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. 2023 Aug 22;10(10):uhad168.
doi: 10.1093/hr/uhad168. eCollection 2023 Oct.

Efficient large fragment deletion in plants: double pairs of sgRNAs are better than dual sgRNAs

Guoning Zhu  1 , Lingling Zhang  1 , Liqun Ma  1 , Qing Liu  2 , Kejian Wang  2 , Jinyan Li  1 , Guiqin Qu  1 , Benzhong Zhu  1 , Daqi Fu  1 , Yunbo Luo  1 , Hongliang Zhu  1
Affiliations

Efficient large fragment deletion in plants: double pairs of sgRNAs are better than dual sgRNAs

Guoning Zhu et al. Hortic Res. .
No abstract available

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Figures

Figure 1
Figure 1
DPS improves deletion efficiency of large fragments in the tomato genome compared to dual sgRNAs. A Large fragment deletion of SlyPDS in the MMEJ strategy. The Pf/Pr primer pair detected large fragment deletions, and the predicted MHS are labeled as pMHS, while the actual repair sites are labeled as MHS. B Large fragment deletion of lncRNA2155 using DPS in transient transformation of tomato cotyledons. C Large fragment deletion of slyTAGL1 using DPS in transient transformation of tomato cotyledons. D Target sites on SlyPDS were used to analyse the effect of the distance between pairs of target sites on the efficiency of large fragment deletion. Four target site groups were established: CR-2 T (sgRNA1, sgRNA8), CR-3 k (sgRNA1, sgRNA2, sgRNA3, sgRNA4), CR-6 k (sgRNA1, sgRNA2, sgRNA5, sgRNA6), and CR-9 k (sgRNA1, sgRNA2, sgRNA7, sgRNA8). The P outside and P inside primer pair were used for qPCR analysis to determine the efficiency of large fragment deletion. E Statistics of large fragment deletion reads in next generation sequencing. F qPCR analysis showed that DPS significantly improved the efficiency of large fragment deletion. The bars represent mean values ± SD. G Target sites on SlyPDS were used to analyse the effect of the internal spacing of single target site pairs on the efficiency of large fragment deletion. Three target site groups were established: CR-100 bp (sgRNA1, sgRNA2, sgRNA7, sgRNA8), CR-150 bp (sgRNA1, sgRNA3, sgRNA6, sgRNA8), and CR-200 bp (sgRNA1, sgRNA4, sgRNA5, sgRNA8). H qPCR analysis showed that the efficiency of large fragment deletion decreased with the increase in fragment length in a single target site pair. The bars represent mean values ± SD.
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