CRISPR/Cas9和λ-Red基因敲除技术在大肠杆菌中的应用比较

Comparative Study of CRISPR/Cas9 and λ-Red Gene Knockout Techniques in Eschrichia coli

摘要: λ-Red和CRISPR/Cas9基因敲除技术已被广泛应用于染色体基因敲除。本研究分别采用λ-Red和CRISPR/Cas9技术对大肠杆菌BL21菌株染色体丙氨酸消旋酶基因alr进行了敲除，并比较了其敲除效率。对于Red敲除技术而言，首先构建由alr上下游同源臂和卡那抗性盒FLT-kan^R-FLT组成的打靶片段Lalr-FLT-kan^R-FLT-Ralr，转化E. coli BL21/pKD46感受态细胞，在pKD46表达的Red重组酶作用下发生同源重组，使染色体alr被卡那抗性盒FLT-kan^R-FLT替换；然后，质粒pCP20转化上述发生了alr替换的E. coli菌株感受态细胞，在pCP20表达的FLP重组酶作用下发生位点特异性重组，删除卡那抗性基因kan^R；最后，对温敏型质粒进行消除，获得alr敲除突变株E. coli BL21 Δalr-Red。对于CRISPR/Cas9技术而言，首先构建敲除质粒pTargetF-alr，制备打靶供体DNA片段L-alr-R；接着，pTargetF-alr和L-alr-R共转化E. coli BL21/pCas感受态细胞，在pTargetF-alr编码的sgRNA序列引导下，质粒pCas编码的Cas9蛋白结合到alr基因切割靶点DNA，通过对双链断裂的同源重组修复实现对alr基因的无痕敲除，获得alr敲除突变株E. coli BL21 Δalr-CRISPR。实验结果显示：使用基于短、长同源臂靶打靶片段的Red技术，E. coli BL21 Δalr-Red阳性转化子检出率分别为5%和55%；而使用CRISPR/Cas技术，E. coli BL21 Δalr-CRISPR阳性转化子检出率为95%。实验结果表明：在E. coli中，使用短同源臂靶打靶片段的Red敲除技术，转化子脱靶率超高，这使阳性转化子鉴定工作量大增；使用长同源臂打靶片段的Red敲除技术，较为容易检测到阳性转化子；使用CRISPR/Cas9技术，敲除效率显著优于λ-Red技术。

Abstract: CRISPR/Cas9 and λ-Red techniques have been wildly used for chromosomal gene knockout. In this study, the alanine racemase gene alr on the genome of Escherichia coli BL21 strain was deleted using the two gene knockout methods, respectively, and the efficiencies of knockout were compared. For λ-Red technique, firstly, the targeting fragments Lalr-FLT-kan^R-FLT-Ralr consisting of the upstream and downstream homologous arms of the gene alr and the kanamycin-resistant cassette FLT-kan^R-FLT were prepared, transformed into the E. coli BL21/pKD46 competent cells, and chromosomal alr was replaced by FLT-kan^R-FLT by pKD46-Red enzymes-mediated homologous recombination; secondly, kan^R was deleted by pCP20-FLP enzyme-mediated site-specific recombination; finally, temperature-sensitive plasmids were eliminated, generating the mutant strain E. coli BL21 Δalr-Red. For CRISPR/Cas9 technique, firstly, knockout plasmid pTargetF-alr was constructed and targeting donor fragment L-alr-R was prepared; secondly, pTargetF-alr and L-alr-R were co-transformed into the E. coli BL21/pCas competent cells; scarless knockout of alr was achieved by cleavage of alr by sgRNA guiding-Cas9 protein and sequential double strands breakage repair based on homologous recombination, generating the mutant strain E. coli BL21 Δalr-CRISPR. The experimental results showed that the rates of the E. coli BL21 Δalr-Red positive transformants were 5% and 55% using short homologous arms and long homologous arms λ-Red techniques, respectively, while the rate of the E. coli BL21 Δalr-CRISPR positive transformants was 95% using CRISPR/Cas9 techniques. These results demonstrate that in E. coli, short homologous arms λ-Red technique leads to a superhigh off-target rate of transformants, which would increase work intensity for positive transformants screening; positive transformants are relatively easy to obtain using long homologous arms λ-Red technique; the knockout rate of CRISPR/Cas9 technique is significantly higher than that of λ-Red technique.