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Acta Agronomica Sinica ›› 2024, Vol. 50 ›› Issue (10): 2425-2434.doi: 10.3724/SP.J.1006.2024.43012

• CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS •     Next Articles

Evaluation of editing efficiency of different CRISPR-Cas12f systems

HUANG Ling-Zhi1,2(), FU Xiao2, QI Xian-Tao2, LIU Chang-Lin2, XIE Chuan-Xiao2, WU Peng-Hao1, REN Jiao-Jiao1,*(), ZHU Jin-Jie2,*()   

  1. 1College of Agriculture, Xinjiang Agricultural University, Urumqi 830052, Xinjiang, China
    2Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing 100081, China
  • Received:2024-03-15 Accepted:2024-06-20 Online:2024-10-12 Published:2024-07-11
  • Contact: *E-mail: renjiaojiao789@sina.com;E-mail: zhujinjie@caas.cn
  • Supported by:
    National Key Research and Development Program of China(2023YFD1202901)

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Abstract:

CRISPR/Cas12f proteins belonging to the Type V-F family are reported to be only 1/4 to 1/3 the size of Cas9 protein molecules, providing a significant advantage in viral vector delivery. However, the CRISPR/Cas12f system for gene editing in plants has been reported to have lower editing activity, limiting its broader application in plant research. In this study, we compared the editing activities of OsCas12f, SpCas12f, and UnCas12f in three different systems: in vitro digestion, yeast, and transient expression in maize protoplasts. The results showed that the editing activities of OsCas12f and SpCas12f proteins were comparable in terms of in vitro digestion of Cas12f/sgRNA complexes, while no substrate digestion activity was detected for UnCas12f. In the yeast mutant eGFP expression restoration assay, OsCas12f exhibited an editing efficiency of over 95% at the two tested loci, which was comparable to Cas12i.3. On the other hand, SpCas12f achieved editing efficiencies of 1.63% and 3.20% at the two sites, respectively, representing the next highest effect. However, UnCas12f showed minimal editing activity. Furthermore, by transiently expressing maize protoplasts, we compared the editing efficiencies of OsCas12f and SpCas12f at endogenous maize loci. It was found that OsCas12f successfully mediated targeted editing at two loci with editing efficiencies of 2.72% and 1.97%, respectively, while SpCas12f only mediated targeted editing at one locus with an editing efficiency of 1.09%. Deletion of bases was the predominant type of mutation introduced by Cas12f proteins at the target loci, with deletion lengths ranging from -9 to -17 base pairs. These comprehensive results indicate that OsCas12f can serve as a versatile tool for developing plant microgene editors and related technologies.

Key words: Cas12f, sgRNA/Cas12 ribonucleoprotein complex, protoplast, editing efficiency

Table 1

Primer sequences used in this paper"

引物名称
Primer name		 引物序列
Primer sequence (5'-3')
原核表达载体构建引物Prokaryotic expression vector construction primers
OsCas12f F CAAGGCCATGGCTGATATCGGATCCATGGACTATAAGGATCATGACG
OsCas12f R TGGTGGTGCTCGAGTGCGGCCGCAAGCTTTTACTTCTTCTTTTTAGCTTGTCC
SpCas12f F CGACAAGGCCATGGCTGATATCGGATCCATGGATTATAAAGATCATGACGGTG
SpCas12f R GGTGGTGGTGCTCGAGTGCGGCCGCAAGCTTCTATTTTTTCTTTTTTGCCTGC
UnCas12f F CGACAAGGCCATGGCTGATATCGGATCCATGGATTATAAAGATCATGACGGTG
UnCas12f R GGTGGTGGTGCTCGAGTGCGGCCGCAAGCTTCTATTTCTTCTTTTTAGCTTGC
酵母表达载体构建引物Yeast expression vector construction primers
pGADT7 Cas12f F1 AAGAAGAGAAAGGTCGAATTGGGTACCAAGCGTCCCGCTGCAACGAAA
pGADT7 Cas12f R1 GGGTTCCGGATCGCGGCCGCCCGGTAGAGGTGTGGTCAATAAGAG
pGADT7 Cas12f F2 GAGGAGTTTACGTCCAGCCAAGCTAGCTCTTTGAAAAGATAATGTATGA
pGADT7 Cas12f R2 GGGTTCCGGATCGCGGCCGCCCGGTAGAGGTGTGGTCAATAAGAG
pGADT7 sgRNA T1 F GAGAGAAAGGTCGAATTGGGTACCAAGCGTCCCGCTGCAACGAAA
pGADT7 sgRNA T1 R ATTAAGGGTTCCGGATCGCGGCCGCAAAAAAAACTCCTTGAGCCCCGTGCCCA
pGADT7 sgRNA T2 F AAGAGAAAGGTCGAATTGGGTACCAAGCGTCCCGCTGCAACGAAA
pGADT7 sgRNA T2 R GGGTTCCGGATCGCGGCCGCAAAAAAAATGCACCGCGGCCGCAACCGTTCAAGT
pGBKT7-eGFP F ACTGTAGCCCTAGACTTGATAGCCATCATCATATCGAAGTTTC
pGBKT7-eGFP R TTAGCTTGGCTGCAAGCGCGCCTAGTACAGCTCGTCCATGCCG
植物表达载体构建引物Plant expression vectors construct primers
pUC19 Cas12f F1 CAGCTATGACCATGATTACGCCAAGCTTATCGAGCAGCTGGCTTGTGGGGAC
pUC19 Cas12f R1 TCACGACGTTGTAAAACGACGGCCAGTGAATTCCTTATCTTTAATCATATTCC
pUC19 Cas12f sgRNA F2 ATTGATTGACAACGGATCCCCGGGTACCATGGAGTCAAAGATTCAAATAGAG
pUC19 Cas12f sgRNA R2 GTCACGACGTTGTAAAACGACGGCCAGTGAATTCCTTATCTTTAATCATATTCC
向导RNA分子体外转录引物Primers for transcription of RNA molecules in vitro
T7 sgRNA scaffold F ATTCTAATACGACTCACTATAGGGAGGGCCGACTTCCCGGCCCAAAATCGAGACAGTAGCC
T1 sgRNA scaffold R CTCCTTGAGCCCCGTGCCCACCTTCAAGCCGCTTTCGCGGCTCATGCACGG
T2 sgRNA scaffold R TGCACCGCGGCCGCAACCGTTCAACCTTCAAGCCGCTTTCGCGGCTCATGCACGG
PCR/RNP体外酶切底物扩增引物Poole/Enpp in vitro digestion substrate amplification primers
T1F CTTGACTACTACCCTCCCACC
T1R TGGTTAACTCCCCGGACCAA
T2F CCATTCCGGTATCGCTTGCT
T2R GACCCATCTGACACCGATCA
玉米原生质体靶位点扩增引物 Primers for amplification of maize protoplasts target sites
T1F1 GGAGTGAGTACGGTGTGCTTCAACTCGACTCCAGCA
T1R1 GAGTTGGATGCTGGATGGGTCGGTGCTCACCTTGAGGC
T2F1 GGAGTGAGTACGGTGTGCCCCGGACTCCAAGTACTGC
T2R1 GAGTTGGATGCTGGATGGGTACAGCGAGTGGTTCTGGA

Fig. 1

Prokaryotic expression and protein purification of different CRISPR-Cas12f proteins A: schematic diagram of the prokaryotic expression vector for CRISPR-Cas12f; B: induced expression of three types of CRISPR-Cas12f proteins at 28℃ and 37℃; C: 12% SDS-PAGE gel electrophoresis image of three CRISPR-Cas12f proteins purified by Ni-NTA chromatography."

Fig. 2

Substrate cleavage efficiency of three CRISPR-Cas12f proteins at different temperatures A: PCR/RNP digestion efficiency of Cas12f at 28℃; B: PCR/RNP digestion efficiency of Cas12f at 37℃; C: PCR/RNP digestion efficiency of Cas12f at 45℃; D: PCR/RNP digestion efficiency of Cas12f at 55℃."

Fig. 3

Substrate cleavage efficiency of three CRISPR-Cas12f proteins at different time points A: PCR/RNP digestion efficiency of Cas12f at 0.5 h; B: PCR/RNP digestion efficiency of Cas12f at 1 h; C: PCR/RNP digestion efficiency of Cas12f at 2 h; D: PCR/RNP digestion efficiency of Cas12f at 3 h."

Fig. 4

Editing efficiency of the three CRISPR-Cas12f systems in yeast A: schematic diagram of yeast transformation vector; B: schematic diagram of the editing efficiency of CRISPR-Cas12f system assessed by fluorescent protein recovery rate; C: statistics of yeast editing efficiency with different CRISPR-Cas12f."

Fig. 5

eGFP expression recovery mediated by OsCas12f system A: Cas12i.3-mediated restoration of eGFP expression, a positive control; B: no sgRNA fails to mediate restoration of eGFP in the OsCas12f system; C: sgRNA-T1 mediates restoration of eGFP using OsCas12f system; D: sgRNA-T2 mediates restoration of eGFP using OsCas12f system."

Table 2

Editing activity of maize endogenous loci mediated by transient expression of OsCas12f and SpCas12f"

系统
System		 靶位点突变
Target mutation (5°-3°)		 突变类型
Mutation
type		 突变数量
Mutation number		 比例
Percentage
(%)
OsCas12f-T1 ATCG$\underline{\underline{\text{TTC}}}$ TGGGCACGGGGCTCAAGGAGTGCATCCT WT 6734 91.91
ATCG$\underline{\underline{\text{TTC}}}$TGGGCACGGGGc-----------ATCCT -11 bp 199 2.72
ATCG$\underline{\underline{\text{TTC}}}$TGGGCACGGGGCt----------ATCCT -10 bp 176 2.40
ATCG$\underline{\underline{\text{TTC}}}$TGGGCACGGGg--------------CCT -14 bp 136 1.86
ATCG$\underline{\underline{\text{TTC}}}$TGGGCACGGGGCt---------CATCCT -9 bp 82 1.12
OsCas12f-T2 GCCACA TGCACCGCGGCCGCAACCGTTCAA$\underline{\underline{\text{GAAA}}}$GCCTGTGGAAGCGCA WT 7655 98.03
GCCACATGCa-----------ACCGTTCAA$\underline{\underline{\text{GAAA}}}$GCCTGTGGAAGCGCA -11 bp 154 1.97
SpCas12f-T2 GCCACA TGCACCGCGGCCGCAACCGTTCAA$\underline{\underline{\text{GAAA}}}$GCCTGTGGAAGCGCA WT 7506 98.91
GCCACATGCACCGCGGCCGCA---GTG$\underline{\underline{\text{GAA-a}}}$-------------CGCA -17 bp 83 1.09
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