BioVector? pKCCpf1 Streptomyces Genome Editing Plasmid / pKCCpf1 鏈霉菌基因組編輯質(zhì)粒

一 產(chǎn)品基本信息與遺傳學背景

• 質(zhì)粒名稱：pKCCpf1 鏈霉菌 CRISPR-Cpf1 基因組編輯質(zhì)粒。

• 開發(fā)背景與來源：由中國科學院上海生命科學研究院蔣衛(wèi)紅研究員團隊開發(fā)。pKCCpf1 屬于專門針對鏈霉菌屬（Streptomyces）設計的全功能一體化（All-in-one）多重基因組編輯與轉錄抑制載體系統(tǒng)。

• 核心分子構件與技術特征：

  • FnCpf1 核酸酶（Cas12a）：該質(zhì)粒攜帶來源于新弗朗西斯氏菌（Francisella novicida）的 Cpf1（現(xiàn)改稱為 Cas12a）核酸酶編碼序列。Cpf1 屬于第2類V型 CRISPR-Cas 系統(tǒng)，相比傳統(tǒng)的 SpCas9，它具有獨特的遺傳學優(yōu)勢：

    • PAM 識別位點：識別富含 T 的原 spacer 相鄰基序（PAM），對于 FnCpf1 其標準識別位點為 5'-TTV-3'（其中 V 為 A、G、C，在鏈霉菌中可部分放寬至 5'-YTV-3'，Y = T/C）。這極大地擴充了高 GC 含量的鏈霉菌基因組中的靶點選擇范圍。

    • 切割末端形態(tài)：FnCpf1 切割 DNA 后產(chǎn)生帶有 4-5個堿基突出端的粘性末端（Staggered Cuts），相比 SpCas9 產(chǎn)生的平末端，更利于通過同源定向修復（HDR）進行精確的片段插入或大片段敲除。

    • crRNA 加工簡化：Cpf1 自身兼具加工 pre-crRNA 的內(nèi)切核糖核酸酶活性，僅需一段 19 nt 的直接重復序列（Direct Repeat, DR）和 23 nt 的引導序列（Spacer）即可工作，無需額外的 tracrRNA。這使得在同一個載體上構建串聯(lián)的 crRNA 陣列（Array）變得極為簡便，極適合多基因同時敲除（Multiplex Genome Editing）。

  • 骨架基礎：基于鏈霉菌經(jīng)典穿梭質(zhì)粒 pKC1139 骨架進行工程化改造。

• 抗性篩選標記：

  • 大腸桿菌及鏈霉菌雙重抗性：阿普拉霉素抗性（Apramycin, AmR / AprR），常規(guī)工作濃度為 50 ug/mL。

• 復制子與溫敏特性：

  • 攜帶 pSG5 溫敏型復制子（Temperature-sensitive replicon）。在 30 攝氏度環(huán)境下可穩(wěn)定復制，當溫度提高至 37-39 攝氏度時，質(zhì)粒復制被徹底抑制，這一特性便于在完成基因編輯后通過高溫培養(yǎng)將質(zhì)粒從鏈霉菌宿主中徹底消除（Plasmid Curing）。

二 核心科研價值與轉化醫(yī)學/工業(yè)應用

在工業(yè)微生物學領域，鏈霉菌是天然產(chǎn)物（如抗生素、抗腫瘤藥物和免疫抑制劑）的最核心來源，pKCCpf1 是突破其基因編輯瓶頸的利器：

1. 攻克 SpCas9 基因重組毒性與死角：

   傳統(tǒng)的 SpCas9 系統(tǒng)在某些鏈霉菌（如吸水鏈霉菌 S. hygroscopicus）中由于其高水平的結構性表達具有極強的細胞毒性，或者因高 GC 基因組缺乏 5'-NGG-3' 靶點而無法有效工作。pKCCpf1 作為完美的替代工具，剪切效率高、毒性低，且專門針對高 GC 富 T 區(qū)域。

2. 多重基因聯(lián)合敲除與代謝通路重塑（Multiplex Engineering）：

   利用 Cpf1 獨特的單導向 RNA 串聯(lián)加工特性，科研人員可在 pKCCpf1 上一次性裝載多個不同的 Spacer 靶點以及同源修復臂（HDR 供體段）。在天藍色鏈霉菌（S. coelicolor）等模式株中，單基因或雙基因的精確敲除效率可高達 75% - 95%，極大加速了多主效次級代謝產(chǎn)物合成基因簇（BGCs）的批量失活與底盤細胞（Chassis Strain）的工程化減產(chǎn)改造。

3. CRISPRi 介導的多基因精細轉錄抑制：

   通過將 FnCpf1 突變轉化為失去剪切活性但保留 DNA 結合能力的死 Cpf1（ddCpf1），該質(zhì)?？芍苯愚D換為高效的集成式 CRISPRi 干擾平臺。在無需破壞基因組的前提下，對鏈霉菌內(nèi)多個競爭性代謝支路基因實施溫和的轉錄抑制，從而將前體代謝流集中導向目標抗生素的合成。

三 實驗室質(zhì)粒轉化、接合轉移、擴增與保存標準步驟

1. 擴增菌株與培養(yǎng)基配置

• 大腸桿菌克隆宿主：常規(guī)克隆及常規(guī)質(zhì)粒維持推薦使用 TOP10、DH5a 或 Mach1 感受態(tài)細胞。

• 大腸桿菌接合轉移宿主：由于鏈霉菌具有極強的限制修飾系統(tǒng)，純化出的質(zhì)粒無法直接轉化鏈霉菌。必須先將 pKCCpf1 轉化入專用的甲基化缺陷型大腸桿菌供體菌 ET12567 (攜帶 pUZ8002 輔助質(zhì)粒) 中，才能與鏈霉菌進行三親或雙親接合轉移（Conjugation）。

• 大腸桿菌培養(yǎng)體系：LB 肉湯/固體瓊脂，添加最終工作濃度為 50 ug/mL 的阿普拉霉素（Apramycin）。

2. 大腸桿菌轉化與擴增步驟

1. 取出 50 uL 大腸桿菌 TOP10（或經(jīng)阿普拉霉素/氯霉素/卡那霉素篩選的 ET12567/pUZ8002）感受態(tài)細胞置于冰上融化。

2. 加入 1 uL 純化的 pKCCpf1 質(zhì)粒 DNA（全長約 11,146 bp，屬于中大質(zhì)粒），輕彈混勻，冰浴 30 分鐘。

3. 置于 42 攝氏度水浴中精確熱擊 45 秒，隨后立即插回冰中迅速冷卻 2 分鐘。

4. 向管內(nèi)加入 500 uL 無抗 LB 肉湯，置于 37 攝氏度搖床內(nèi)以 200 rpm 復蘇勻速搖菌 60 分鐘（阿普拉霉素抗性表達較慢，建議確保復蘇時間足 1 小時）。

5. 4000 rpm 離心棄去部分上清，留 100 uL 液體涂布于含 50 ug/mL 阿普拉霉素的 LB 固體平板上。

6. 置于 37 攝氏度 培養(yǎng)箱中倒置培養(yǎng) 14 - 16 小時，等待單菌落長出。

3. 質(zhì)粒提取與鏈霉菌接合轉移簡要流程

1. 挑選大腸桿菌單菌落，接種至 5 - 10 mL 含阿普拉霉素的液體 LB 中，37 攝氏度過夜搖菌，利用標準高純度質(zhì)粒提取試劑盒提取質(zhì)粒 DNA。

2. 將重組好的 pKCCpf1 轉化入大腸桿菌供體菌 ET12567/pUZ8002 中，并在含有阿普拉霉素（50 ug/mL）、氯霉素（25 ug/mL）和卡那霉素（50 ug/mL）的三抗 LB 平板上篩選出正確的供體單菌落。

3. 接合轉移關鍵控制：將處于對數(shù)生長期的 ET12567 工程菌與鏈霉菌的新鮮孢子（或復蘇中的菌絲體）按比例混合，涂布于添加了 $MgCl_2$ 的大豆粉甘露醇（MS）固體平板上。在 30 攝氏度（允許質(zhì)粒穩(wěn)定復制的常溫）下孵育 16 - 20 小時后，表面覆蓋含有阿普拉霉素（最終工作濃度約 50 ug/mL）和萘啶酮酸（Nalidixic acid，25 ug/mL，用于特異性徹底殺死大腸桿菌供體菌）的無菌水層。

4. 繼續(xù)在 30 攝氏度 下培養(yǎng) 3 - 5 天，長出的抗性接合子即為質(zhì)粒成功導入的鏈霉菌菌株。

5. 篩選與消除（Curing）：在 30 攝氏度下誘導 Cpf1 剪切并完成同源修復后，將菌株接種至不含阿普拉霉素的 ISP2 或 MS 培養(yǎng)基中，置于 37 - 40 攝氏度高溫下連續(xù)搖菌/傳代培養(yǎng)，促使溫敏型骨架 pKCCpf1 停止復制并自發(fā)流失。隨后通過平行平板對比篩選出阿普拉霉素敏感、且經(jīng) PCR 驗證編輯成功的干凈突變株。

4. 質(zhì)粒及大腸桿菌甘油菌長期保存

• 質(zhì)粒 DNA 保存：提取出的純化 pKCCpf1 質(zhì)粒分裝后置于 -20 攝氏度 或 -80 攝氏度超低溫冰箱中，可穩(wěn)定保存數(shù)年，避免反復凍融。

• 大腸桿菌工程菌保存：取擴增旺盛的過夜菌液 800 uL，加入 200 uL 滅菌高純無菌甘油（最終甘油工作濃度約 20%），在凍存管中徹底混勻，立即投入 -80 攝氏度 超低溫冰箱內(nèi)長期保存。

Part 2 English Section

I General Information and Genetic Architecture

• Plasmid Designation: pKCCpf1 Streptomyces CRISPR-Cpf1 Genome Editing Shuttle Vector.

• Development History & Source: Engineered and deposited by the research group of Professor Weihong Jiang at the Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences (CAS).pKCCpf1 functions as a comprehensive, all-in-one multiplex genome editing and transcriptional interference platform tailored specifically for the genus Streptomyces.

• Core Molecular Architecture & Engineering Modules:

  • FnCpf1 Endonuclease (Cas12a): The vector expresses the codon-optimized endonuclease sequence encoding FnCpf1 derived from Francisella novicida. Classed as a Class 2 Type V CRISPR-Cas system, Cpf1 offers pivotal biological advantages over conventional SpCas9 within actinomycete systems:

    • PAM Recognition Modalities: Cpf1 recognizes a T-rich Protospacer Adjacent Motif (PAM), mapping predominantly to 5'-TTV-3' sequences (where V = A/G/C, and can adapt partially to 5'-YTV-3' profiles in Streptomyces niches). This drastically multiplies candidate targeting coordinates across the hyper-GC-rich genomes characteristic of streptomycetes.

    • Staggered Cleavage Geometry: Unlike the blunt-ended double-strand breaks (DSBs) produced by SpCas9, FnCpf1 introduces a 4-5 nucleotide cohesive staggered overhang. These sticky ends significantly enhance the efficiency of homology-directed repair (HDR) path integration for precise sequence deletion or large biosynthetic gene cluster (BGC) substitutions.

    • Autonomous crRNA Processing: Cpf1 possesses an intrinsic, independent endoribonuclease activity dedicated to trimming its own pre-crRNA transcripts.Operating without the requirement of a secondary trans-activating crRNA (tracrRNA), a simple minimal array comprising a 19-nt Direct Repeat (DR) linked to a 23-nt target Spacer is fully functional. This architectural minimalism streamlines the configuration of extensive multiplexed crRNA strings on a single vector for simultaneous poly-genic targeting (Multiplex Genome Editing).

  • Backbone Matrix: Synthesized directly over the molecular frame of the classical Streptomyces shuttle plasmid pKC1139.

• Selective Resistance Elements:

  • Dual E. coli/Streptomyces Selection Tag: Apramycin resistance gene (AmR / AprR), routinely deployed at a work concentration of 50 ug/mL.

• Replicon Mechanics & Temperature-Sensitive Profiles:

  • Armed with the pSG5 temperature-sensitive replicon. The plasmid replicates with high fidelity at a permissive temperature of 30°C. Shifting the incubation matrix up to a restrictive threshold of 37°C–39°C completely arrests plasmid replication kinetics. This allows for straightforward plasmid curing from the host cells post-editing by continuous thermal subculturing.

II Strategic Research Value and Industrial/Translational Fields

Within industrial microbiology pipelines, members of the genus Streptomyces generate over two-thirds of clinically utilized natural product secondary metabolites (antibiotics, anti-tumor drugs, immunosuppressants). pKCCpf1 dismantles historical genetic engineering roadblocks in these hosts:

1. Bypassing Cas9-Induced Toxic Shock & Target Blindspots:

   Constitutive expression of conventional SpCas9 triggers fatal genomic auto-toxicity in distinct wild actinomycete strains (e.g., Streptomyces hygroscopicus) or fails completely due to a dearth of mandatory 5'-NGG-3' motifs within high-GC matrices. pKCCpf1 serves as a high-efficiency alternative, maintaining minimized baseline toxicity combined with rigorous structural fidelity across T-rich target arrays.

2. Multiplex Functional Knockouts & Bottom-Up Chassis Optimization:

   Leveraging the autonomous processing traits of Cpf1 crRNA loops, investigators can stitch multiple distinct Spacers and corresponding homologous repair donor fragments (HDR cassettes) directly into pKCCpf1. In benchmark hosts such as Streptomyces coelicolor, singular or dual locus disruption metrics yield 75% to 95% clean recombination efficiency, accelerating the systematic deletion of competing indigenous biosynthetic gene clusters (BGCs) to construct standardized, clear metabolic底盤 (chassis lineages).

3. CRISPRi-Driven Multiplex Multi-Valent Transcriptional Repression:

   By transitioning the system to utilize a catalytically inactive "dead" Cpf1 variant (ddCpf1), pKCCpf1 scales into an integrated CRISPRi multiplex silencing platform. Investigators can drive multi-point down-regulation of native metabolic diversion paths simultaneously without altering the host chromosome topology, maximizing precursor flux redirection toward desired specialized target products.

III Bacterial Transformation, Intergeneric Conjugation, Proliferation, and Storage Protocols

1. Host Strains and Medium Configuration

• E. coli Cloning Host: Standard laboratory lineages configured for cloning maintenance, including TOP10, DH5a, or Mach1 competent cells.

• Intergeneric Conjugation Donor Host: Because Streptomyces species express highly restrictive endogenous DNA methylation defense barriers, purified naked plasmid DNA cannot be directly transformed into wild spores. The assembled pKCCpf1 must be transformed into the methylation-deficient E. coli donor strain ET12567 harboring the non-transmissible conjugative helper plasmid pUZ8002 prior to initiating biparental or triparental intergeneric conjugation.

• Bacterial Selective Medium Matrix: Standard Lysogeny Broth (LB) liquid formulas or solid agar matrices supplemented with 50 ug/mL Apramycin.

2. E. coli Competent Transformation Routine

1. Retrieve an aliquot of 50 uL competent E. coli TOP10 cells (or antibiotic-validated ET12567/pUZ8002 cells) gently onto a chilled ice bed.

2. Deliver 1 uL of the purified pKCCpf1 construct (~11,146 bp) directly into the cell suspension. Mix by smooth flicking (do not vortex) and incubate on ice for 30 minutes.

3. Transfer the tube into a calibrated water bath set precisely at 42°C for a rigorous heat-shock window of 45 seconds. Instantly plunge the tube back into the ice bed for 2 minutes without agitation.

4. Inoculate the shocked cells with 500 uL of sterile, antibiotic-free liquid LB broth. Proliferate in an orbital shaking incubator at 37°C running at 200 rpm for 60 minutes of out-growth recovery (Apramycin resistance marker expression profiles develop slowly; ensure a full 1-hour recovery window is completed).

5. Concentrate the cells via centrifugation at 4,000 rpm for 3 minutes. Decant excess supernatant, resuspend the pellet in the remaining ~100 uL volume, and spread evenly onto pre-warmed selective LB agar plates supplemented with 50 ug/mL Apramycin.

6. Incubate inverted at 37°C for 14–16 hours until distinct colonies materialize.

3. Plasmid Extraction and Intergeneric Conjugation Key Workflow

1. Pick a singular, well-isolated E. coli colony from the selective plate, inoculate into 5–10 mL of Apramycin-supplemented liquid LB broth, and shake at 37°C overnight. Extract the pKCCpf1 plasmid using a standard centrifugal mini-prep kit.

2. Transform the sequence-verified pKCCpf1 vector into E. coli ET12567/pUZ8002, selecting robust single transformants on LB agar plates containing Apramycin (50 ug/mL), Chloramphenicol (25 ug/mL), and Kanamycin (50 ug/mL).

3. Conjugation Protocol Milestones: Blend active log-phase ET12567 donor cultures thoroughly with freshly harvested Streptomyces spores (or germinated mycelial fragments) at precise empirical ratios. Plate the mixture onto Mannitol Soy Flour (MS) agar surfaces fortified with $MgCl_2$. Incubate at the permissive baseline of 30°C for 16–20 hours, then overlay the lawn evenly with a sterile water layer containing Apramycin (final target plate concentration 50 ug/mL) and Nalidixic acid (25 ug/mL, administered to selectively eliminate the E. coli donor mass completely).

4. Resume incubation strictly at 30°C for 3–5 days until primary exconjugant colonies breakthrough the selection overlay.

5. Curing the Plasmids: After cultivating the exconjugants at 30°C to induce Cpf1-mediated genomic double-strand cleavage and subsequent HDR repair synthesis, inoculate the validated clones into non-selective liquid growth media (e.g., ISP2 or MS broth). Incubate the culture within a shaking incubator set to a restrictive thermal zone of 37°C–40°C across consecutive generation passages to halt the pSG5 replication machinery. Streak the cells onto non-selective agar and perform parallel replicate picking to isolate clean, Apramycin-sensitive, PCR-validated target mutants.

4. Indefinite Storage Protocols

• Purified Plasmid Retention: Store recovered aliquots of purified pKCCpf1 DNA within sterile nuclease-free water at -20°C or -80°C. Prevent recurrent freeze-thaw processing.

• Bacterial Glycerol Stock Stabilization: Blend 800 uL of active, late-log phase selective E. coli culture with 200 uL of sterile analytical-grade high-purity glycerol (yielding a final ~20% glycerol freezing matrix) inside a cryovial. Vortex briefly to unify, and store immediately within an ultra-low -80°C freezer for stable maintenance stretching across years.

The Alt-R CRISPR-Cpf1 System provides an exceptional alternative tool for genome editing across distinct bacterial and mammalian lines where traditional Cas9 PAM parameters cannot be met.

BioVector NTCC質(zhì)粒載體菌株細胞蛋白抗體基因保藏中心

電話:400-800-2947

工作QQ/微信同號:1843439339

網(wǎng)址

http://www.nedfriskphoto.com