酵母基因工程中CRISPR-Cas 系统的使用

—— 金珠

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。

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。

in Saccharomyces cerevisiae

• CRISPR-Cas system for genome engineering.

1. CRISPR-Cas directed CAN1 mutagenesis

2. CRISPR-Cas stimulated homologous recombination with donor DNA and transient gRNA PCR cassette

3. CRISPR-Cas stimulated homologous recombination with donor DNA and gRNA expression plasmid

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 )

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.

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

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

Electroporation

• increase homologous recombination rates of ssDNA and dsDNA donors by

5-fold and 130-fold

3. homologous recombination withdonor DNA and gRNA expression plasmid

• In BY4741 cells containing a centromeric plasmid constitutively expressing Cas9

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

(near 100%) canavanine resistant

DISCUSSION