Methods and compositions for targeted editing of polynucleotides
A targeting and nucleic acid sequence technology, applied in the field of targeted editing and composition for polynucleotides, capable of solving problems such as increasing Cas9 targeting and/or mutagenesis efficacy
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example 1
[0183] Example 1: Improved genome editing efficiency by mutation of crRNA and tracrRNA
[0184] This example describes mutation of crRNA and / or tracrRNA to improve genome editing efficiency. These mutations generally fall into four regions. The first mutation targets the RNA polymerase III pause signal. These mutations typically target two regions of the dual guide RNA complex. Two of these regions contain the "UUUU" motif in RNA. The continuous sequence of T's in the DNA template is the pause signal for RNA polymerase III. The first poly-U motif is located downstream of the crRNA prospacer. Point mutations were made to disrupt this pause signal. Such as figure 1 As shown in , mutants d2 to d7 were generated to determine how disruption of this first motif would affect genome editing efficiency. "d1" is as figure 1 The sequence of the crRNA without the indicated sequence for the prespacer sequence (SEQ ID NO: 113) and its corresponding tracrRNA (SEQ ID NO: 114) is sho...
example 2
[0187] Example 2: Targeted Mutagenesis in Rice
[0188] To test the genome editing efficiency of the dual guide constructs described above, targets were identified in the genome of rice (rice or Oryza sativa). The target gene is DENSE AND ERECT PANICLE 1 (DEP1). The japonica dep1 mutant contains a 625 bp deletion near the 3' end of DEP1. The mutant has dense and erect ears, higher grain number and lower plant height compared to wild type (Huang et al., 2009, Nat Genet 41:494-497). Indica rice has a wild-type copy of the DEP1 gene. For the examples described herein, DEP1 was targeted for mutation, and all crRNA constructs described herein contained 5'-ACTGCAGTGCGTGCTGCGC-3' (SEQ ID NO:45), which encodes the prospacer sequence of the crRNA molecules described herein . Those of ordinary skill in the art will appreciate that the present invention is not limited to the sequence of the prospacer or its corresponding DNA target, and that mutations of the crRNA and tracrRNA mole...
example 3
[0196] Example 3: Further Optimization of Dual Guide RNA Molecules
[0197] Additional mutated constructs were generated to determine whether the DNA could be further optimized by mutagenesis of crRNA and / or tracrRNA, particularly in regions where they interact to form duplexes, or by insertion of additional RNA elements Targeting RNA duplex structures. Based on the results shown in Table 1, a number of these constructs were constructed.
[0198] Construct 24127 encodes a crRNA molecule (SEQ ID NO:28) containing the d11 mutation, and containing the above and figure 1 The tracrRNA molecule (SEQ ID NO 29) of both the d9 5' deletion mutation and the d11 mutation described in . Construct 24128 also encodes a tracrRNA with a d9 deletion mutation. In addition, the crRNA and tracrRNA of construct 24128 have been mutated to increase the 10 of the duplex at the 3' end of the crRNA (see SEQ ID NO:30) and the 5' end of the tracrRNA (see SEQ ID NO:31). GC content in nucleotides.
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