Kinetics and fidelity of the repair of Cas9-induced double-strand DNA breaks. Brinkman, E.K. et al

Figure 1: We established a clonal K562 cell line with a stably integrated construct that encodes a tightly controlled inducible Cas9 nuclease. Western blot analysis of Cas9 at various time points after induction of Cas9 by Shield-1. It shows that Cas9 accumulates gradually after induction. Figure 3, 5, S3, S6: We established a variant of the ligation-mediated PCR assay for the quantification of DNA breaks at a defined location (Chailleux et al., 2014; Dai et al., 2000; Garrity and Wold, 1992). For the detection of broken DNA, we first denature the DNA and subject it to a primer extension reaction using a primer near the break site. This ensures that all cleavage sites are converted into blunt ends, even if resection of the broken ends has occurred. Next, an adaptor is ligated to the blunted DNA end, followed by PCR with one primer near the break site and a second primer that is complementary to the adaptor sequence. When analyzed on agarose gels, the samples from cells treated with sgRNA and Shield-1 yielded a band of the expected size. Figure 6: in vitro digestion by CRISPR. By PCR we first produced double-stranded DNA fragments of 600 – 1,000 bp of 3 loci in the genome. We then incubated each fragment in vitro with the respective Cas9/sgRNA complex to induce DSBs, and investigated the reaction products by agarose electrophoresis. Some samples Cas9 was subsequently denatured by two different heat treatment protocols (20 min 80 ˚C or 5 min 96 ˚C). DNA was incubated for either 2 hours or overnight.

图1：我们构建了一株稳定整合有编码受严密调控的诱导型Cas9核酸酶序列的克隆化K562细胞系。采用免疫印迹（Western blot）分析经Shield-1诱导Cas9后的不同时间点的Cas9蛋白表达情况，结果显示Cas9蛋白在诱导后呈渐进式积累。
图3、图5、补充图S3及补充图S6：我们优化了连接介导PCR（ligation-mediated PCR, LM-PCR）检测方法，用于对特定位点的DNA断裂进行定量分析（Chailleux等人，2014；Dai等人，2000；Garrity与Wold，1992）。该检测的具体流程为：首先对DNA进行变性处理，随后在断裂位点附近使用引物进行引物延伸反应，此举可确保所有切割位点均被转化为平端，即便断裂末端发生了末端切除修饰亦不例外。接下来，将接头连接至平端化的DNA末端，随后使用断裂位点附近的一条引物与接头序列互补的另一条引物进行PCR扩增。经琼脂糖凝胶电泳分析后，经sgRNA与Shield-1处理的细胞样本可出现预期大小的特异性条带。
图6：CRISPR体外酶切实验。我们首先通过PCR扩增得到基因组中3个位点的600~1000 bp双链DNA片段，随后将各片段与对应的Cas9/sgRNA复合物进行体外孵育以诱导双链断裂（double-strand break, DSB），并通过琼脂糖凝胶电泳对反应产物进行检测。部分样本中的Cas9随后通过两种不同的热处理方案进行变性处理：80℃孵育20分钟，或96℃孵育5分钟。DNA与复合物的孵育时间分别设置为2小时及过夜。