酵母基因工程中 CRISPR- Cas 系统的使用

1. 酵母基因工程中CRISPR-Cas系统的使用 ——金珠

2. CRISPR-Cas系统 • CRISPR （Clustered Regularly Interspaced Short Palindromic Repeats）常间回文重复序列丛集 CRISPR 是一个特殊的DNA 重复序列家族, 广泛分布于细菌和古细菌基因组中。CRISPR 位点通常由短的高度保守的重复序列(repeats)组成, 重复序列的长度通常21~48 bp, 由于具有回文序列,可以形成发卡结构, 重复次数最高可达250 次。重复序列之间被26~72 bp间隔序列(spacer)隔开。外源DNA整合到CRISPR重复片段的基因组内，但随后CRISPR间隔区可以帮助细胞识别和阻止这些外源DNA在以后攻击细胞。

4. Cas基因 在CRISPR 位点附近, 存在一系列CRISPR相关(CRISPR-associated, Cas)基因。编码的蛋白具有核酸相关的功能域。有几种不同的Cas基因，能够产生不同功能的蛋白质，包括核酸内切酶、解旋酶、核酸结合和转录调控作用的酶。 文中使用的是CRISPR-CasⅡ系统，CAS9基因来自Streptococcus pyogenes（化脓性链球菌），利用Cas9的核酸酶特性，和CRISPR RNA一起形成复合物，来识别靶核酸序列并导致其降解。

6. in Saccharomycescerevisiae • CRISPR-Cas system for genome engineering. • CRISPR-Cas directed CAN1 mutagenesis • CRISPR-Cas stimulated homologous recombination with donor DNA and transient gRNA PCR cassette • CRISPR-Cas stimulated homologous recombination with donor DNA and gRNA expression plasmid

7. 1. CRISPR-Cas directed CAN1 mutagenesis Cas gene: In BY4733 cells containing a centromeric plasmid constitutively expressing Cas9 under the Gal-L promoter • Design gRNA expressing plasmid ( p426 )

8. The Cas9 gene • The Cas9 gene was a codon-optimized version originally constructed for expression in human cells。 • p415 Gal-L and p414 TEF1p plasmids were each cut with XhoI and XmaI, and the backbone containing the promoter and terminator was gel purified. • Then Gibson assembled into the vector using the NEB Gibson Assembly kit.

9. Showing robust and specific RNA-guided endonuclease activity at targeted endogenous genomic loci in yeast

11. 2. homologous recombination with donor DNA and transient gRNA PCR cassette • Use ade2-101 allele gene to repair in Strain VL6-48 cells • ade2-101gene is essential for adenine(腺嘌呤) biosynthesis • Electroporation : gRNA cassette and 90 mer donor single-stranded or double-stranded oligonucleotide

12. Electroporation

13. increase homologous recombination rates of ssDNA and dsDNA donors by 5-fold and 130-fold

14. 3. homologous recombination withdonor DNA and gRNA expression plasmid • In BY4741 cells containing a centromeric plasmid constitutively expressing Cas9

15. co-transformed : gRNA CAN1.Y expression plasmid and Donor DNA or A 1.4 kb KanMX cassette (conferring G418 resistance) • plated without dilution on SC-uracil and SC-tryptophan • 10-5dilutions onto Yeast Peptone Adenine Dextrose (YPAD)for two days. • replica to Selective Plates with canavanine plates andYPAD plates with 100 mg/ml G418 antibiotic

16. (near 100%) canavanine resistant

17. DISCUSSION