BioVector® pKCCpf1 Streptomyces Genome Editing Plasmid / pKCCpf1 链霉菌基因组编辑质粒

一 产品基本信息与遗传学背景

• 质粒名称：pKCCpf1 链霉菌 CRISPR-Cpf1 基因组编辑质粒。

• 开发背景与来源：由中国科学院上海生命科学研究院蒋卫红研究员团队开发。pKCCpf1 属于专门针对链霉菌属（Streptomyces）设计的全功能一体化（All-in-one）多重基因组编辑与转录抑制载体系统。

• 核心分子构件与技术特征：

  • FnCpf1 核酸酶（Cas12a）：该质粒携带来源于新弗朗西斯氏菌（Francisella novicida）的 Cpf1（现改称为 Cas12a）核酸酶编码序列。Cpf1 属于第2类V型 CRISPR-Cas 系统，相比传统的 SpCas9，它具有独特的遗传学优势：

    • PAM 识别位点：识别富含 T 的原 spacer 相邻基序（PAM），对于 FnCpf1 其标准识别位点为 5'-TTV-3'（其中 V 为 A、G、C，在链霉菌中可部分放宽至 5'-YTV-3'，Y = T/C）。这极大地扩充了高 GC 含量的链霉菌基因组中的靶点选择范围。

    • 切割末端形态：FnCpf1 切割 DNA 后产生带有 4-5个碱基突出端的粘性末端（Staggered Cuts），相比 SpCas9 产生的平末端，更利于通过同源定向修复（HDR）进行精确的片段插入或大片段敲除。

    • crRNA 加工简化：Cpf1 自身兼具加工 pre-crRNA 的内切核糖核酸酶活性，仅需一段 19 nt 的直接重复序列（Direct Repeat, DR）和 23 nt 的引导序列（Spacer）即可工作，无需额外的 tracrRNA。这使得在同一个载体上构建串联的 crRNA 阵列（Array）变得极为简便，极适合多基因同时敲除（Multiplex Genome Editing）。

  • 骨架基础：基于链霉菌经典穿梭质粒 pKC1139 骨架进行工程化改造。

• 抗性筛选标记：

  • 大肠杆菌及链霉菌双重抗性：阿普拉霉素抗性（Apramycin, AmR / AprR），常规工作浓度为 50 ug/mL。

• 复制子与温敏特性：

  • 携带 pSG5 温敏型复制子（Temperature-sensitive replicon）。在 30 摄氏度环境下可稳定复制，当温度提高至 37-39 摄氏度时，质粒复制被彻底抑制，这一特性便于在完成基因编辑后通过高温培养将质粒从链霉菌宿主中彻底消除（Plasmid Curing）。

二 核心科研价值与转化医学/工业应用

在工业微生物学领域，链霉菌是天然产物（如抗生素、抗肿瘤药物和免疫抑制剂）的最核心来源，pKCCpf1 是突破其基因编辑瓶颈的利器：

1. 攻克 SpCas9 基因重组毒性与死角：

   传统的 SpCas9 系统在某些链霉菌（如吸水链霉菌 S. hygroscopicus）中由于其高水平的结构性表达具有极强的细胞毒性，或者因高 GC 基因组缺乏 5'-NGG-3' 靶点而无法有效工作。pKCCpf1 作为完美的替代工具，剪切效率高、毒性低，且专门针对高 GC 富 T 区域。

2. 多重基因联合敲除与代谢通路重塑（Multiplex Engineering）：

   利用 Cpf1 独特的单导向 RNA 串联加工特性，科研人员可在 pKCCpf1 上一次性装载多个不同的 Spacer 靶点以及同源修复臂（HDR 供体段）。在天蓝色链霉菌（S. coelicolor）等模式株中，单基因或双基因的精确敲除效率可高达 75% - 95%，极大加速了多主效次级代谢产物合成基因簇（BGCs）的批量失活与底盘细胞（Chassis Strain）的工程化减产改造。

3. CRISPRi 介导的多基因精细转录抑制：

   通过将 FnCpf1 突变转化为失去剪切活性但保留 DNA 结合能力的死 Cpf1（ddCpf1），该质粒可直接转换为高效的集成式 CRISPRi 干扰平台。在无需破坏基因组的前提下，对链霉菌内多个竞争性代谢支路基因实施温和的转录抑制，从而将前体代谢流集中导向目标抗生素的合成。

三 实验室质粒转化、接合转移、扩增与保存标准步骤

1. 扩增菌株与培养基配置

• 大肠杆菌克隆宿主：常规克隆及常规质粒维持推荐使用 TOP10、DH5a 或 Mach1 感受态细胞。

• 大肠杆菌接合转移宿主：由于链霉菌具有极强的限制修饰系统，纯化出的质粒无法直接转化链霉菌。必须先将 pKCCpf1 转化入专用的甲基化缺陷型大肠杆菌供体菌 ET12567 (携带 pUZ8002 辅助质粒) 中，才能与链霉菌进行三亲或双亲接合转移（Conjugation）。

• 大肠杆菌培养体系：LB 肉汤/固体琼脂，添加最终工作浓度为 50 ug/mL 的阿普拉霉素（Apramycin）。

2. 大肠杆菌转化与扩增步骤

1. 取出 50 uL 大肠杆菌 TOP10（或经阿普拉霉素/氯霉素/卡那霉素筛选的 ET12567/pUZ8002）感受态细胞置于冰上融化。

2. 加入 1 uL 纯化的 pKCCpf1 质粒 DNA（全长约 11,146 bp，属于中大质粒），轻弹混匀，冰浴 30 分钟。

3. 置于 42 摄氏度水浴中精确热击 45 秒，随后立即插回冰中迅速冷却 2 分钟。

4. 向管内加入 500 uL 无抗 LB 肉汤，置于 37 摄氏度摇床内以 200 rpm 复苏匀速摇菌 60 分钟（阿普拉霉素抗性表达较慢，建议确保复苏时间足 1 小时）。

5. 4000 rpm 离心弃去部分上清，留 100 uL 液体涂布于含 50 ug/mL 阿普拉霉素的 LB 固体平板上。

6. 置于 37 摄氏度 培养箱中倒置培养 14 - 16 小时，等待单菌落长出。

3. 质粒提取与链霉菌接合转移简要流程

1. 挑选大肠杆菌单菌落，接种至 5 - 10 mL 含阿普拉霉素的液体 LB 中，37 摄氏度过夜摇菌，利用标准高纯度质粒提取试剂盒提取质粒 DNA。

2. 将重组好的 pKCCpf1 转化入大肠杆菌供体菌 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 平板上筛选出正确的供体单菌落。

3. 接合转移关键控制：将处于对数生长期的 ET12567 工程菌与链霉菌的新鲜孢子（或复苏中的菌丝体）按比例混合，涂布于添加了 $MgCl_2$ 的大豆粉甘露醇（MS）固体平板上。在 30 摄氏度（允许质粒稳定复制的常温）下孵育 16 - 20 小时后，表面覆盖含有阿普拉霉素（最终工作浓度约 50 ug/mL）和萘啶酮酸（Nalidixic acid，25 ug/mL，用于特异性彻底杀死大肠杆菌供体菌）的无菌水层。

4. 继续在 30 摄氏度 下培养 3 - 5 天，长出的抗性接合子即为质粒成功导入的链霉菌菌株。

5. 筛选与消除（Curing）：在 30 摄氏度下诱导 Cpf1 剪切并完成同源修复后，将菌株接种至不含阿普拉霉素的 ISP2 或 MS 培养基中，置于 37 - 40 摄氏度高温下连续摇菌/传代培养，促使温敏型骨架 pKCCpf1 停止复制并自发流失。随后通过平行平板对比筛选出阿普拉霉素敏感、且经 PCR 验证编辑成功的干净突变株。

4. 质粒及大肠杆菌甘油菌长期保存

• 质粒 DNA 保存：提取出的纯化 pKCCpf1 质粒分装后置于 -20 摄氏度 或 -80 摄氏度超低温冰箱中，可稳定保存数年，避免反复冻融。

• 大肠杆菌工程菌保存：取扩增旺盛的过夜菌液 800 uL，加入 200 uL 灭菌高纯无菌甘油（最终甘油工作浓度约 20%），在冻存管中彻底混匀，立即投入 -80 摄氏度 超低温冰箱内长期保存。

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底盘 (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.

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