要用Bsa I切sgRNA质粒和连接反应,更加简便,不会产生上述的产物IV和V。即设计Р 6Р 的2条靶点引物3’端有15-17 nt分别与U3/U6启动子和sgRNA配对,用PCR扩增出片段后进行第二轮overlapping PCR。Р 2ndР Figure 5. Procedures for generation of a sgRNA expression cassette containing a target sequence by overlapping PCR. The chimeric primers with target sequence strands are given in Table S2. The first PCR can be carried out in two separated reactions with U-F/U#T#- and gRT#+/gR-R primer pair, respectively, or in one reaction with the all 4 primers. U# indicates a given promoter, and T#+ and T#- indicate strands of a target sequence.Р 7Р 引物设计例子Р 靶点第1碱基与转录起始碱基相同(regular target)Р 19 ntР gRT#+GGATGA(下划线与sgRNA配对)Р (#为靶点特异编号)Р OsU6aT#-Р OsU6bT#-Р OsU6cT#-GAР (下划线与OsU6a 配对)GA(下划线与OsU6b 配对)GA(下划线与OsU6c 配对)Р GAOsU3 配对)Р Target sequenceР TgRT#+Р OsU3T#-TAGGCGACGTCGTCGTGTTGT(下划线与
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