Investigation of CRISPR/Cas9-induced SD1 rice mutants highlights the importance of molecular characterization in plant molecular breeding
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Abstract: Although Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated 9 (Cas9) system has been widely used for basic research in model plants, its application for applied breeding in crops has faced strong regulatory obstacles, due mainly to a poor understanding of the authentic output of this system, particularly in higher generations. In this study, different from any previous studies, we investigated in detail the molecular characteristics and production performance of CRISPR/Cas9-generated SD1 (semi-dwarf 1) mutants from T2 to T4 generations, of which the selection of T1 and T2 was done only by visual phenotyping for semidwarf plants. Our data revealed not only on- and off-target mutations with small or lager indels but also exogenous elements in T2 plants. All indel mutants passed stably to T3 or T4 without additional modifications independent on the presence of Cas9, while some lines displayed unexpected hereditary patterns of Cas9 or some exogenous elements. In addition, effects of various SD1 alleles on rice height and yield differed depending on genetic backgrounds. Taken together, our data showed that the CRISPR/Cas9 system is effective in producing homozygous mutants for functional analysis, but it may be not as precise as expected in rice, and that early and accurate molecular characterization and screening must be carried out for generations before transitioning of the CRISPR/Cas9 system from laboratory to field.
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Key words:
- CRISPR/Cas9 /
- Plant height /
- Yield
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Fig. 1. The molecular characteristics of CRISPR/Cas9-induced SD1 mutants in rice. A: Diagram summary of the experimental design and the final output of CRISPR/Cas9-induced SD1 mutants. B: Sequencing results of the identified 20 genotypes in CRISPR/Cas9-induced SD1 T2 plants. WT, wild type; d#, deletion of # bp; i#, insertion of # bp; r#, replacement of # bp; chi, chimera. Ellipsis (…) indicates the occurrence of large chromosomal deletion.C: Summary of off-targets detected in SD1 T2 mutants.
Fig. 2. Effects of mutated SD1 alleles on plant height and yield in T3 and T4 generations. A: Height; B: Yield. 9815B, JIAODA138, or HUAIDAO1055 represents different genetic backgrounds. Values are mean ± SD (standard deviation of the mean, n = 10). Mean comparison was carried out using XLSTAT 2018 software, with different letters representing significant difference at P < 0.01.
Table 1. Signatures and segregations of CRISPR/Cas9-induced SD1 mutants in rice.
Line Background T2 T3 T4 Signature Target genotype Cas9 Mutation segregation Cas9 Mutation segregation Cas9 Q10 9815B Homozygote i1/i1 + nt nt nt nt Q11 9815B Homozygote d7/d7 + nt nt nt nt Q13 9815B Homozygote dL/dL + nt nt nt nt Q14 9815B Homozygote d7/d7 + nt nt nt nt Q16 9815B Heterozygote d1/WT + nt nt nt nt Q18 9815B Homozygote i1/i1 + 10i1/i1 10+ 10i1/i1 2+/8– Q21 9815B Heterozygote i1/WT + 3i1/i1:5h:2WT 8+/2– 10i1/i1 10– Q23 9815B Homozygote d24/d24 + 10d24/d24 10+ 10d24/d24 1+/9– Q26 9815B Homozygote i1/i1 – 10i1/i1 10– nt nt Q27 9815B Homozygote d257 + 10d257 10+ 10d257 2+/8- Q30 9815B Homozygote i1/i1 + 10i1/i1 9+/1– 10i1/i1 10– Q31 9815B Homozygote d63/d63 + 10d63/d63 7+/3– 10d63/d63 10– Q34 9815B Homozygote d4/d4 + 10d4/d4 10– nt nt Q36 9815B Homozygote d7/d7 – 10d7/d7 10– nt nt Q41 9815B Chimera d263,i194,r1 + 10d263/i194/r1 9+/1– 10d263/i194/r1 10– Q46 JIAODA138 Homozygote i1/i1 – 10i1i1 10– nt nt Q48 JIAODA138 Homozygote i1/i1 – 10i1/i1 10– 10i1/i1 10– Q56 JIAODA138 Homozygote i1/i1 + 10i1/i1 6+/4– 10i1/i1 10– Q60 JIAODA138 Homozygote i1/i1 + 10i1/i1 4+/6– 10i1/i1 10– Q62 JIAODA138 Biallele r1/i1 + 2r1/r1:5r1/i1:3i1/i1 6+/4– 10i1/i1 10– Q71 HUAIDAO1055 Homozygote i5/i5 + 10i5/i5 5+/5– 10i5/i5 10– Q73 HUAIDAO1055 Homozygote d7/d7 – 10d7/d7 10– 10d7/d7 10– Q74 HUAIDAO1055 Biallele r1/i1 + 3r1/r1:5r1/i1:2i1/i1 7+/3– 10i1/i1 10– Q76 HUAIDAO1055 Homozygote d1/d1 – 10d1/d1 10– 10d1/d1 10– Q79 HUAIDAO1055 Homozygote d19/d19 + nt nt nt nt Q86 HUAIDAO1055 Chimera d3,i1,r3 + nt nt nt nt Q89 HUAIDAO1055 Homozygote d63/d63 + nt nt nt nt Q97 HUAIDAO1055 Homozygote d2/d2 + nt nt nt nt Q103 HUAIDAO1055 Chimera d382,i1,r1 + nt nt nt nt Q107 HUAIDAO1055 Chimera d382,i1,r1 + nt nt nt nt Q115 HUAIDAO1055 Chimera WT,d382,i1 + nt nt nt nt +, Cas9 detected; –, Cas9 not detected; nt, not tested. d#, deletion with # bp; i#, insertion with # bp; r#, replacement of # bp; h, heterozygous; WT, wild-type; #d, #i, #r, #+, #−, number of lines with identified deletion, insertion, replacement, presence of, and absence of Cas9, respectively. 
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Table 2. Signatures and segregations of detected exogenous elements in CRISPR/Cas9-induced SD1 mutants in rice.
Line T2 T3 T-DNA element Vector backbone element T-DNA element Vector backbone element HPT 35S NOS LacZ HPT 35S NOS LacZ Q18 + + + – 10+ 10– 10+ 10– Q21 + + + – 8+/2– 2+/8– 2+/8– 10– Q23 + + + – 10+ 10– 10+ 10– Q26 – – – – 10– 10– 10– 10– Q27 + + + – 10+ 10– 10+ 10– Q30 + + + – 9+/1– 10– 9+/1– 10– Q31 + + + + 7+/3– 10– 4+/6– 10– Q34 + + + + 10– 10– 10– 10– Q36 – – – – 10– 10– 10– 10– Q41 + + + + 9+/1– 6+/4– 9+/1– 10– Q46 – – – – 10– 10– 10– 10– Q48 – – – – 10– 10– 10– 10– Q56 + + + – 6+/4– 10– 6+/4– 10– Q60 + + + – 6+/4– 10– 6+/4– 10– Q62 + + + – 4+/6– 10– 3+/7– 10– Q71 + + + – 7+/3– 10– 7+/3– 10– Q73 – – – – 10– 10– 10– 10– Q74 + + + – 7+/3– 1+/9– 6+/4– 10– Q76 – – – – 10– 10– 10– 10– + and –, presence and absence of detected corresponding exogenous elements, respectively; nt, not tested; #+, #–, numbers of lines with detected exogenous elements; CRISPR, Clustered Regularly Interspaced Short Palindromic Repeats; Cas9, CRISPR-associated 9.
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