TIM, a targeted insertional mutagenesis method utilizing CRISPR/Cas9 in Chlamydomonas reinhardtii
TIM, a targeted insertional mutagenesis method utilizing CRISPR/Cas9 in Chlamydomonas reinhardtii
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DOI：
10.1371/journal.pone.0232594
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发表时间：
2020-05-13
期刊：
影响因子：
3.7
通讯作者：
Witman, George B.
中科院分区：
文献类型：
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作者：
Picariello, Tyler;Hou, Yuqing;Witman, George B.
突变体的产生和随后的分析对于理解基因和蛋白质的功能至关重要。在这里，我们描述了 TIM，一种针对模式生物莱茵衣藻的高效、经济、基于 CRISPR 的靶向插入诱变方法。 TIM 利用 Cas9 引导 RNA (gRNA) 核糖核蛋白 (RNP) 与外源双链（供体）DNA 一起递送到细胞中。供体 DNA 包含基因特异性同源臂和一个完整的抗生素抗性基因，该基因插入 Cas9 产生的双链断裂处。优化该方法的多个参数后，我们能够在两种不同细胞壁菌株中的六个不同基因中产生六个突变体，突变效率范围为 40% 至 95%。此外，这些高效率允许在单个实验中同时靶向两个单独的基因。 TIM 在许多参数方面都很灵活，可以使用电穿孔或玻璃珠方法来传递 RNP 和供体 DNA。 TIM 在莱茵衣藻中实现的突变率比之前报道的任何基于 CRISPR 的方法都要高得多，并且有望对许多（如果不是全部）非必需核基因有效。
Generation and subsequent analysis of mutants is critical to understanding the functions of genes and proteins. Here we describe TIM, an efficient, cost-effective, CRISPR-based targeted insertional mutagenesis method for the model organism Chlamydomonas reinhardtii. TIM utilizes delivery into the cell of a Cas9-guide RNA (gRNA) ribonucleoprotein (RNP) together with exogenous double-stranded (donor) DNA. The donor DNA contains gene-specific homology arms and an integral antibiotic-resistance gene that inserts at the double-stranded break generated by Cas9. After optimizing multiple parameters of this method, we were able to generate mutants for six out of six different genes in two different cell-walled strains with mutation efficiencies ranging from 40% to 95%. Furthermore, these high efficiencies allowed simultaneous targeting of two separate genes in a single experiment. TIM is flexible with regard to many parameters and can be carried out using either electroporation or the glass-bead method for delivery of the RNP and donor DNA. TIM achieves a far higher mutation rate than any previously reported for CRISPR-based methods in C. reinhardtii and promises to be effective for many, if not all, non-essential nuclear genes.