGG | -3 bp | D3a* |
GGAGATCCTCGGGCCCTTTCTGGG | + 1 bp | I1a* |
GGAGATCCTCGGGCCCT—CTGGG | -1 bp | D1a* |
T1 | GGAGATCCTCGGGC———TCTGGG | -3 bp | D3b |
GGAGATCCTCGGGCCAT—CTGGG | R;-1 bp | R; D1b |
GGAGATCCTCGGGCC——TCTGGG | -2 bp | D2 |
GGAGATCCTCGGG———————GGG | -7 bp | D7 |
GGAGATCCTCGGGCC——······—— | -23 bp | D23 |
TATT——·······················——CCAC | -60 bp | D60 |
GTGG——·······················——GGTG | -200 bp | D200 |
| Target sequence 2 (5’-3’) | In/Del | Symbol |
WT | GTGGACCTTTGAGAGGTTACTGG | / | / |
T0 | GTGGACCTTTGAGAGGTTTACTGG | + 1 bp | I1b* |
GTGGACCTT—————————ACTGG | -9 bp | D9* |
T1 | GTGG——··························——GGTG | -200 bp | D200 |
| Target sequence 3 (5’-3’) | In/Del | Symbol |
WT | GGTAATGATGGCGCCTGCGGCGG | / | / |
T0 | ———·····················———GCGG | -21 bp | D21 |
T1 | GGTAATGATGGCGCCTGGCGGCGG | + 1 bp | I1c |
GGTAATGATGGCGCCTGACGGCGG | + 1 bp | I1d |
GGTAATGATGGCGCCT—CGGCGG | -1 bp | D1c |
I/D, insertions and deletions. R, replacement. Different I/D results are numbered 1(then 2, 3 ...). The PAM sequence, base insertions and base replacements are bolded, base deletions are marked with dashes. These symbols linked with Fig. 3a.
Table 2
The DNA sequence of mutations displayed in the CRISPR/Cpf1 system.
| Sequence (5’-3’) | In/Del | Symbol |
WT M1 | AGAGCCAGAGCGAGAGC AGAGCCAGAGCCAGAGCCAGAGCGAGAGC | + 12 bp | I12* |
WT M2 | GTATATACACTGCTCGCTCTT GTATATATACAATGCTCGCTCTT | R1;+2 bp | Ra; I2* |
WT M3 | CGGTGGTGGTGGTGCCGAA CGGTGGTGGTG———CCGAA | -3 bp | D3 |
WT M4 | TCCCTTTCTTGACCTTTGCTT TCCCTT—CTTGACCTTTGCTT | -1 bp | D1a |
WT M5 | ACCTTTGCTTGGAACCATTGA ACCTTTGCATG—AACCATTGA | R2;-1 bp | Rb; D1b |
WT M6 | TCTGGGAGCTGCTACCACCG TCTGGGAGCTGCTAC—ACCG | -1 bp | D1c |
WT M7 | CCGAC··············AGAGCGAG CCGAC—·······—AGAGCGAG | -200 bp | D200 |
WT M8 | GCTGCTGGTCATGGTGACGG GCTGCT—GTCATGGTGACGG | -1 bp | D1d |
WT M9 | TTTGAGAGGTTACTGGAAGAGGAG TTTGAGAGGTTACTGAAAGAGGAG | R3 | Rc |
WT M10 | CGGTGGTGGTGGTGCCGAAC CGGTGGTGGTG—TGCCGAAC | -1 bp | D1e |
I/D, insertions and deletions. R, replacement. Different I/D results are numbered 1(then 2, 3 ...). The PAM sequence, base insertions and base replacements are bolded, base deletions are marked with dashes. These symbols linked with Fig. 3b.
To summarize, we managed to perform multiplex gene editing with the CRIPSR/Cpf1 system with multiple crRNAs and compared with that of the CRISPR/Cas9 system. Our results showed that the editing efficiency based on multiple gRNAs of the CRISPR/Cas9 system was superior to that of multiple crRNAs of the CRISPR/Cpf1 system.
CRIPSR/Cpf1 generated more types of new mutations in the T2 generation
According to the sequencing results in the T0 and T1 generations, the CRISPR/Cpf1 system showed relatively poor editing efficiency and uncommon editing patterns. Compared to other CRISPR systems, whose editing sites were usually on-target regions, its editing sites located between the two targets. Moreover, it precisely protected the target sites from cleavage and could implement continuous editing in the next generation.
To further explore the value of the CRISPR/Cpf1 system in maize, we further examined four ears of each line edited by the CRISPR/Cpf1 system in the T2 generation. Half of ears of line 1 and 2 contained editing results, while other ears were not. No editing events were found in line 3 ears. To evaluate the editing efficiency of the CRISPR/Cpf1 system, we selected eight kernels of each ear for plantation. We found that the CRISPR/Cpf1 system created more types of new mutations (Fig. 3b, Additional file 1: Fig. S4a, Fig. S4c). For instance, a 1 bp deletion was displayed between the 1st and 2nd targets and between the 2nd and 3rd targets as well as on the 1st target (Fig. 3b, Table 2). It also created a base replacement on the 1st target and 2nd target (Fig. 3b, Table 2). Surprisingly, there was a 200 bp longer fragmental deletion including the 1st target (Fig. 3b, Table 2). In short, for the CRISPR/Cpf1 system, we observed more types of new mutations in the next generation.