一种从任意DNA来源大规模制备gRNA文库的通用方案

摘要

生成大规模gRNA文库的方法应具备简单、高效且成本低廉的特点。本文介绍了一种基于靶DNA酶切的gRNA文库制备方案。该方法称为CORALINA（通过可控核酸酶活性实现全面gRNA文库构建），为昂贵的定制寡核苷酸合成提供了替代方案。

摘要

CRISPR/Cas9 系统在基因组和表观基因组编辑中的广泛应用源于其简单性和可适应性。一种效应分子（Cas9 核酸酶或无核酸酶活性的 dCas9 融合蛋白）通过一种称为向导 RNA（gRNA）的小型合成 RNA 被靶向至基因组中的特定位点。CRISPR 系统的双组分特性使其可用于筛选研究，因为含有数千个独立 gRNA 表达盒的质粒文库可在单次实验中对基因组中多个不同位点进行功能探究。

迄今为止，用于文库构建的gRNA序列几乎完全依赖于寡核苷酸合成，这种方法限制了文库中序列的复杂性，且成本相对较高。本文详细介绍了一种名为CORALINA（通过可控核酸酶活性实现全面gRNA文库生成）的方法，这是一种简单且经济高效的gRNA文库构建技术，基于对输入DNA的酶切消化来生成高度复杂的gRNA文库。由于CORALINA文库可由任何来源的DNA制备，因此具备丰富的定制化选择，能够支持多种多样的CRISPR相关筛选实验。

引言

将细菌的 CRISPR/Cas9 系统改造为一种分子靶向工具，引发了分子生物学领域最近的一场革命。以往从未有过如此简便的方法可在基因组特定位点上操控染色质。CRISPR 的常见应用包括靶向基因突变¹、基因组编辑²、表观基因组编辑³、转录激活以及基因沉默⁴。CRISPR 系统的一个显著优势在于，其应用并不局限于已被深入研究的候选位点，因为 gRNA 文库使得无偏倚的筛选成为可能。这有助于在无需任何先验实验知识的情况下发现基因组中的功能位点。然而，目前 gRNA 文库的构建主要依赖于寡核苷酸合成，且可供购买的非人源或非小鼠来源的 gRNA 文库，或靶向开放阅读框以外区域的文库选择十分有限。因此，尽管 CRISPR 筛选已展现出极强的应用潜力^5,6,7,8，但其全部潜力尚未被充分挖掘。

为了克服传统gRNA生成方法的局限性，近年来已发展出两种新策略。这两种策略均基于对目标DNA进行可控的酶切消化，而非依赖定制寡核苷酸合成。CORALINA⁹采用微球菌核酸酶，而目前唯一可替代的方法CRISPR-EATING¹⁰则利用限制性内切酶（HpaII、ScrFI、BfaI和MmeI）。重要的是，这两种技术均可应用于任意输入DNA，将其作为gRNA原间隔序列的来源。尽管CRISPR-EATING方法采用策略减少了克隆的gRNA中其靶位点后不含有必需的S.pyogenes PAM（原间隔序列邻近基序）的数量，但它仅能生成特定区域内所有可能功能性gRNA的一小部分。相比之下，CORALINA能够生成来源于序列的所有潜在gRNA，但也包含更高比例的非功能性向导序列。通过可控核酸酶活性进行gRNA文库构建，可简便且经济地为任何物种、任何Cas9蛋白或效应系统生成全面的gRNA文库。此外，CORALINA具有良好的可定制性，通过选择合适的输入材料和载体即可决定文库的类型、大小和内容。本文提供了一套详细的实验方案，可用于从多种DNA来源（图1）——包括细菌人工染色体（BACs）或基因组DNA⁹——构建全面的gRNA文库。本方案所附的代表性结果是将CORALINA方法应用于BAC DNA后获得的。

方案

1. Digestion of DNA with Micrococcal Nuclease

1. Perform an optimization reaction for each new batch of micrococcal nuclease enzyme (MNase).
   NOTE: The number of units of MNase used should be tested (using a serial dilution, Figure 2A). Usually, 5-10 U of MNase digest 1 µg of purified genomic or BAC DNA down to a range of 5-100 bp with the conditions described below.
2. Per reaction, set up 1 µL of 10x MNase Buffer, 1x bovine serum albumin (BSA), 1 µg of target DNA, 1 µL of MNase (0.1-50 units) in a 10 µL reaction volume.
3. Incubate at 37 °C for 15 min.
4. Immediately inactivate the enzyme by adding 1 µL of 500 mM ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA).

2. Separation of DNA Fragments Using Polyacrylamide Gel Electrophoresis (PAGE)

1. Add sample buffer/gel loading dye to DNA samples and load onto a 20% PAGE gel. Load an appropriate DNA ladder for sizing (5 bp DNA Ladder). Do not overload the gel (1 µg DNA per well).
2. Run gels in appropriate 1x TRIS-Borate-EDTA (TBE) running buffer at 150 V (constant) for around 1.5 h or until the lower dye front (bromophenol blue, dark blue color) that travels at around 15 bp, reaches the lower end of the gel.
3. Stain the gel using an ultra-sensitive nucleic acid stain and visualize under UV light.
4. From the gel image, determine the optimum concentration of MNase for digesting DNA down to 20-30 bp in size.
5. Use the optimized concentration of MNase to digest the target DNA by repeating steps 1.2-2.2. Usually, setting up 10-12 reactions (i.e. 10-12 µg of total starting material) will yield enough digested DNA following PAGE gel extraction to proceed to subsequent steps.
6. Using a sterile scalpel, cut the gel next to the marker lane and stain only the part of the gel containing the ladder with fresh 1x TBE running buffer containing 1X of an ultra-sensitive nucleic acid stain. Visualize the DNA ladder and excise MNase-digested DNA fragments in the size range between approximately 18-30 bp using a razor blade.
   NOTE: Avoid exposing the MNase digested fragments to UV light. It is possible to use a blue light source (instead of UV) or take an image of the ladder under UV, print it to scale and use this to guide excision of MNase-digested DNA fragments). This step avoids staining and exposure of DNA fragments to UV light. Always use an unused, sterile disposable scalpel or razor blade for this step to avoid contamination.
7. Transfer the gel slice to a microcentrifuge tube.
8. Stain the remainder of the gel with nucleic acid stain as above, expose to UV light and record an image to keep a record of the gel excision step.

3. Isolation of DNA Fragments from PAGE-gels Using the Crush and Soak Method

NOTE: This step has been adopted from Sambrook et al.¹¹

1. Prepare PAGE gel solubilization buffer (1.88 mL of 4 M ammonium acetate, 150 µL of 1 M magnesium acetate, 30 µL of 0.5 M ethylenediaminetetraacetic acid (EDTA) (pH 8) in ultrapure H₂O to a total volume of 15 mL).
2. Crush the excised gel slice against the wall of the micro-centrifuge tube using a sterile pipette tip.
3. Add 2 gel volumes of PAGE solubilization buffer and incubate at 37 °C for 16 h on a rotating platform.
4. Centrifuge the samples for 1 min at maximum speed in a microcentrifuge. Transfer the supernatant to a new microcentrifuge tube, taking care not to transfer any crushed gel pieces.
5. Add 0.5 volumes of PAGE solubilization buffer to the gel pellet, vortex, and repeat the centrifugation (step 3.4). Combine the supernatants.
6. Extract the DNA fragments using standard phenol-chloroform extraction.
   CAUTION: Phenol is toxic if it comes into contact with skin or if swallowed. Safety precautions such as gloves, protective eyewear, a lab coat, and working in a fume hood are critical. Dispose of all phenol-containing waste according to the institute's regulations.
   1. Add one volume of phenol:chloroform:isoamyl alcohol (25:24:1) to the sample and vortex thoroughly.
   2. Centrifuge for 10 min at maximum speed (16,000 x g) in a tabletop centrifuge at room temperature. Carefully transfer the upper aqueous phase to a fresh microcentrifuge tube. Take care not to carry over any phenol during pipetting.
   3. Repeat steps 3.6.1 and 3.6.2 once.
   4. Add a small amount (0.2 µL) of glycogen, 0.1 volumes of 3 M sodium acetate (pH 5.2) and 2 volumes of 100% ethanol (EtOH).
   5. Vortex and incubate at -20 °C for several hours or overnight.
   6. Centrifuge for 30 min at maximum speed at 4 °C using a tabletop centrifuge.
   7. Carefully remove the supernatant and wash the DNA pellet with 70% EtOH.
   8. Centrifuge for 30 min at maximum speed at 4 °C using a tabletop centrifuge.
   9. Remove the supernatant and air-dry the DNA pellet. Make sure all the EtOH has evaporated but be careful not to over-dry the pellet.
   10. Dissolve DNA pellet in 12 µL of H₂O.
       NOTE: PAGE gel extraction is very inefficient. For every 10 µg of starting DNA digested with MNase, expect to recover 1-20 ng of purified fragments after gel extraction. Control amount and integrity of fragments by loading 1/6^th (2 µL) on a PAGE gel (Figure 2C).

4. End Repair of MNase-digested, Gel-purified Fragments

1. Set up the following reaction using a DNA blunting kit: 10 µL of purified DNA from step 3.6.10, 1.5 µL of 10X blunting buffer, 1.5 µL of 1 mM dNTP mix, 0.6 µL of blunting enzyme, 1.4 µL of H₂O.
2. Incubate at 22 °C for 30 min, and then heat-inactivate the enzyme by incubation at 70 °C for 10 min.
3. Add 85 µL of H₂O and perform a reaction clean-up using standard phenol/chloroform- extraction and EtOH-precipitation (as described in section 3.6). Proceed immediately to linker ligation.

5. Linker Generation

NOTE: Linkers need to be amplified in parallel with section 3 to be able to proceed immediately with linker ligation. Primer sequences used below must be appropriate for the chosen gRNA expression vector. Those presented here have been designed for the vector pgRNA-pLKO.1.⁹ For amplification of the 5' linker from pgRNA-pLKO.1, use the primer sequences 5'-linker-F (TTGGAATCACACGACCTGGA) and 5'-linker-R (CGGTGTTTCGTCCTTTCCAC), yielding a 689 bp amplicon. For amplification of the 3' linker from pgRNA-pLKO.1, use the primers 3'-linker-F: (GTTTTAGAGCTAGAAATAGCAAGTTAAAATA) and 3'-linker-R: (ACTCGGTCATGGTAAGCTCC), which yield an 848 bp amplicon.

1. PCR-amplify the adapter sequences from the gRNA expression vector (using reagents of choice and custom primer sequences, if necessary). For pgRNA-pLKO.1 set up the following 50 µL PCR reaction: 25 µL of PCR master mix, 2.5 µL of primer F (10 µM), 2.5 µL of primer R (10 µM), 0.1 ng of gRNA expression vector (pgRNA-pLKO.1) in H₂O.
2. Incubate reactions on a thermocycler using the following conditions: 1 cycle 98 °C for 30 s, 32 cycles 98 °C for 10 s, 59 °C for 10 s, 72 °C for 30 s, 1 cycle 72 °C for 10 min.
3. Purify PCR reactions using solid phase reversible immobilization beads according to the manufacturer's instructions. Elute in 30 µL of H₂O.
4. To enforce directional ligation of the linker to the end-repaired, MNase-digested DNA fragments, digest linkers with appropriate restriction enzymes (here HindIII and SacII). Set up the following reactions:
   1. For digestion of 5' linker with HindIII, use 30 µL of purified 5' linker amplicon from step 5.3., 5 µL of Buffer, 3 µL of HindIII (20U/µL), and 12 µL of H₂O.
   2. For digestion of 3' linker with SacII, use 30 µL of purified 3' linker amplicon from step 5.3., 5 µL of Buffer, 3 µL of SacII (20 U/µL), and 12 µL of H₂O.
5. Incubate digests at 37 °C for 3 h.
6. Add DNA gel loading dye and run the restriction enzyme digests on a 1% agarose gel. Excise the bands at 637 bp (5' linker digest) and 295 bp (3' linker digest).
7. Purify the DNA from the excised gel pieces using a gel extraction kit.

6. Linker Ligation and Amplification of Inserts

1. Set up a 14 µL ligation reaction using equimolar amounts of MNase-digested, end-repaired fragments and linker sequences.
   NOTE: Linker to fragment ratios may be optimized. It is recommended to include a no-fragment control (NFC) reaction.
   1. Use 5 ng of MNase-digested fragments (end-repaired and purified), 120 ng of 5'linker (HindIII digest, purified), 55 ng of 3' linker (SacII digest, purified), 1.4 µL of T4 ligase Buffer, and 1.4 µL of concentrated T4 DNA ligase in H₂O.
2. Incubate the ligation reactions at 16 °C for 16 h. Do not heat-inactivate the enzyme. Proceed immediately to nick-translation.
   NOTE: The end-repaired MNase-digested fragments provide the 5' phosphates necessary for ligation of linkers, as the linker themselves are un-phosphorylated.
3. To the ligation reaction (and the NFC reaction) add the following: 25 µL of Taq 2X master mix (capable of nick translation), 2.5 µL of primer Linker-Minus450-F (10 µM, GGGCAAGTTTGTGGAATTGG), 2.5 µL of primer Linker-Plus275-R (10 µM, AAGTGGATCTCTGCTGTCCC) and 6 µL of H₂O.
4. Include a no-template control (NTC). Incubate reactions on a thermocycler using the following conditions: 1 cycle (nick translation): 72 °C for 20 min, 1 cycle: 95 °C for 5 min, 3-4 cycles: 95 °C for 15 s, 58 °C for 15 s, 72 °C for 30 s, 1 cycle: 72 °C for 5 min.
5. Perform a reaction clean-up using solid phase reversible immobilization beads with a sample to bead ratio of 1:1. Elute in 40 µL of H₂O.
6. Further amplify the desired fragment (5' linker + MNase fragment +3' linker) using appropriate PCR reagents and the following primers:
   1. Use 12.5 µL of PCR master mix, 1.25 µL of primer Linker-Minus450-F (10 µM, GGGCAAGTTTGTGGAATTGG), 1.25 µL of primer Linker-Plus275-R (10 µM, AAGTGGATCTCTGCTGTCCC), 2.5 µL of purified nick translation product from step 6.4. and 7.5 µL of H₂O. Use the following conditions: 1 cycle: 98 °C for 30 s, 10-16 cycles: 98 °C for 10 s, 63° C for 10 s, 72° C for 15 s, 1 cycle: 72 °C for 10 min.
      NOTE: If necessary, several reactions can be set up in parallel to ensure there is enough PCR product for subsequent steps. To visualize small amounts of the PCR product on an agarose gel, set up additional reactions as in step 6.5 and increase the cycle number from 15 to 32. This reaction can be used as quality control, if amplicons are not visible after 15 cycles. Include also a no template control (NTC).

7. Size Selection

NOTE: This step separates MNase-fragments with the correctly attached 5' and 3' linker from fragments with two 5' or two 3' linkers based on size.

1. Combine all 15-cycle PCR reactions from step 6.5., add DNA loading dye and run on a 0.8% agarose gel. Excise the prominent band at 869 bp and purify DNA using a gel extraction kit.
2. Quantify the amount of DNA (e.g. using a spectrophotometer).

8. Cloning of PCR-amplified Fragments into the gRNA Expression Vector by Gibson Assembly

1. Prepare assembly master mix as follows:
   NOTE: The following steps are adapted from Gibson et al.¹².
   1. Create 6 mL isothermal reaction buffer by combining 3 mL of 1 M Tris(hydroxymethyl)aminomethane (Tris)-HCl pH 7.5, 300 µL of 1 M MgCl, 60 µL of 100 mM deoxyguanosine triphosphate (dGTP), 60 µL of 100 mM deoxyadenosine triphosphate (dATP), 60 µL of 100 mM deoxythymidine triphosphate (dTTP), 60 µL of 100 mM deoxycytidine triphosphate (dCTP), 300 µL of 1 M dithiothreitol (DTT), 1.5 g of polyethylene glycol (PEG)-8000, 300 µL of 100 mM nicotinamide adenine dinucleotide (NAD) in ultrapure H₂O.
      NOTE: This buffer can be aliquoted and stored at -20 °C.
   2. Create 1.2 mL assembly master mix by combining 320 µL of 5X isothermal reaction buffer, 3 µL of 10 U/µL T5 exonuclease, 20 µL of 2 U/µL DNA polymerase, 160 µL of 40 U/µL DNA ligase in ultrapure H₂O. Use 15 µL of assembly master mix with 5 µL of insert.
      NOTE: The assembly master mix can be aliquoted and stored at -20 °C, where it is stable for more than a year and can tolerate multiple freeze-thaw cycles. The chosen amount of T5 exonuclease is ideal for use with long overhangs.
2. Vector backbone digestion
   NOTE: Make sure to digest a sufficient amount of vector as input for the desired number of assembly reactions in step 8.3.
   1. Per reaction, add the following: 1.5 µg of gRNA expression vector (pgRNA-pLKO.1), 5 µL of buffer, 1.5 µL of AgeI (5U/µL), and 38.5 µL of H₂O. Incubate digest at 37 °C for 2 h.
3. Dephosphorylation of the linearized vector.
   NOTE: This step is advised, but not strictly necessary. T5 exonuclease in the master mix will mostly remove the AgeI overhangs before the Taq DNA ligase has had a chance to act. Therefore, excessive re-ligation of the vector is not expected.
   1. Add 2.5 µL of shrimp alkaline phosphatase enzyme (rSAP, 1 U/µL).
   2. Incubate at 37 °C for 30 min, and then inactivate the enzyme by incubating at 65 °C for 5 min.
4. Perform DNA purification
   NOTE: It is recommended to perform an agarose gel extraction step. Alternatively, column- or bead purification can be used. It is important to check that the vector digestion is complete, e.g. by agarose gel electrophoresis. For comparison, undigested vector should be run in parallel.
5. Quantify the amount of purified digested vector in the sample.
6. Set up the assembly with 2-fold molar excess of inserts to vector. Per 20 µL reaction use 100 ng of vector (AgeI digest from step 8.5), 12.2 ng of insert (from step 7.1.), 15 µL of assembly master mix from step 8.1.2. in H₂O.
7. Incubate at 50 °C for 1 h.
8. Purify the reactions using column purification. Resuspend the DNA in 75 µL of H₂O (or appropriate volume depending on scale of electroporation).
   NOTE: The transformation efficiency is greatly dependent on DNA purity. Additional purification steps (e.g. phenol/chloroform extraction) might improve efficiency.

9. Preparation of Electro-competent TG1 E. coli Cells

1. Ensure that all centrifuge bottles and flasks are free from detergents by rinsing and subsequent filling with distilled water before autoclaving.
   NOTE: This step helps to remove any impurities which may affect transformation efficiency. Water should be discarded immediately before use. Alternatively, use of disposable centrifugation bottles might be advisable.
2. Ensure that centrifuge bottles, tubes and solutions used for the preparation of electrocompetent cells are chilled on ice prior to use. It is best to conduct the following steps in a cold room to minimize temperature fluctuations which may affect the transformation efficiency.
3. Prepare 2TY medium. To 16 g of bacto tryptone, 10 g of yeast extract, and 5 g of NaCl, add distilled water to 1 L, mix and autoclave. Store medium at room temperature.
4. Prepare 2TY-agar coated bioassay dishes and 10 cm Petri dishes containing the appropriate antibiotic. Store plates at 4 °C.
   NOTE: The choice of antibiotic depends on the nature of the gRNA expression vector, use 100 µg/mL ampicillin for pgRNA-pLKO.1.
5. Scrape from a glycerol stock of TG1 cells to inoculate 10 mL of 2TY medium (without antibiotics).
6. Incubate culture at 37 °C overnight (~16 h) with shaking at 225 rpm.
7. Inoculate 1 L of 2TY medium (without antibiotics) with the 10 mL of overnight culture (1/100 dilution) and divide it equally between two 2 L flasks (containing baffles).
8. Incubate culture at 37 °C, 225 rpm until an OD600 nm of 0.55 is reached (approximately after 1.5 - 2 h). Use a spectrophotometer to check OD600 nm regularly.
9. Chill cultures on ice for 30 min.
10. Split the culture equally between four 500 mL centrifuge bottles (pre-chilled on ice).
11. Centrifuge for 15 min at 4,000 x g at 4 °C in a pre-chilled centrifuge.
12. Decant supernatants and add 1 volume (i.e. 250 mL) of pre-chilled ice-cold sterile distilled H₂O to each of the centrifuge bottles. Resuspend the bacterial pellet by swirling or inverting the bottle (or by gentle pipetting, if necessary).
    NOTE: It is easier to resuspend the pellet by first adding a small volume of water. Ensure the pellet is completely resuspended eventually.
13. Centrifuge for 15 min at 4,000 x g at 4 °C.
14. Repeat the wash two times (steps 9.12. and 9.13). Remove the supernatant. Be careful when decanting as the bacterial pellet becomes increasingly loose after washing.
15. Resuspend the pellet in 50 mL of sterile, ice-cold 10% glycerol and transfer it to a pre- chilled 50 mL centrifuge tube.
16. Centrifuge cells for 15 min at ~4,000 x g at 4 °C. Carefully remove the supernatant.
17. Gently resuspend the bacteria in 2 mL of ice-cold sterile 10% glycerol.
18. Keep on ice if the cells are to be used immediately for electroporation.
    NOTE: The cells can be frozen in aliquots of 50 µL in 0.5 mL tubes in a dry-ice ethanol bath and stored at -80 °C, but this is not recommended.

10. Electroporation of TG1 Electrocompetent E. coli Cells

NOTE: Electroporation is one of the bottlenecks in comprehensive library generation. To preserve the library representation, it is recommended to conduct as many individual electroporation reactions as necessary/practicable and to perform the quality control steps described below (10.6. and 10.8.).

1. Aliquot the purified reactions (from step 8.8) into sterile and pre-chilled PCR tubes and keep them on ice (1 µL per tube). Chill 1 mm gap electroporation cuvettes on ice.
2. Add 25 µL of freshly prepared TG1 cells directly to one aliquot of DNA and immediately transfer the mixture into an electroporation cuvette. Flick or tap the cuvette to ensure the cells/DNA mix is distributed along the length of the cuvette chamber (without any trapped air or bubbles).
3. Place the cuvette in the slide chamber and start the appropriate electroporation program (e.g. 1 pulse of 1.8 kV (EC1)).
   NOTE: The time constant should lie between 5.7 and 6.0 ms. In case the electroporator arcs, flick the cuvette, ensure there are no air bubbles in the chamber and try again.
4. Immediately add 975 µL of room temperature 2TY medium to the cuvette.
5. Using a transfer pipette, move the electroporated bacteria to a 50 mL tube. Repeat from step 10.2. and collect all cells transformed with one library in one 50 mL tube.
6. Document the total volume.
7. Quality control step
   1. To quantify the competence of the freshly generated electro-competent cells, perform a separate electroporation reaction using a defined quantity of uncut plasmid DNA (e.g. 10 pg pUC19 control plasmid). Transfer this to a 1.5 mL microfuge tube.
      NOTE: The competency should be at least 10¹⁰ colony-forming units (cfu) per µg DNA. Freshly prepared cells usually perform better than this.
8. Incubate transformed bacteria at 37 °C for 60 min shaking at 225 rpm.
9. Quality control step:
   1. Plate a defined small amount of bacteria transformed with the library (e.g. 10 µL of a 10-100 fold dilution) on a 10 cm agar plate with the appropriate antibiotic selection.
      NOTE: Knowing the total culture volume (from step 10.6.), the obtained colony number can be used to estimate the total number of colonies for the entire library. 20-30 fold representation of the library should ideally be maintained.
10. Disperse the remainder of the bacteria onto 2TY agar coated bioassay dishes containing the appropriate antibiotic selection.
    NOTE: The volume of the culture can be reduced by centrifugation at ~4,000 x g for 10 min (or until a visible pellet has formed and the supernatant appears clear). This reduces the time plates need to dry after spreading of the culture. Use of plates rather than liquid culture minimizes disproportionate growth of individual colonies.¹³
11. Spread an appropriate volume of the pUC19 control electroporation reaction on an additional 10 cm agar dish with appropriate antibiotic selection.
12. Incubate agar plates overnight (16 h) at 37 °C.
13. Count obtained colonies on the control plates (pUC19 and control library plate) to estimate CFU/µg DNA for the bacteria as well as library complexity.

11. Extraction of Plasmid DNA

1. Add 10 mL of 2-TY media to the overnight plates, scrape the bacterial layer off the plate using a disposable spreader and collect it in a 50 mL tube. Repeat a few times until all of the plate appears clean.
2. Extract DNA using a Plasmid Maxi kit (2-3 columns needed per Bio-assay plate).

结果

利用本方案，已从人和小鼠基因组DNA⁹以及BAC DNA成功构建了CORALINA gRNA文库（图1）。为了获得适合克隆至gRNA表达载体的输入DNA片段，必须确定限制性核酸酶消化的最佳条件。在微球菌核酸酶（MNase）消化条件优化中的典型结果如图2A所示。当核酸酶用量不足（0.1、2、3、4、4.5或5单位）时，在目标大小范围（10–100 bp）内几乎无明显产物生成；而5.5–7.5单位仍产生平均长度过长的片段。酶用量较高（50单位）时，在10分钟内即导致输入DNA过度降解。因此，选择了一个中间用量（10单位）。随后将消化反应放大，以产生足量的消化片段用于后续纯化与克隆（图2B）。尽管建议根据片段大小盲选DNA条带，并仅依赖DNA ladder进行定位，以尽量减少DNA暴露于紫外光下，但凝胶可在切割后染色，用于消化效果与切割质量的质控。图2B展示了一个典型的聚丙烯酰胺凝胶（PAGE）示例，其中已切取20–30 bp之间的DNA片段。经凝胶纯化的MNase消化片段被上样至20% PAGE凝胶，以验证片段大小选择及纯化的成功（图2C）。本方案兼容自定义接头序列的使用，可将MNase消化的片段克隆至任意选定的gRNA表达载体中。本研究中采用gRNA-PLKO⁹作为载体骨架。接头序列通过标准PCR从gRNA表达载体中扩增获得。图2D展示了一个典型的接头扩增结果，其中未出现额外、错误或无模板的扩增产物。接下来，使用限制性内切酶对扩增的接头进行消化，以确保接头能够以正确方向连接至MNase消化的DNA片段上。图2D显示了5'和3'接头在分别经HindIII和SacII酶切前后的琼脂糖凝胶电泳结果，表明接头已完全消化为预期大小的片段（分别为637 bp和295 bp）。凝胶图像右侧部分记录了酶切后接头片段的切胶回收过程。在完成接头片段的凝胶回收后，下一步是将接头与末端修复后的MNase消化片段进行连接。由于接头序列是使用未磷酸化的引物通过PCR生成的，因此接头自身不应发生自连。只有经过末端修复的MNase消化DNA片段才提供连接反应所需的磷酸基团。连接产物经切刻平移（nick translation）后，通过PCR进行扩增。为避免过度PCR扩增可能引起的偏差，从而影响文库中gRNA序列的代表性，总扩增循环数限制在20个循环以内。PCR完成后，扩增产物在琼脂糖凝胶上难以观察，因此需另设32个循环的对照PCR以检测产物（但该对照产物不用于文库构建）。该对照PCR的结果如图2E所示。此步骤可用于优化连接反应，并确保反应体系中不存在PCR假象，后者有时会在“无片段对照”（NFC）中出现。图2E展示了在片段与接头序列以等摩尔（1:1）比例进行连接反应后，经扩增获得的目标扩增子（5'接头 + DNA片段 + 3'接头，长度：869 bp）。

图1：gRNA文库制备建议时间线。 CORALINA提供了一种简单且成本效益高的策略，可从任何生物体的多种不同DNA来源中生成全面的gRNA文库。该方案可在一周的工作时间内完成。接头生成可与DNA末端修复并行进行。制备电转化感受态细菌需要两天时间，包括过夜培养步骤，因此应在组装反应设置之前开始。 请点击此处查看该图的放大版本。

图2：实验流程中的关键步骤。 (A) 对BAC DNA进行可控消化，可产生不同大小的片段。图中展示了MNase消化的优化结果。纯化的BAC DNA用不同量的MNase处理10分钟。10 U的MNase可生成所需长度（20–30 bp）的DNA片段。(B) 通过从聚丙烯酰胺凝胶中切胶回收，选择20至30 bp大小的片段。纯化的BAC DNA用10 U MNase处理10分钟。图像为切胶后拍摄。(C) 对凝胶纯化片段的质量控制。凝胶纯化后，取六分之一纯化的MNase片段上样至20% PAGE凝胶，以检测片段大小选择和纯化是否成功。(D) 扩增用于组装的接头序列，并对接头进行限制性内切酶消化以确保定向克隆。5'和3'接头分别经PCR扩增后，用HindIII和SacII酶切。设置无模板对照（NTC），以排除PCR假象和DNA污染。左侧：分析性上样；右侧：制备性上样。图像为凝胶切胶后拍摄。(E) 可通过增加PCR循环数（32次）进行PCR检测，以分析接头与DNA片段的连接是否成功，并设置以H₂O作为模板的无模板对照（NTC），或以前一步缺口平移反应的NTC作为模板（NTC NT）进行对照。必须包含无片段对照（NFC），即在连接和缺口平移反应中省略MNase片段的扩增对照。只有将MNase片段与接头DNA结合的样品才能扩增出预期产物（869 bp）。请点击此处查看该图的放大版本。

讨论

CORALINA 可通过受控的核酸酶消化靶 DNA 并对产生的双链片段进行大规模克隆，从而构建大规模的 gRNA 文库。统计推断表明，使用本方案已成功克隆的独立 gRNA 序列数量远超 10⁷ 个⁹。CORALINA 可在多个方面进行定制。模板 DNA 的选择决定了目标区域以及所生成文库的最大复杂度。使用本方案，此前已从人和小鼠的基因组 DNA 成功构建 CORALINA 文库⁹。本文展示的代表性结果描述了从纯化的 BAC DNA 构建 CORALINA 文库的过程。通过选择不同的 gRNA 表达载体和连接子序列，可进一步实现定制化。我们此前已测试了三种不同长度的连接子组合用于 Gibson 组装，其效率差异较小⁹。

由于CORALINA gRNA的间隔序列源自整体消化的DNA，其长度通常并非精确的20 bp，而是呈现一定的长度分布，其均值取决于MNase消化的参数以及从PAGE凝胶中切取的片段大小。图2B 和 C 所示的代表性示例中，片段的中位长度介于19至27 bp之间。根据我们的经验，所生成的gRNA间隔序列能够忠实地保留原始片段的长度⁹。由于过短的片段（短于20 bp）会导致gRNA的脱靶率升高，应予以避免；而较长的片段在后续应用中通常问题较小，已有研究表明，间隔序列长达45 bp的gRNA仍具有功能活性⁹。

CORALINA 实验方案中最关键的两个步骤是 MNase 消化片段的大小选择和克隆步骤。若产生的片段过短（例如，平均长度低于 18 bp），或引入过多空的 gRNA 表达载体，则会导致文库失效。因此，优化 MNase 消化步骤（图 2A）、监测片段切离情况（图 2B、C）、确认 gRNA 载体骨架被完全消化，并在整个实验流程中设置不含片段的对照组，均至关重要。同时必须特别注意保持 gRNA 文库的代表性。文库构建过程中常见的瓶颈之一是将质粒高效转入细菌以进行扩增。因此，需要大量具有高转化效率的感受态细菌以及大量的独立电穿孔操作，才能获得足够数量的 gRNA 克隆。

在未来几十年内，为了充分挖掘基于CRISPR的筛选方法的全部潜力，必须开发新的gRNA文库构建策略。目前迫切需要成本低廉、操作简便且可定制的方法来生成大规模文库，这是使筛选技术能够应用于更多模式系统以及不同CRISPR工程方法的前提条件。CORALINA为此迈出了第一步。其潜在应用十分广泛，尤其适用于构建基因组的全面文库、来源于非主流模式生物的cDNA文库、高度特异性的文库，以及联合使用多种具有不同PAM要求的CRISPR蛋白的实验设计。

与其他方法不同，CORALINA 能够从输入的 DNA 中生成所有可能的 gRNA。然而，该方法的一个缺点是文库中也包含了缺乏所需 PAM 序列的 gRNA，这一特点与另一种用于生成 gRNA 文库的酶法 CRISPR-EATING 共有（表 1）。选择理想的 gRNA 文库构建方法取决于计划进行的筛选实验的具体要求，尤其是目标区域的性质（基因区、调控区、基因间区）和大小（单个位点、多个区域或全基因组范围）。我们认为，在需要分析大量非编码区或调控区、序列信息不完整或不可靠（如非传统模式系统、物种混合样本（例如 微生物组）或实验获得的输入材料）、联合使用不同的 CRISPR 核酸内切酶，或对较短且明确的位点进行饱和分析（例如 由 BAC 克隆代表的区域）时，使用 CORALINA 具有特殊优势。

材料

本文使用的材料清单

姓名	公司	目录编号	评论
500 mM EGTA	Sigma Aldrich	03777-10G	1.4.，Mnase 的失活
Novex Hi-Density TBE 上样缓冲液	Thermo Fisher Scientific	LC6678	2.1.
Novex® TBE 凝胶，20%，10 孔	Thermo Fisher Scientific	EC6315BOX	2.1.，预制 20% PAGE 凝胶
O'RangeRuler 5 bp DNA 标准品	Thermo Fisher Scientific	SM1303	2.1.
Novex® TBE 电泳缓冲液	Thermo Fisher Scientific	LC6675	2.1.，PAGE 凝胶电泳缓冲液
一次性无菌手术刀	VWR	233-5363	2.3.，可使用其他等效试剂
SYBR Green I 核酸染料（DMSO 中 1000 倍浓缩液）	Sigma Aldrich	S9430	2.3. + 2.5.，Thermo Fisher Scientific 亦有供应（S7563）
UltraPure 苯酚:氯仿:异戊醇（25:24:1）	Thermo Fisher Scientific	15593-031	3.6.1. + 4.3.，可使用其他等效试剂
糖原	Sigma	10901393001	3.6.4.，可使用其他等效试剂
3M 乙酸钠，pH 5.2	Thermo Fisher Scientific	R1181	3.6.4.，可使用其他等效试剂
乙醇			3.6.4. + 9.1.8.，分子生物学级
快速平末端修复试剂盒	New England Biolabs	E1201	4.1.
乙酸铵	Sigma	A1542	3.1.，可使用其他等效试剂
乙酸镁	Sigma	M5661	3.1.，可使用其他等效试剂
0.5 M EDTA（pH 8.0）	VWR	MOLEM37465520（或 Promega V4231）	2.2. + 3.1.，可使用其他等效试剂
Agencourt AMPure XP 磁珠	Beckman Coulter	A63881	5.3. + 6.5.
凝胶回收试剂盒	QIAGEN	28704	5.7. + 7.1. + 8.4.，可使用其他等效试剂
高浓度 T4 DNA 连接酶	New England Biolabs	M0202T	6.1. + 8.1.2.
Long Amp Taq 2X 预混液	New England Biolabs	M0287S	6.3.
含 HF 缓冲液的 Phusion 高保真 PCR 预混液	New England Biolabs	M0531S	5.1. + 6.6.，可使用其他等效试剂
HindIII	New England Biolabs	R0104S	5.4.1.
SacII	New England Biolabs	R0157S	5.4.2.
AgeI	New England Biolabs	R0552S	8.2.1.
Tris 碱	Sigma	93362	8.1.1.
2M MgCl₂	Sigma	93362	8.1.1.
dGTP、dATP、dCTP、dTTP	New England Biolabs	N0446S	8.1.1.
DTT	Sigma	DTT-RO	8.1.1.
PEG-8000	Sigma	P5413	8.1.1.
NAD	Sigma	N6522	8.1.1.
T5 核酸外切酶	New England Biolabs	M0363S	8.1.2.
Phusion DNA 聚合酶	New England Biolabs	M0530S	8.1.2.
Taq DNA 连接酶	New England Biolabs	M0208L	8.1.2.
rSAP	New England Biolabs	M0371S	8.3.1.
TG1 感受态细胞	Lucigen	60502-1	9.1.
1 mm 间隙电穿孔比色皿	VWR	732-2267	10.2.
生物检测皿（聚苯乙烯，245 mm × 245 mm × 25 mm）	Fisher Scientific	DIS-988-010M	9.4.
NaCl	Sigma	S7653	9.3.
胰蛋白胨	BD	211705	9.3.
酵母提取物	BD	212750	9.3.
琼脂	Sigma	A1296	9.4.
甘油	Sigma	G5516	9.17.
MNAse	New England Biolabs	M0247S	1.1.
Nanodrop	Thermo Fisher Scientific	ND-2000	全文
MicroPulser	Bio-Rad	165-2100	10.2.
电穿孔比色皿	Bio-Rad	732-2267	10.2.
250 ml 离心管	Corning	430776	9.1–9.9.