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作物学报 ›› 2024, Vol. 50 ›› Issue (10): 2425-2434.doi: 10.3724/SP.J.1006.2024.43012

• 作物遗传育种·种质资源·分子遗传学 •    下一篇

不同CRISPR-Cas12f系统的编辑效率比较

黄灵芝1,2(), 符晓2, 祁显涛2, 刘昌林2, 谢传晓2, 吴鹏昊1, 任姣姣1,*(), 朱金洁2,*()   

  1. 1新疆农业大学农学院, 新疆乌鲁木齐 830052
    2中国农业科学院作物科学研究所, 北京 100081
  • 收稿日期:2024-03-15 接受日期:2024-06-20 出版日期:2024-10-12 网络出版日期:2024-07-11
  • 通讯作者: *任姣姣, E-mail: renjiaojiao789@sina.com;朱金洁, E-mail: zhujinjie@caas.cn
  • 作者简介:E-mail: 1993253522@qq.com
  • 基金资助:
    国家重点研发计划项目(2023YFD1202901)

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 Published:2024-10-12 Published online: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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摘要：

来自Type V-F家族的CRISPR/Cas12f蛋白报道仅为Cas9蛋白分子的1/4到1/3大小, 在病毒载体的递送上具有重要优势。然而, CRISPR/Cas12f系统介导植物基因编辑的报道较少, 编辑活性相对较低, 限制了该系统在植物上的进一步应用。本研究分别在体外酶切、酵母以及玉米原生质体瞬时表达3个体系中比较了OsCas12f、SpCas12f及UnCas12f的编辑活性。 结果表明, 基于Cas12f/sgRNA的体外酶切, OsCas12f与SpCas12f蛋白的编辑活性相当, 未检测到UnCas12f对底物酶切活性; 在酵母突变eGFP的恢复表达试验中, OsCas12f在2个测试位点对eGFP蛋白的表达恢复效率达到95%以上, 效率与Cas12i.3相当; SpCas12f介导的2个位点eGFP蛋白表达恢复效率分别是1.63%与3.20%, 效果次之; UnCas12f蛋白几乎无编辑活性; 玉米原生质体瞬时表达比较OsCas12f和SpCas12f介导的玉米内源位点的编辑效率, 发现OsCas12f对2个位点的编辑效率分别为2.72%和1.97%, 而SpCas12f仅能介导其中1个位点的定点编辑, 编辑效率为1.09%。Cas12f蛋白在靶位点处引入的突变类型以碱基的缺失为主, 缺失碱基长度在-9~ -17 bp之间。综上, OsCas12f可以作为植物微型基因编辑器及衍生技术开发的底盘工具酶。

关键词: Cas12f, sgRNA/Cas12核糖核蛋白复合体, 原生质体, 编辑效率

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

表1

本研究所用的引物序列"

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

图1

不同CRISPR-Cas12f蛋白的原核表达及蛋白纯化 A: CRISPR-Cas12f原核表达载体结构示意图; B: 3种CRISPR-Cas12f蛋白在28℃和37℃下的诱导表达; C: 3种CRISPR-Cas12f蛋白Ni-NTA纯化后12% SDS-PAGE凝胶电泳图。"

图2

3种CRISPR-Cas12f蛋白在不同温度下的底物酶切效率 A: 28℃时Cas12f的PCR/RNP酶切效率; B: 37℃时Cas12f的PCR/RNP酶切效率; C: 45℃时Cas12f的PCR/RNP酶切效率; D: 55℃时Cas12f的PCR/RNP酶切效率。"

图3

3种CRISPR-Cas12f蛋白在不同时间下底物酶切效率 A: Cas12f在0.5 h时PCR/RNP酶切效率; B: Cas12f在1 h时PCR/RNP酶切效率; C: Cas12f在2 h时PCR/RNP酶切效率; D: Cas12f在3 h时PCR/RNP酶切效率。"

图4

3种CRISPR-Cas12f系统的酵母编辑效率 A: 酵母转化载体示意图; B: 通过荧光蛋白恢复率评估CRISPR-Cas12f系统编辑效率原理图; C: 不同CRISPR-Cas12f介导的酵母eGFP恢复效率统计。"

图5

OsCas12f系统介导的eGFP表达恢复 A: Cas12i.3介导的eGFP表达恢复, 为阳性对照; B: 无sgRNA不能介导OsCas12f系统恢复eGFP; C: sgRNA-T1介导OsCas12f系统恢复eGFP; D: sgRNA-T2介导OsCas12f系统恢复eGFP。"

表2

OsCas12f及SpCas12f瞬时表达介导的玉米内源位点编辑"

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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