Department of Genetics, Yale University School of Medicine, New Haven, CT 06510, USA.
Department of Genetics, Yale University School of Medicine, New Haven, CT 06510, USA; Yale Stem Cell Center, Yale University School of Medicine, New Haven, CT 06510, USA; Yale Cancer Center, Yale University School of Medicine, New Haven, CT 06510, USA.
Methods. 2018 Nov 1;150:11-18. doi: 10.1016/j.ymeth.2018.06.014. Epub 2018 Jun 28.
The impact of the CRISPR-Cas biotechnological systems has recently broadened the genome editing toolbox available to different model organisms further with the addition of new efficient RNA-guided endonucleases. We have recently optimized CRISPR-Cpf1 (renamed Cas12a) system in zebrafish. We showed that (i) in the absence of Cpf1 protein, crRNAs are unstable and degraded in vivo, and CRISPR-Cpf1 RNP complexes efficiently mutagenize the zebrafish genome; and (ii) temperature modulates Cpf1 activity especially affecting AsCpf1, which experiences a reduced performance below 37 °C. Here, we describe a step-by-step protocol on how to easily design and generate crRNAs in vitro, purify recombinant Cpf1 proteins, and assemble ribonucleoprotein complexes to carry out efficient mutagenesis in zebrafish in a constitutive and temperature-controlled manner. Finally, we explain how to induce Cpf1-mediated homology-directed repair using single-stranded DNA oligonucleotides. In summary, this protocol includes the steps to efficiently modify the zebrafish genome and other ectothermic organisms using the CRISPR-Cpf1 system.
CRISPR-Cas 生物技术系统的影响最近通过添加新的高效 RNA 导向内切酶，进一步拓宽了可供不同模式生物使用的基因组编辑工具包。我们最近在斑马鱼中优化了 CRISPR-Cpf1（重命名为 Cas12a）系统。我们表明：（i）在没有 Cpf1 蛋白的情况下，crRNA 在体内不稳定并降解，而 CRISPR-Cpf1 RNP 复合物可有效地使斑马鱼基因组发生突变；（ii）温度调节 Cpf1 的活性，特别是影响 AsCpf1，其在 37°C 以下性能降低。在这里，我们描述了一个逐步的方案，说明如何在体外轻松设计和生成 crRNA，纯化重组 Cpf1 蛋白，并组装核糖核蛋白复合物，以在恒定和温度控制的方式下在斑马鱼中进行有效的突变。最后，我们解释了如何使用单链 DNA 寡核苷酸诱导 Cpf1 介导的同源定向修复。总之，该方案包括使用 CRISPR-Cpf1 系统高效修饰斑马鱼基因组和其他变温动物的步骤。
