Division of Cell Biology and Imaging, Department of Radiology, University of Massachusetts Medical School, Worcester, Massachusetts, United States of America.
Department of Anatomy and Structural Biology, Interdisciplinary Graduate School, University of Yamanashi, Chuo, Yamanashi, Japan.
PLoS One. 2020 May 13;15(5):e0232594. doi: 10.1371/journal.pone.0232594. eCollection 2020.
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.
产生突变体并对其进行后续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，这是一种针对模式生物莱茵衣藻的高效、经济、基于 CRISPR 的靶向插入性诱变方法。TIM 利用 Cas9 指导 RNA（gRNA）核糖核蛋白（RNP）和外源双链（供体）DNA 递送入细胞。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，该基因在 Cas9 产生的双链断裂处插入。在优化了该方法的多个参数后，我们能够在两种不同的有细胞壁的菌株中对六个不同基因中的六个基因产生突变体，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不同的基因。TIM 在许多参数方面具有灵活性，并且可以使用电穿孔或玻璃珠法来递送 RNP 和供体 DNA。TIM 实现的突变率远远高于 CRISPR 方法在莱茵衣藻中报道的任何方法，并且有望对许多（如果不是全部）非必需核基因有效。
