針對雙歧桿菌屬開發基因編輯技術以調控代謝途徑：破壞限制修飾系統促進基因編輯效率

Abstract

腸道微生物群中的雙歧桿菌屬(Bifidobacterium)被視為益生菌，與人類健康息息相關，不僅能夠改善腸道健康，還能增強免疫力。為了深入探討雙歧桿菌是如何影響人類宿主、釐清是否有特定分子在此扮演重要角色，針對雙歧桿菌特定基因和代謝路徑的調控至關重要。然而，雙歧桿菌內限制修飾(RM)系統會將外來未甲基化的DNA切除，造成轉形(transformation)效率大幅降低，導致難以在雙歧桿菌屬中開發基因工程技術。本篇論文中，我們成功在雙歧桿菌屬中建立了一系列CRISPR-Cas9基因鹼基編輯器(cytosine base editor systems, cBEST)，藉由精準調控鹼基編輯器和sgRNA的表現量來編輯雙歧桿菌屬中特定的基因。為了解決轉形效率差的問題，我們在龍根菌(Bfidobacterium longum NCIMB 8809)以及青春雙歧桿菌(Bifidobacterium adolescentis DSM 20083)中利用cBEST技術消除限制酶或甲基化特定DNA序列來躲避限制修飾系統，我們成功將轉型效率提高至三十萬倍，而基因編輯成功率則提高至100%。接著，為了驗證cBEST方法能精準調控特定的代謝路徑，我們透過分析不同變異株的甲硫胺酸(methionine)循環路徑，了解DNA甲基化與甲硫腺苷(5’-methylthioadenosine)調控的關係。我們也成功在不同雙歧桿菌屬中編輯膽鹽水解酶(bsh)，並透過LC-MS/MS定量分析，證明我們能精準調控雙歧桿菌屬的膽酸代謝路徑。整體而言，我們成功利用CRISPR-Cas9基因編輯技術在雙歧桿菌屬中精準調控代謝途徑，並提供策略去躲避限制修飾系統，我們相信此方法必能成為研究腸道微生物強而有力的工具。

Intestinal microbiota members of the Bifidobacterium genus are increasingly recognized as probiotic potential and therapeutics applications to improve human health. However, the paucity of genetic tools and the prevalence of restriction modification (RM) systems limit the genetic manipulation in Bifidobacterium. In this study, we established a series of CRISPR/Cas9 cytosine base editor systems (cBESTs) with fine tuning base editor and sgRNA expression that are portable across multiple Bifidobacteria. We showed that bypassing RM systems by either eliminating restriction endonucleases or matching DNA methylation patterns in B. longum NCIMB 8809 and B. adolescentis DSM 20083 significantly improved both transformation (up to 300,000-fold) and editing efficiencies (up to 100%). Furthermore, we successfully manipulated methionine cycle pathway to elucidate the correlation between DNA methylation and 5’-methylthioadenosine regulation through MetK-deficient and RM-disrupted strains. Finally, we achieved the genetic knockout of the conserved bile salt hydrolase (bsh) gene in B. longum NCIMB 8809, B. longum DSM 20219 and B. infantis DSM 20088, effectively deactivating bile acids deconjugation. In summary, the ability to efficiently engineer Bifidobacterium genomes will definitely open new avenues for research and applications towards improving human health.