The CRISPR-associated Cas12b system is the third most efficient CRISPR tool for targeted genome editing in plants after Cas9 and Cas12a. Although the genome editing ability of AaCas12b has been previously investigated in rice, its off-target effects in plants are largely not known. In this study, we first engineered single-guide RNA (sgRNA) complexes with various RNA scaffolds to enhance editing frequency. We targeted EPIDERMAL PATTERNING FACTOR LIKE 9 (OsEPFL9) and GRAIN SIZE 3 (OsGS3) genes with GTTG and ATTC protospacer adjacent motifs, respectively. The use of two Alicyclobacillus acidoterrestris scaffolds (Aac and Aa1.2) significantly increased the frequency of targeted mutagenesis. Next, we performed whole-genome sequencing (WGS) of stably transformed T0 rice plants to assess off-target mutations. WGS analysis revealed background mutations in both coding and noncoding regions with no evidence of sgRNA-dependent off-target activity in edited genomes. We also showed Mendelian segregation of insertion and deletion (indel) mutations in T1 generation. In conclusion, both Aac and Aa1.2 scaffolds provided precise and heritable genome editing in rice.
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References
1.
TengF, CuiT, FengG, et al.Repurposing CRISPR-Cas12b for mammalian genome engineering. Cell Discov, 2018; 4(1); doi: 10.1038/s41421-018-0069-3
2.
StreckerJ, JonesS, KoopalB, et al.Engineering of CRISPR-Cas12b for human genome editing. Nat Commun, 2019; 10(1); doi: 10.1038/s41467-018-08224-4
LiuL, ChenP, WangM, et al.C2c1-sgRNA complex structure reveals RNA-guided DNA cleavage mechanism. Mol Cell, 2017; 65(2):310–322; doi: 10.1016/j.molcel.2016.11.040
5.
AtkinsA, ChungC-H, AllenAG, et al.Off-target analysis in gene editing and applications for clinical translation of CRISPR/Cas9 in HIV-1 therapy. Front Genome Ed, 2021; 3(August):1–26; doi: 10.3389/fgeed.2021.673022
6.
TangX, LiuG, ZhouJ, et al.A Large-scale whole-genome sequencing analysis reveals highly specific genome editing by both Cas9 and Cpf1 (Cas12a) nucleases in rice. Genome Biol, 2018; 19(1):1–13; doi: 10.1186/s13059-018-1458-5
7.
WangX, TuM, WangY, et al.Whole-genome sequencing reveals rare off-target mutations in CRISPR/Cas9-edited grapevine. Hortic Res, 2021; 8(1); doi: 10.1038/s41438-021-00549-4
8.
FengZ, MaoY, XuN, et al.Multigeneration analysis reveals the inheritance, specificity, and patterns of CRISPR/Cas-induced gene modifications in Arabidopsis. Proc Natl Acad Sci U S A, 2014; 111(12):4632–4637; doi: 10.1073/pnas.1400822111
9.
NekrasovV, WangC, WinJ, et al.Rapid generation of a transgene-free powdery mildew resistant tomato by genome deletion. Sci Rep, 2017; 7(1):1–6; doi: 10.1038/s41598-017-00578-x
10.
LiJ, ManghwarH, SunL, et al.Whole genome sequencing reveals rare off-target mutations and considerable inherent genetic or/and somaclonal variations in CRISPR/Cas9-edited cotton plants. Plant Biotechnol J, 2019; 17(5):858–868; doi: 10.1111/pbi.13020
11.
ZhangY, RenQ, TangX, et al.Expanding the scope of plant genome engineering with Cas12a orthologs and highly multiplexable editing systems. Nat Commun, 2021; 12(1):1–11; doi: 10.1038/s41467-021-22330-w
12.
WuF, QiaoX, ZhaoY, et al.Targeted mutagenesis in Arabidopsis thaliana using CRISPR-Cas12b/C2c1. J Integr Plant Biol, 2020; 62(11):1653–1658; doi: 10.1111/jipb.12944
13.
WangQ, AlariqiM, WangF, et al.The application of a heat-inducible CRISPR/Cas12b (C2c1) genome editing system in tetraploid cotton (G. hirsutum) plants. Plant Biotechnol J, 2020; 18(12):2436–2443; doi: 10.1111/pbi.13417
14.
GaoY, ZhaoY. Self-processing of ribozyme-flanked RNAs into guide RNAs in vitro and in vivo for CRISPR-mediated genome editing. J Integr Plant Biol, 2014; 56(4):343–349; doi: 10.1111/jipb.12152
15.
LiZ, ZhangD, XiongX, et al.A potent Cas9-derived gene activator for plant and mammalian cells. Nat Plants, 2017; 3(12):930–936; doi: 10.1038/s41477-017-0046-0
16.
PanC, WuX, MarkelK, et al.CRISPR-Act3.0 for highly efficient multiplexed gene activation in plants. Nat Plants, 2021; 7(7):942–953; doi: 10.1038/s41477-021-00953-7
17.
TangX, LowderLG, ZhangT, et al.A CRISPR-Cpf1 system for efficient genome editing and transcriptional repression in plants. Nat Plants, 2017; 3(February):1–5; doi: 10.1038/nplants.2017.18
18.
YouQ, ZhongZ, RenQ, et al.CRISPRMatch: An automatic calculation and visualization tool for high-throughput CRISPR genome-editing data analysis. Int J Biol Sci, 2018; 14(8):858–862; doi: 10.7150/ijbs.24581
19.
PinelloL, CanverMC, HobanMD, et al.Analyzing CRISPR genome-editing experiments with CRISPResso. Nat Biotechnol, 2016; 34(7):695–697; doi: 10.1038/nbt.3583
20.
MurrayMG, ThompsonWF. Rapid isolation of high molecular weight plant DNA. Nucleic Acids Res, 1980; 8(19):4321–4326; doi: 10.1093/nar/8.19.4321
21.
LiuQ, WangC, JiaoX, et al.Hi-TOM: A platform for high-throughput tracking of mutations induced by CRISPR/Cas Systems. Sci China Life Sci, 2019; 62(1):1–7; doi: 10.1007/s11427-018-9402-9
22.
RandallLB, SretenovicS, WuY, et al.genome- and transcriptome-wide off-target analyses of an improved cytosine base editor. Plant Physiol, 2021; 187(1):73–87; doi: 10.1093/plphys/kiab264
23.
JiangH, LeiR, DingSW, et al.Skewer: A fast and accurate adapter trimmer for Next-Generation Sequencing paired-end reads. BMC Bioinformatics, 2014; 15(1):1–12; doi: 10.1186/1471-2105-15-182
24.
LiH, DurbinR. Fast and accurate long-read alignment with burrows-wheeler transform. Bioinformatics, 2010; 26(5):589–595; doi: 10.1093/bioinformatics/btp698
25.
LiH, HandsakerB, WysokerA, et al.The Sequence alignment/map format and SAMtools. Bioinformatics, 2009; 25(16):2078–2079; doi: 10.1093/bioinformatics/btp352
26.
McKennaA, HannaM, BanksE, et al.The Genome Analysis Toolkit: A MapReduce framework for analyzing next-generation DNA sequencing data. Genome Res, 2010; 20(9):1297–1303; doi: 10.1101/gr.107524.110
27.
WilmA, AwPPK, BertrandD, et al.LoFreq: A sequence-quality aware, ultra-sensitive variant caller for uncovering cell-population heterogeneity from high-throughput sequencing datasets. Nucleic Acids Res, 2012; 40(22):11189–11201; doi: 10.1093/nar/gks918
28.
CibulskisK, LawrenceMS, CarterSL, et al.Sensitive detection of somatic point mutations in impure and heterogeneous cancer samples. Nat Biotechnol, 2013; 31(3):213–219; doi: 10.1038/nbt.2514
29.
KoboldtDC, ZhangQ, LarsonDE, et al.VarScan 2: Somatic mutation and copy number alteration discovery in cancer by exome sequencing. Genome Res, 2012; 22(3):568–576; doi: 10.1101/gr.129684.111
30.
YeK, SchulzMH, LongQ, et al.Pindel: A pattern growth approach to detect break points of large deletions and medium sized insertions from paired-end short reads. Bioinformatics, 2009; 25(21):2865–2871; doi: 10.1093/bioinformatics/btp394
31.
BaeS, ParkJ, KimJS. Cas-OFFinder: A fast and versatile algorithm that searches for potential off-target sites of Cas9 RNA-guided endonucleases. Bioinformatics, 2014; 30(10):1473–1475; doi: 10.1093/bioinformatics/btu048
32.
LiuG, YinK, ZhangQ, et al.Modulating chromatin accessibility by transactivation and targeting proximal dsgRNAs enhances Cas9 editing efficiency in vivo. Genome Biol, 2019; 20(1):1–11; doi: 10.1186/s13059-019-1762-8
33.
BigelyteG, YoungJK, KarvelisT, et al.Miniature type V-F CRISPR-Cas nucleases enable targeted dna modification in cells. Nat Commun, 2021; 12(1):1–8; doi: 10.1038/s41467-021-26469-4
34.
RenQ, SretenovicS, LiuG, et al.Improved plant cytosine base editors with high editing activity, purity, and specificity. Plant Biotechnol J, 2021; 19(10):2052–2068; doi: 10.1111/pbi.13635
35.
JinS, ZongY, GaoQ, et al.Cytosine, but not adenine, base editors induce genome-wide off-target mutations in rice. Science, 2019; 364(6437):292–295; doi: 10.1126/science.aaw7166
36.
JinS, FeiH, ZhuZ, et al.Rationally designed APOBEC3B cytosine base editors with improved specificity. Mol Cell, 2020; 79(5):728–740.e6; doi: 10.1016/j.molcel.2020.07.005
37.
WuY, RenQ, ZhongZ, et al.Genome-wide analyses of PAM-relaxed Cas9 genome editors reveal substantial off-target effects by ABE8e in rice. Plant Biotechnol J, 2022; 1–13; doi: 10.1111/pbi.13838
38.
GürelF, ZhangY, SretenovicS, et al.CRISPR-Cas nucleases and base editors for plant genome editing. aBIOTECH, 2020; 1(1):74–87; doi: 10.1007/s42994-019-00010-0
39.
HassanMM, ZhangY, YuanG, et al.construct design for CRISPR/Cas-based genome editing in plants. Trends Plant Sci, 2021; 26(11):1133–1152; doi: 10.1016/j.tplants.2021.06.015
40.
JosephsEA, KocakDD, FitzgibbonCJ, et al.Structure and specificity of the RNA-guided endonuclease Cas9 during DNA interrogation, target binding and cleavage. Nucleic Acids Res, 2015; 43(18):8924–8941; doi: 10.1093/nar/gkv892
41.
BrinerAE, DonohouePD, GomaaAA, et al.Guide RNA functional modules direct Cas9 activity and orthogonality. Mol Cell, 2014; 56(2):333–339; doi: 10.1016/j.molcel.2014.09.019
42.
MaS, LvJ, FengZ, et al.Get ready for the CRISPR/Cas system: A beginner's guide to the engineering and design of guide RNAs. J Gene Med, 2021; 23(11); doi: 10.1002/jgm.3377
43.
YangH, GaoP, RajashankarKR, et al.PAM-dependent target DNA recognition and cleavage by C2c1 CRISPR-Cas endonuclease. Cell, 2016; 167(7):1814–1828.e12; doi: 10.1016/j.cell.2016.11.053
44.
ZalatanJG, LeeME, AlmeidaR, et al.Engineering complex synthetic transcriptional programs with CRISPR RNA scaffolds. Cell, 2015; 160(1–2):339–350; doi: 10.1016/j.cell.2014.11.052
45.
ZhangY, MalzahnAA, SretenovicS, et al.The emerging and uncultivated potential of CRISPR technology in plant science. Nat Plants, 2019; 5(8):778–794; doi: 10.1038/s41477-019-0461-5
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