Improving the efficiency of the CRISPR-Cas12a system with tRNA-crRNA arrays

Xixun Hu, Xiangbing Meng, Jiayang Li, Kejian Wang*, Hong Yu*

aState Key Laboratory of Rice Biology, China National Rice Research Institute, Chinese Academy of Agricultural Sciences, Hangzhou 310006,Zhejiang,China

bState Key Laboratory of Plant Genomics and National Center for Plant Gene Research (Beijing), Institute of Genetics and Developmental Biology,The Innovative Academy of Seed Design,Chinese Academy of Sciences,Beijing 100101,China

cUniversity of Chinese Academy of Sciences,Beijing 100049,China

Keywords:crRNA CRISPR-Cas12a tRNA-crRNA array Genome editing Editing efficiency

ABSTRACT CRISPR-Cas12a offers a convenient tool for multiplex genome editing in rice. However, the CRISPR-Cas12a system displays variable editing efficiency across genomic loci,with marked influence by CRISPR RNAs(crRNAs).To improve the efficiency of the CRISPR-Cas12a system for multiplex genome editing,we identified various architectures and expression strategies for crRNAs. Transformation of binary vectors loaded with engineered CRISPR-Cas12a systems into rice calli and subsequent sequencing revealed that a modified tRNA-crRNA array not only efficiently achieved rice multiplex genome editing, but also successfully edited target sites that were not edited by the crRNA array. This improvement contributes to the application of the CRISPR-Cas12a system in plant genome editing, especially for genomic loci that have hitherto been difficult to edit.

1.Introduction

CRISPR-Cas12a (also known as Cpf1) is becoming the prevalent genome editing tool in animals and plants, owing to its unique features [1-3]. It differs from CRISPR-Cas9, which recognizes G-rich (mostly NGG) protospacer adjacent motifs(PAMs); CRISPR-Cas12a recognizes T-rich PAM regions [4],greatly expanding the range of genome editing. In addition,Cas12a generates sticky ends at PAM-distal bases, allowing more accurate and efficient operations mediated by CRISPRCas12a, such as gene replacement based on recombination repair.For multiplex genome editing,Cas12a can process precrRNAs, allowing different crRNAs to be arranged as an array under a promoter. Moreover, the short crRNA of CRISPRCas12a increases flexibility in vector construction. Thus,CRISPR-Cas12a offers advantages over CRISPR-Cas9 in multiplex editing[2,3].

Editing efficiency is crucial for the application of genome editing techniques. To achieve efficient multiplex genome editing with CRISPR-Cas12a, two crRNA-expressing systems,the tRNA-crRNA array and the crRNA array, have been developed in both rice and human cells [1-3]. The tRNAcrRNA array, previously developed for the CRISPR-Cas9 system in multiplex genome editing, employs the tRNA maturation process to release crRNAs [5]. The crRNA array produces crRNAs depending on the pre-crRNA processing ability of Cas12a [3]. Our previous study successfully employed tRNA-crRNA arrays in a rice CRISPR-Cas12a system[1],and similar systems in mammalian cells[6]increased the efficiency of genome editing.However,the tRNA-crRNA array faces a serious problem. The 3′ end of crRNA is crucial for accurately interrogating DNA targets by DNA-RNA pairing.Several mismatches in this region will dramatically weaken the efficiency of CRISPR-Cas12a [1]. In the tRNA-crRNA array,the tRNA maturation process will leave 1-4 residual bases at the 3′ end of crRNAs lying between two tRNAs [5], and will thereby reduce the efficiency of the CRISPR-Cas12a system.The present study aimed to modify the tRNA-crRNA system to overcome this deficiency.

2. Materials and methods

2.1. Screening and designing crRNAs

Target sites (H1-H3) for FnCas12a (Cas12a from Francisella novicida) were selected from previously reported studies [2,7].Target sites that failed to be edited (N1-N4) were from our previous study [1], in which none of them was modified.Matured crRNA in our test included a 19-nt matured DR(direct repeat)and a 23-nt spacer.The longer DR included a 36-nt pre-DR and a 23-nt spacer,and the longer spacer crRNA included a 19-nt DR and a 30-nt spacer (Fig. 1-A). The DR or pre-DR sequences were based on original sequences in their CRISPRCas12a systems.

2.2. Binary vector construction and generation of transgenic rice

For the crRNA expression cassette, crRNAs were directly arrayed in series in the crRNA array (Fig.1-B),whereas tRNAcrRNA units,which included a crRNA between two tRNAs(Fig.1-C), were generated and organized in series in tRNA-crRNA array. All arrays were synthesized and constructed under a rice U3 promoter to build crRNA expression cassettes. The binary vector used contained a crRNAs expression cassette and a Cas12a expression cassette, in which a rice-optimized sequence of Cas12a was under the OsACTIN1 promoter. As a host plant, the rice cultivar Nipponbare was used. Transformation was performed by Agrobacterium-mediated method with the strain EHA105[8].

2.3. Detection of mutations

Total DNA of leaves was extracted by the cetyltrimethyl ammonium bromide method[1].DNA fragments were amplified by PCR with KOD FX DNA Polymerase (TOYOBO, Osaka,Japan) and specific primers are listed in Table S1. To detect mutations around target sites, Sanger sequencing was employed, and multiple peaks produced by heterozygous biallelic mutations, monoallelic mutations, and chimeras were decoded by the DSD method[9].

3. Results

3.1. Longer crRNAs exert different effects on rice genome editing efficiencies

Previous studies revealed that additive bases in the spacer or direct repeat (DR) of crRNA could work as protecting nucleotides for the spacer that paired with target DNA or to improve the nuclease activity of crRNA-Cas12a complexes in rice [10].However in human cells, matured crRNA, which is shorter,performed better in multiplex editing [3]. We first compared the efficiency of three kinds of crRNA: matured crRNA (19-nt DR and 23-nt spacer),longer spacer crRNA(19-nt DR and 30-nt spacer), and longer DR crRNA (36-nt DR and 23-nt spacer), to investigate the effects of various crRNA architectures in rice(Fig. 1-A). We generated the crRNA array to express crRNAs targeting three reported target sites (H1-H3) on OsRLK-798,OsRLK-802, and OsALS (Fig. 1-B, D) [2,7]. The rice ACTIN1(ACT1)promoter was used to express FnCas12a because it was more efficient than the 35S promoter with the CRISPR-Cas9 system in our previous study [11]. After selection of hygromycin B and regeneration, we obtained transgenic seedlings of 46 lines with matured crRNAs, 41 lines with the longer spacer crRNA,and 48 lines with the longer DR crRNA in three replicates (Table S2). Sequencing results showed that the editing efficiencies of the longer spacer crRNA and the longer DR crRNA were increased at H1, reduced at H2 and showed no changes at H3 compared to the editing efficiency of matured crRNAs (Figs. 1-E, S1). The influence of crRNA architecture on efficiency was also evident in mutation-type analysis(Table S3).Besides,double-mutation rates rose,while triple-mutation rates decreased with longer-spacer crRNAs or longer-DR crRNAs.These results suggested that longer-spacer crRNAs exert stronger effects than longer-DR crRNAs.

3.2. A modified tRNA-crRNA array is efficient for multiplex genome editing in rice

Previous studies in rice revealed that AACA of the 5′ end of tRNA sequences limited the targets suitable for genome editing [1], hindering the application of this system in Cas12a-mediated genome editing. Because RNase P and RNase Z recognize tRNA structures rather than its sequences[12,13], we hypothesized that the AACA is not essential for tRNA excision. Accordingly, we shortened the tRNA by removing the AACA from the 5′ end of tRNA sequence (Fig.S2)and extended the spacer from 19 nt to 23 nt.

Compared with the previous tRNA-crRNA array which would add extra bases to the 3′ end of crRNAs, we expected that this shortened tRNA-crRNA array could produce completed crRNAs and crRNAs with slightly shortened 3′ ends that would completely match DNA targets and effectively induce the cleavage activity of Cas12a. We accordingly generated modified tRNA-crRNA arrays for expressing crRNAs, where each crRNA was inserted between two tRNAs(Fig.1-C).With FnCas12a,all targets showed high(from 29.2%to 55.6%) mutation rates (Fig. 1-F). The overall editing efficiencies of modified tRNA-crRNA array were comparable to that of the crRNA array at a single target site,although the crRNA array generated more homozygotes and heterozygous bialleles (Table S3, Fig. S3). The modified tRNA-crRNA array also generated more double mutations,while the crRNA array generated more triple mutations (Fig. 1-F, Table S4). Taken together, these results showed that the tRNA-crRNA array was efficient for single and multiplex genome editing in rice.

Fig.1- Editing efficiencies of FnCas12a with different architectures and expression strategies of crRNA at H1,H2,and H3.(A)Various crRNA architectures.Red rhombus indicates the full-length DR(36 nt),while the half rhombus indicates the matured DR sequence(19 nt).The light and dark blue circles indicate respectively a matured spacer(23 nt)and lengthened spacer(30 nt).(B)and(C)Expression strategies of crRNAs,the crRNA array(B)and tRNA-crRNA array(C).crRNAs containing two parts shown in Panel A are indicated by rectangles,and different colors are used for highlighting different crRNAs.The purple letter T and gray pentagon indicate respectively tRNA and poly-T.(D)Locations of H1,H2,and H3.PAMs are highlighted in red.(E)Editing efficiencies of lengthened crRNAs.(F) Editing efficiencies of different expression strategies of crRNAs.Values are mean ± SEM for three independent experiments.Asterisks indicate significant differences by two-tailed Student's t-test(*P ＜0.1, **P ＜0.05).

3.3. Modified tRNA-crRNA arrays overcome the failure of genome editing

In our previous study, four target sites (N1-N4) failed to be edited with CRISPR-LbCas12a using an unmodified tRNAcrRNA array [1] (Fig. 2-A, Table S1). In the present study, N1 and N2 were successfully edited with CRISPR-FnCas12a using the crRNA array, with mutation rates of 5.9% and 13.0%,respectively. One plant showed double mutations (3.7%) (Fig.2-B, Table S5). Use of modified tRNA-crRNA arrays in the CRISPR-FnCas12a system improved editing efficiency even further,with three of four target sites(N1,N2 and N3)showing mutations (Fig. S4) at rates of 28.8% in N1, 17.8% in N2, and 1.5% in N3. Six plants displayed double mutations (9.3%), a higher rate than that from the crRNA array(Fig.2-B,Table S5).However, no mutation was detected at N4, and most mutations at N1-N3 produced by FnCas12a were monoalleles with either the crRNA or the tRNA-crRNA array(Table S6).

To further confirm this result, a similar test was performed with LbCas12a. Transformation generated 85 lines with crRNA arrays and 75 lines with tRNA-crRNA arrays in three replicates.DNA sequencing showed that no mutation was detected with the crRNA array among the four target sites,whereas two target sites were successfully edited using the tRNA-crRNA array(19.8%in N1 and 1.3% in N2) (Fig. 2-C, Table S7). All of these results demonstrate that the modified tRNA-crRNA array can substantially improve the genome editing performance of the CRISPRCas12a system at target sites that are difficult to edit.

4. Discussion

At present,CRISPR-Cas12a is the prevalent system used for plant genome editing[14,15].In particular,the application of FnCas12a in plants expanded the available PAMs of CRISPR-Cas12a systems from TTTV to TTV PAMs[7,16].Owing to its lack of a requirement of trans-activating crRNA, the expressional cassette of a crRNA targeting multiple genes can be fairly short, facilitating its construction and expression. Although the CRISPR-Cas12a system offers advantages in vector construction for multiplex genome editing,its efficiency varies across target sites,impeding its application in rice. The crRNA array was firstly reported by Zetsche et al.for CRISPR-Cas12a system and rapidly became the system of choice for multiplex genome editing [3]. The crRNA array is relatively short,given that it requires no other sequence allowing Cas12a to recognize the structure of DR and process it for single crRNA.It thus eases the synthesis and assembly of more crRNAs.Our previous study reported genome editing with Cas12a using a tRNA-crRNA array in rice [1]. In the tRNA-crRNA array,each crRNA was inserted in the interval between two tRNAs.After transcription, the tRNA would form a secondary structure that could be recognized and cleaved by RNase P and Z to release crRNAs. Previous studies in mammalian cells also showed the tRNA-crRNA can enhance the efficiency of genome editing [6].Besides, the tRNA was also used for simplifying CRISPR-Cas9 or Cas12a systems,with a tRNA-gRNA array assembled behind Cas9 or Cas12a under the same promoter[17,18].

Fig.2- Efficiencies of FnCas12a and LbCas12a with two expression strategies of crRNAs at target sites difficult to edit.(A)Genomic loci of N1 to N4.PAMs are highlighted in red.(B)Efficiencies of FnCas12a at N1-N4.(C)Efficiencies of LbCas12a at N1-N4.Values are mean ± SEM for three independent experiments. Asterisks indicate significant differences by two-tailed Student's t-test(*P ＜0.1,**P ＜0.05).

To increase efficiency of the CRISPR-Cas12a system,especially at target sites that have been hard to edit, we investigated several architectures and expression strategies of crRNAs. Longer crRNAs, whether with longer DR or longer spacer, produced different effects at different target sites, in apparent dependence on the position of crRNAs. In the present study, we also tested two expression strategies for crRNAs: the crRNA array and the tRNA-crRNA array. Both displayed high efficiency in rice multiplex genome editing.However, in comparison with the crRNA array, the tRNAcrRNA array showed higher efficiencies at previously uneditable target sites, possibly owing to different abundances of Cas12a and RNase P/Z as well as to tRNA's internal promoter elements (boxes A and B). Adequate RNase P/Z allows tRNA-crRNA arrays to be processed more effectively and form more functional complexes of Cas12a-crRNA. The boxes A and B can directly recruit RNA polymerase III(Pol III),increasing the transcription of tRNA-crRNA arrays [5]. In summary, our result offers a better solution for simultaneously targeting rice genomic loci that have hitherto been difficult to edit.

Declaration of Competing Interest

The authors declare that they have no competing interests to disclose.

Acknowledgments

This work was funded by the National Key Research and Development Program of China (2016YFD0101800), the Agricultural Science and Technology Innovation Program of Chinese Academy of Agricultural Sciences, and the National GMO New Variety Breeding Program of China(2016ZX08011-001).

Appendix A. Supplementary data

Supplementary data for this article can be found online at https://doi.org/10.1016/j.cj.2019.06.007.