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Dense sgRNA Library Construction Using a Molecular Chipper Approach
使用分子碎片机方法构建密集覆盖的sgRNA文库

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Abstract

Genetic screens using single-guide-RNA (sgRNA) libraries and CRISPR technology have been powerful to identify genetic regulators for both coding and noncoding regions of the genome. Interrogating functional elements in noncoding regions requires sgRNA libraries that are densely covering, and ideally inexpensive, easy to implement and flexible for customization. We present a Molecular Chipper protocol for generating dense sgRNA libraries from genomic regions of interest. This approach utilizes a combination of random fragmentation and a Type III restriction enzyme to derive a dense coverage of sgRNA library from input DNA.

Keywords: Molecular chipper(分子碎片机), sgRNA library(sgRNA文库), CRISPR-Cas9(CRISPR-Cas9), Non-coding genome(非编码基因组), Reporter screen(报告基因筛选)

Background

Genome editing using Streptococcus pyogenes (sp) Cas9 and sgRNA libraries is a powerful tool to screen for functional genetic regulators in mammalian cells by generating biallelic loss-of-function sequence alterations (Wiedenheft et al., 2012; Mali et al., 2013; Koike-Yusa et al., 2014; Shalem et al., 2014; Wang et al., 2014; Zhou et al., 2014). Cas9 binds sgRNA, which can be designed to target Cas9 toward a defined locus in the genome. The nuclease activity of Cas9 cuts target DNA locus, leading to double-stranded DNA breaks, which upon DNA repair through non-homologous end-joining pathway frequently results in short deletions at the locus of interest.
   The powerful genomic editing capacity of the CRISPR-Cas9 system has led to the use of sgRNA libraries to interrogate protein-coding genes as well as noncoding regions. Several sgRNA libraries for protein-coding genes and/or limited numbers of non-coding genes have been reported in functional screening, through sgRNA enrichment, to identify genes and networks regulating specific cellular functions (Koike-Yusa et al., 2014; Shalem et al., 2014; Wang et al., 2014; Zhou et al., 2014; Canver et al., 2015; Sanjana, 2016). Several non-coding sgRNA libraries consisting of 703-18,000 sgRNAs densely covering regulatory regions of genes of interest, such as BCL11A, Tdgf1a and drug-resistance regulating genes, were also reported in gene-specific functional screens for distal and proximal regulating elements (Canver et al., 2015; Rajagopal et al., 2016; Korkmaz et al., 2016; Sanjana, 2016). These sgRNA libraries were all produced by careful bioinformatics design, oligonucleotide synthesis on microarray, and cloning of oligonucleotide pool(s) into vectors. This synthetic approach has been very useful, but requires computational expertise for genome-wide sgRNA design and expensive microarray synthesis, and thus is challenging for most laboratories.
   Enzymatically generated sgRNA libraries covering regions of repetitive genomic sequences or loci are useful for CRISPR-Cas9 imaging of genomic sequences or loci (Lane et al., 2015). Due to lack of high-density (~111 bp), such sgRNA libraries are not reported in screening for functional non-coding regions. Another enzymatic method was reported to generate high-density (~20 bp) sgRNA library from cDNA (Arakawa, 2016). This type of sgRNA library consists of cell source-specific, differentially expressed sequences, thus, was neither reported for applications in functional screening.
   Without prior knowledge of the locations of critical noncoding-element-containing regions, functional mapping of noncoding genomic regions requires sgRNA libraries that densely populate regions of interest. The ideal method requires flexibility for adjusting the scale of sgRNA production to easily cope with this need. We describe here a detailed protocol of the Molecular Chipper approach that processes any input DNA piece(s) to generate a near base-resolution sgRNA library densely covering the input DNA of interest.

Materials and Reagents

  1. Pipette tips (prefer ones with filters to minimize contamination, such as those from Denville Scientific)
  2. Tubes (Denville Scientific, catalog number: C2170 )
  3. Petri dishes (Corning, Falcon®, catalog number: 351029 )
  4. NEB 5-alpha Electrocompetent E. coli (New England Biolabs, catalog number: C2989K )
  5. Retroviral vector pSUPER-CRISPR that contains and a puromycin selection marker and a U6 promoter to drive expression of sgRNA that is cloned at BamHI-HindIII sites (for details, see Cheng et al., 2016b).
    Note: Please write us to request for this material.
  6. T4 DNA ligase at 2000,000 U/ml (New England Biolabs, catalog number: M0202T ). Use in ligations where T4 DNA ligase is required in excess within a small volume, such as that described in step 2
  7. T4 DNA ligase at 400,000 U/ml (New England Biolabs, catalog number: M0202S )
  8. T4 polynucleotide kinase (New England Biolabs, catalog number: M0201S )
  9. Distilled water (AmericanBio, catalog number: AB02123-00500 )
  10. QIAquick PCR Purification Kit (QIAGEN, catalog number: 28104 )
  11. Agarose (AmericanBio, catalog number: AB00972 )
  12. Ethidium bromide, 10 mg/ml (Sigma-Aldrich, catalog number: E1510 )
  13. 3 M sodium acetate, pH 5.2
  14. 100% ethanol (AmericanBio, catalog number: AB00515-00500 )
  15. 70% ethanol (AmericanBio, catalog number: AB04010-00500 )
  16. 1 kb plus DNA standard (Thermo Fisher Scientific, InvitrogenTM, catalog number: 10787018 )
  17. Agarose, low melting point (AmericanBio, catalog number: AB00981 )
  18. 10 bp DNA standard (Thermo Fisher Scientific, InvitrogenTM, catalog number: 10821015 )
  19. NEBNext End Repair Module (New England Biolabs, catalog number: E6050S )
  20. 10,000x SYBR Safe DNA Gel Stain (Thermo Fisher Scientific, InvitrogenTM, catalog number: S33102 )
  21. QIAEX II Gel Extraction Kit (QIAGEN, catalog number: 20021 )
  22. QIAquick Gel Extraction Kit (QIAGEN, catalog number: 28704 )
  23. EcoP15I-adaptor oligonucleotide pair: sense aaaactcgagcagcagtggatccG and anti-sense /5phos/Cggatccactgctgctcgag (Integrated DNA Technologies; 25 nmole DNA oligo scale; Standard desalting purification). The anti-sense oligo has a 5’-phosphase modification
  24. 100 bp DNA standard (New England Biolabs, catalog number: N3231S )
  25. EcoP15I enzyme (New England Biolabs, catalog number: R0646L )
  26. PCI: Phenol:Chloroform:Isoamyl Alcohol 25:24:1, saturated with TE (10 mM Tris, pH 8.0, 1 mM EDTA) (Sigma-Aldrich, catalog number: P2069-100ML )
  27. Phenol, saturated with Tris, pH 7.5 (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 17914 )
  28. Chloroform (AmericanBio, catalog number: AB00350-00500 )
  29. PCI (Phenol:chloroform:isoamyl alcohol 25:24:1), Tris saturated (Roche Diagnostics, catalog number: 03117944001 )
  30. 3’ sgRNA backbone adaptor oligo nucleotide pair: sense /5phos/nngttttagagctagaaatagcaagttaaaataaggctagtccgttatcaacttgaaaaagtggcaccgagtcggtgc-tttttttaagctttat and anti-sense ataaagcttaaaaaaagcaccgactcggtgccactttttcaagttgataac-ggactagccttattttaacttgctatttctagctctaaaac (Integrated DNA Technologies, 100 nmole DNA oligo scale, Standard desalting purification). The -sense oligo has a 5’-phosphase modification
  31. BamHI-HF enzyme (New England Biolabs, catalog number: R3136S )
  32. HindIII-HF enzyme (New England Biolabs, catalog number: R3104S )
  33. MiniElute Gel Extraction kit (QIAGEN, catalog number: 28604 )
  34. LB medium (Thermo Fisher Scientific, InvitrogenTM, catalog number: 12795027 )
  35. QIAprep Spin Miniprep Kit (QIAGEN, catalog number: 27104 )
  36. Oligo nucleotide used in Sanger sequencing of cloned-sgRNA: ctccctttatccagccctca (Intergatred DNA Technologies, 25 nmole DNA oligo scale, Standard desalting purification)
  37. Ampicillin (AmericanBio, catalog number: AB00115-00100 )
  38. Tris base (Sigma-Aldrich, catalog number: T6066 )
  39. Ethylenediaminetetraacetic acid (EDTA) (Sigma-Aldrich, catalog number: EDS-100G )
  40. Glacial acetic acid (Sigma-Aldrich, catalog number: 695092 )
  41. Agar (AmericanBio, catalog number: AB01185-00500 )
  42. 50x TAE gel running buffer (see Recipes)

Equipment

  1. Pipettes
  2. 37 °C water bath (Fisher Scientific, model: Model 215 , catalog number: 15-462-15Q)
  3. NanoDrop 2000 (Thermo Fisher Scientific, model: NanoDropTM 2000 , catalog number: ND-2000)
  4. Microcentrifuge (Eppendorf, model: 5254 , catalog number: 022620444)
  5. S220 Focused-ultrasonicator and sonication vials (COVARIS, model: S220 )
  6. Gel Illuminator (UltraSlim LED Illuminator, Maestrogen, catalog number: SLB-01W )
  7. IncuBlock heating block (Danville Scientific, model: I-0259 , catalog number: 08302)
  8. Gel electrophoresis system (Thermo Fisher Scientific, Thermo ScientificTM, model: OwlTM EasyCastTM B1A , catalog number: B1A)
  9. Electroporation System (Bio-Rad Laboratories, model: Gene Pulser XcellTM, catalog number: 1652660 )

Procedure

  1. For large DNA fragments, such as BAC clones, start from step 6. If input DNA are small and composed of multiple pieces, such as individual PCR products, purified DNA pieces are quantified by a NanoDrop 2000 and pooled in an equal molar ratio.
  2. If using an input pool of multiple pieces of DNA, randomly ligated larger products are then generated using T4 DNA ligase in excess. Specifically, for each μg of DNA, the DNA pool are ligated with 4,000 U of T4 DNA ligase (New England Biolabs) in a 50 μl reaction for 3 h at 37 °C. We start with ~20 μg total DNA that have 5’-phosphate groups.
    Note: This step has been added to avoid biasing against regions located near the ends of input DNA pieces (because we perform a size selection after this fragmentation step, and sequences close to the ends would be represented by very small fragments after fragmentation, and thus would be under-represented in the final library).
  3. If directly using PCR products, perform a kinase reaction prior to ligation using T4 polynucleotide kinase (TPK). Specifically, we followed the manufacture NEB’s protocol of a reaction consisting of 300 pmol of PCR DNA, 5 μl of 10x buffer, 5 μl of 10 mM ATP, and 1 μl (10 U) of T4 PNK, supplemented with distilled water to total 50 μl, The reaction is incubated at 37 °C for 30 min, followed by inactivation at 65 °C for 20 min. DNA in the phosphorylation reaction is then purified by QIAGEN PCR DNA purification kit and eluted in 50 μl of distilled water prior to ligation.
  4. Run 10 μl or 200 ng of ligation products on 1% agarose gel with 0.5 μg/ml ethidium bromide in 1x TAE buffer, in order to check sizes of ligation products (usually > 10 kb on average).
  5. The ligated large amount of DNA is purified by ethanol precipitation. Add 10% volume of 3 M sodium acetate, pH 5.2, and 2 volumes of 100% ethanol; mix and then precipitate in -20 °C for one hour; spin down using a pre-cooled microcentrifuge for 10 min, wash with 70% ethanol; air dry for one minute; and re-suspend in 150 μl water.
  6. To generate random DNA fragments, ~14 μg of the input DNA (ligated if originating from multiple pieces) in 120 μl of water is sonicated in a S220 Focused-ultrasonicator for 90 sec to result in fragments peaking at sizes of 400-450 bp (Peak Power = 140 V, Duty Factor = 5, Cycle/Burst = 200 and Average Power = 7). Run at least 2-4 μl (250-500 ng) DNA using 1 kb Plus DNA standard (Invitrogen) on 2% agarose gel with ethidium bromide to check peak sizes of fragmented DNA (Figure 1A).


    Figure 1. DNA gel examples of critical steps in Molecular Chipper procedure. A. Sonicated DNA (2-4 μl or 250-500 ng) in step 6 was visualized on 2% agarose gel with the help of 250 ng of 1 kb Plus DNA standard, showing peak fragment size of 400-450 bp. B. 5 μl (~200 ng) of DNA fragments ligated to adaptor in step 9 was visualized on a 4% LMP agarose gel with 10 bp DNA standard, showing formation of adaptor dimer and trimer and ligated DNA fragment at higher molecular weight. C. The total ligation in step 9 was loaded onto a 1% agarose gel to visualize and to recover ligated DNA fragments in 200-1,000 bp linear DNA range, as detailed in step 10. D. Adaptor-ligated DNA fragments were digested with EcoP15I to release the 38-bp DNA ends and loaded onto 4% LMP agarose gel to purify the 38-bp DNA pool, as detailed in step 11. E. The full-length sgRNA pool digested with BamHI and HindIII was loaded onto 4% LMP agarose gel to view and purify the 115-bp sgRNA DNA pool, as detailed in step 15.
    Note: the ~115-bp DNA band appears faint due to high background.

  7. Sonicated fragments are repaired in a 150 μl End Repair reaction with 15 μl of the NEBnext End Repairing Enzyme Mix, 30 μl of NEBNext End Repair Reaction 10x buffer, supplemented with distilled water to total 300 μl and react at 25 °C for 30 min, followed by 1% agarose gel purification of the 400-450 bp DNA fragments (visualized with 1x SYBR Safe in the gel by a blue-light gel dock, such as the Maestrogen Illuminator, in order to avoid DNA damage under UV light especially short-wave UV light that is usually available in labs, when ethidium bromide is used to visualize DNA) by using QIAEX II Gel Extraction Kit.
  8. To obtain fragment ends from both ends of the random DNA fragments, an EcoP15I-adaptor is first prepared by annealing two oligonucleotides aaaactcgagcagcagtggatccG and /5phos/Cggatccactgctgctcgag (IDT) in equal molar ratio in 1x ligation buffer (NEB) at 10 μM by heating up to 95 °C and then gradually decreasing temperature by 1 °C per minute to 25 °C in a PCR machine or by incubating in boiled water cooling down gradually to room temperature. The annealed DNA adaptor contains an EcoP15I site (in bold) followed by a total 8-bp spacer, including a BamHI site (underlined) and a G (capitalized) at the end for later sgRNA cloning to serve as transcription start site from the U6 promoter.
  9.  ~12 μg of the DNA fragments are ligated, at a ~1:10 molar ratio to 6.0 μg of the above annealed EcoP15I-adaptor with 20,000 U T4 DNA ligase (New England Biolabs) in 300 μl reaction for 3 h at 37 °C. Run 5 μl of ligation on 4% low melting point (LMP) gel with 10-bp DNA standard (Invitrogen) to confirm successful ligation by visualizing formation of 44-bp adaptor dimer (we also saw trimer formation) and the presence of higher MW DNA (Figure 1B).
  10.  The adaptor-ligated DNA fragments in 200-1,000-bp linear standard range are purified from adaptor monomer and other non-specific bands by running them on a 1% agarose gel with NEB 100 bp DNA ladder (Figure 1C) and by using QIAEX II Gel Extraction Kit. 1% gel is important to minimize gel volume to increase yield.
  11.  ~5 μg of the EcoP15I-adaptor-ligated gel-purified DNA is digested by 100 U of EcoP15I enzyme (New England Biolabs) in 300 μl reaction with specified buffer and ATP concentration for exactly 1 h at 37 °C. To check completeness of digestion and efficiency of adaptor ligation in step 9, run 15 μl of the digestion with 10 bp DNA standard (Invitrogen) on 4% low-melting-point agarose gel with ethidium bromide to visualize the 38 bp (expecting ~25-50 ng of the 38 bp DNA fragment; Figure 1D).
  12. After digestion, EcoP15I digestion reaction is cleaned by equal-volume sequential phenol/PCI/chloroform extractions and centrifugation at 18,400 x g for 5 min after each extraction, to get rid of EcoP15I protein bound to DNA potentially. The upper phase is always collected during repeated extraction. The final supernatant is precipitated with ethanol by adding 1/10 volume of 3 M sodium acetate, pH 5.7, and 2-volume of 100% ethanol to precipitate DNA, centrifuging at 18,400 x g for 10 min and washing DNA pellet with 70% ethanol, air-dried and finally re-suspended the DNA pellet in 50 μl of distilled water.
  13. Precipitated digestion products are gel-purified (on 4% low-melting-point agarose gel) to obtain a ~38-bp DNA fragment pool (EcoP15I-adaptor + 19/17 bases from ends of random DNA fragments) by diluting the gel slice with 2 volumes of 1x TAE buffer, melting the gel slice at 70 °C on a heating block, extracting by phenol/PCI/chloroform and dissolving DNA in 20 μl water.
  14. To ligate to the rest of sgRNA backbone, 280 ng of the purified 38-bp DNA pool is ligated in 50 μl reaction with 4,000 U (New England Biolabs, M0202T) of T4 DNA ligase for 3 h at 37 °C, at a 1:5 molar ratio to 2.75 μg of a sgRNA-backbone-adaptor. The sgRNA-backbone-adaptor contains two Ns for binding to overhangs from EcoP15I digestion products, the remaining sgRNA sequence (without the target-recognition domain), a polyT stretch for polymerase III transcriptional termination, and a HindIII site for cloning. This sgRNA-backbone adaptor was prepared by annealing two oligonucleotides below, followed by 4% low-melting-point agarose gel-purification, by using QIAEX II Gel Extraction Kit, to eliminate improperly annealed products.
    Two oligonucleotides: /5phos/nngttttagagctagaaatagcaagttaaaataaggctagtccgttatcaacttgaaaaagtggcaccgagtcggtgc-tttttttaagctttat
    ataaagcttaaaaaaagcaccgactcggtgccactttttcaagttgataacggactagccttattttaacttgctatttctagctctaaaac (IDT).
    See step 8 for details of annealing the two oligonucleotides to prepare for the sgRNA-backbone adaptor DNA.
  15. The ligated sgRNA DNA pool is cleaned by QIAquick PCR Purification Kit, digested in 50 μl with 20 U each of BamHI and HindIII (New England Biolabs) overnight at 37 °C, and gel-purified by running with 10 bp DNA standard (Invitrogen) on 4% low-melting-point agarose (Figure 1E) and by using the MiniElute Gel Extraction Kit to obtain a ~115-bp sgRNA pool. The 115-bp DNA band on gel usually appears faint due to high gel background when running large ligated DNA amount.
  16. This sgRNA pool is quantified by SYBR Safe Gel Stain (Invitrogen) on a fluorometer (total ~10 ng sgRNA pool DNA generated from starting material specified in step 9), and ligated at 3:1 molar ratio with 40 U of T4 DNA ligase (New England Biolabs) per 100 ng total DNA per 10 μl reaction at room temperature overnight into BamHI-HindIII sites of a retroviral vector pSUPER-CRISPR which contains a U6 promoter and a puromycin selection marker (Cheng et al., 2016b).
  17. The ligated DNA is ethanol precipitated (see step 4 for details) and dissolved in 10 μl water.
  18. Ligation products are transformed into NEB5 alpha competent cells in 30 μl aliquots by electroporation using a GenePulser Xcell. The transformed cells from 10 electro-transformations are then pooled together, grown in 10-ml LB medium for 1 h. Several small fractions of transformation are plated to estimate total transformed clones. To estimate the % cloning efficiency, 10-20 colonies are grown up to miniprep DNA using the QIAprep Spin Miniprep Kit, and the cloned sgRNA inserts are confirmed by Sanger sequencing using oligo nucleotide ctccctttatccagccctca. We found that about 80% clones have 19-20 mer sgRNAs cloned. The other 20% clones have much shorter sgRNAs cloned, likely due to non-specific cleavage activity of the EcoP15I enzyme.
  19. The transformation culture is grown overnight in 100 ml of LB medium containing 100 μg ml-1 of ampicillin for plasmid DNA preparation. Properly prepared, a library of > 1 million clones can be easily achieved.
  20. For retrovirus preparation, an example of functional reporter screen, sgRNA enrichment data analysis to identify functional non-coding genomic regions, and examples of mechanistic studies, see our previous publication (Cheng et al., 2016b).

Data analysis

  1. Sanger DNA sequencing data was analyzed using the DNA alignment feature in the ApE program, which can be downloaded and installed free at http://biologylabs.utah.edu/jorgensen/wayned/ape/. To do that, open the two sequences in ApE, choose Align Two Sequences under Tools in the program, specify the two sequence files in the pull-down menu in the new window, and choose OK to compute and generate the new alignment file.
  2. sgRNA library complexity was calculated as total E. coli colony number on plate times dilution factors.

Notes

  1. To better understand different stages of the construction of the sgRNA library, see a flow chart of the entire technical procedure in our previous publication (Cheng et al., 2016b) and a general conceptual flow chart of Molecular chipper-sgRNA library generation in comparison to conventional array-synthesized sgRNA library generation (Cheng et al., 2016a).
  2. The specified, optimized ligation and digestion time is important.
  3. Starting with a large amount of input DNA is critical. We start with minimal 20 μg of input DNA in our original report (Cheng et al., 2016b) and we usually prepare at least 40-60 μg of starting DNA material in case of failure at any intermediate step in the procedure.
  4. 1:5 molar ratio ligation to adaptors is important for efficient ligation of adaptors to both ends of DNA fragments. More adaptors may help to increase the ligation efficiency, but would cause higher background in gel purification, thus difficult to visualize the ligated DNA products.
  5. Obtaining hundreds of nanograms of the 38 bp fragments for ligating to the sgRNA backbone is a must for going forward.
  6. Desired complexity/coverage is achieved by transforming enough high-competency competent cells. We compared available commercial cells and found that NEB 5-alpha Electrocompetent E. coli is among the highest competency.
  7. If there are no 38 bp bands present after the first EcoP15I digestion, 1) Check if the adaptor has annealed well: ligate only the adaptors which should result in the presence of a major band of the monomer and minor non-specific bands on 4% low melting point gel; 2) Check if sonicated DNA are repaired well and if ligation goes well: treat a small fraction of repaired DNA by Taq DNA polymerase in the presence of dNTPs at 72 °C for 5 min, followed by TA cloning (Invitrogen): and many white colonies should appear if end repairing works well and sequencing of the cloned inserts should reveal adaptors at both ends of the cloned fragments; 3) Purify the adaptors from impurities by running on 3-4% LMP gel to reduce background during gel purification of the ligated DNA.
  8. If there are not enough colonies/coverage after transforming the final ligation, 1) Identify a commercial source of competent cell with high transformation efficiency, e.g., electroporation competent cells from NEB; 2) Alternatively, make your own electro-competent cells of higher efficiency; 3) Perform a test transformation to calculate total colony number/complexity/coverage before transforming the rest of ligation to make the whole DNA library.
  9. If cloning efficiency is not high, 1) Digest the full-length ligated sgRNA fragment with BamHI and HindIII for 6 h instead of overnight to minimize star activity; 2) Test the vector quality by cloning a mock BamHI-HindIII fragment and if necessary, a stuffer can be cloned first for efficient preparation of the double-enzyme digested vector.

Recipes

  1. 50x TAE gel running buffer per liter
    242 g Tris base
    18.61 g EDTA
    57.1 ml glacial acetic acid
    Dissolve in distilled water and bring final volume to 1 L

Acknowledgments

This study was supported in part by NIH grants R01CA149109 (to J.L.) and R01GM099811 (to Y.D. and J.L.). The protocol was adapted from previous work (Cheng et al., 2016a and 2016b). The authors declare no conflicts of interest or competing interests that may impact the design and implementation of this protocol.

References

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  3. Cheng, J., Pan, W., Roden, C., Chen, Z., and Lu, J. (2016a). Molecular Chipper: Functional Mapping of the Non-Coding Genome with CRISPR. Next Generat Sequenc & Applic 3: 132.
  4. Cheng, J., Roden, C. A., Pan, W., Zhu, S., Baccei, A., Pan, X., Jiang, T., Kluger, Y., Weissman, S. M., Guo, S., Flavell, R. A., Ding, Y. and Lu, J. (2016b). A Molecular Chipper technology for CRISPR sgRNA library generation and functional mapping of noncoding regions. Nat Commun 7: 11178.
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  6. Korkmaz, G., Lopes, R., Ugalde, A. P., Nevedomskaya, E., Han, R., Myacheva, K., Zwart, W., Elkon, R. and Agami, R. (2016). Functional genetic screens for enhancer elements in the human genome using CRISPR-Cas9. Nat Biotechnol 34(2): 192-198.
  7. Lane, A. B., Strzelecka, M., Ettinger, A., Grenfell, A. W., Wittmann, T. and Heald, R. (2015). Enzymatically generated CRISPR libraries for genome labeling and screening. Dev Cell 34(3): 373-378.
  8. Mali, P., Esvelt, K. M. and Church, G. M. (2013). Cas9 as a versatile tool for engineering biology. Nature methods 10(10): 957-963.
  9. Rajagopal, N., Srinivasan, S., Kooshesh, K., Guo, Y., Edwards, M. D., Banerjee, B., Syed, T., Emons, B. J., Gifford, D. K. and Sherwood, R. I. (2016). High-throughput mapping of regulatory DNA. Nat Biotechnol 34(2): 167-174.
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简介

使用单导向RNA(sgRNA)文库和CRISPR技术的遗传筛选功能强大可以识别基因组编码区和非编码区的遗传调控因子。 在非编码区域中询问功能元件需要密集覆盖的sgRNA文库,理想的便宜,易于实现和灵活定制。 我们提出了一个分子切片方案从感兴趣的基因组区域产生密集的sgRNA文库。 该方法利用随机断裂和III型限制酶的组合从输入DNA导出sgRNA文库的致密覆盖。
【背景】使用化脓性链球菌(sp)的基因组编辑Cas9和sgRNA文库是通过产生双重缺失功能序列改变来筛选哺乳动物细胞功能性遗传调节因子的有力工具(Wiedenheft et al。,2012; Mali et al。,2013; Koike-Yusa等,2014; Shalem等,2014; Wang等,2014; Zhou等,2014)。 Cas9结合sgRNA,其可被设计为将Cas9靶向基因组中定义的基因座。 Cas9的核酸酶活性切割靶DNA位点,导致双链DNA断裂,在通过非同源末端连接途径进行DNA修复时,经常导致感兴趣的基因座短缺失。
CRISPR-Cas9系统强大的基因组编辑能力导致使用sgRNA文库来询问蛋白质编码基因以及非编码区域。通过sgRNA富集功能筛选,报告了几种用于蛋白质编码基因和/或有限数量的非编码基因的sgRNA文库,以鉴定调控特定细胞功能的基因和网络(Koike-Yusa等,2014; Shalem et al。 2014; Wang等,2014; Zhou等,2014; Canver等,2015; Sanjana,2016)。还报道了几个非编码的sgRNA文库,其密集地覆盖感兴趣的基因的调控区,如BCL11A,Tdgf1a和耐药调节基因的703-18,000个sgRNA,也被用于远端和近端调节元件的基因特异性功能筛选(Canver et al。,2015; Rajagopal et al。,2016; Korkmaz et al。,2016; Sanjana,2016)。这些sgRNA文库均通过仔细的生物信息学设计,微阵列上的寡核苷酸合成和寡核苷酸库的克隆到载体中产生。这种合成方法非常有用,但是需要针对全基因组sgRNA设计和昂贵的微阵列合成的计算专长,因此对于大多数实验室来说是具有挑战性的。
酶切产生的覆盖重复基因组序列或基因座区域的sgRNA文库可用于基因组序列或基因座的CRISPR-Cas9成像(Lane等,2015)。由于缺乏高密度(〜111bp),在功能非编码区的筛选中没有报道这样的sgRNA文库。据报道另一种酶法从cDNA中产生高密度(〜20bp)sgRNA文库(Arakawa,2016)。这种类型的sgRNA文库由细胞来源特异性差异表达的序列组成,因此,在功能筛选中没有报道应用。
没有关于非关键非编码元件区域的位置的先前知识,非编码基因组区域的功能映射需要密集地填充感兴趣区域的sgRNA文库。理想的方法需要灵活调整sgRNA生产的规模,以便轻松应对这一需求。我们在这里描述了分子切片方法的详细方案,其处理任何输入的DNA片段以产生密集覆盖感兴趣的输入DNA的近碱基分辨率sgRNA文库。

关键字:分子碎片机, sgRNA文库, CRISPR-Cas9, 非编码基因组, 报告基因筛选

材料和试剂

  1. 移液器提示(更喜欢使用过滤器以最大限度地减少污染,例如来自Denville Scientific)
  2. 管(Denville Scientific,目录号:C2170)
  3. 培养皿(Corning,Falcon ®,目录号:351029)
  4. NEB 5-alpha Electrocompetent E。大肠杆菌(New England Biolabs,目录号:C2989K)
  5. 逆转录病毒载体pSUPER-CRISPR含有嘌呤霉素选择标记和U6启动子,以驱动在HindⅢ位点上克隆的sgRNA的表达(详见参见Cheng等人,2016b)。
    注意:请写信请求本材料。
  6. T4 DNA连接酶,2000,000 U/ml(New England Biolabs,目录号:M0202T)。用于需要T4 DNA连接酶在小体积中过量的连接,如步骤2中所述的
  7. T4 DNA连接酶在40万U/ml(New England Biolabs,目录号:M0202S)
  8. T4多核苷酸激酶(New England Biolabs,目录号:M0201S)
  9. 蒸馏水(AmericanBio,目录号:AB02123-00500)
  10. QIAquick PCR纯化试剂盒(QIAGEN,目录号:28104)
  11. 琼脂糖(AmericanBio,目录号:AB00972)
  12. 溴化乙锭,10mg/ml(Sigma-Aldrich,目录号:E1510)
  13. 3 M醋酸钠,pH 5.2
  14. 100%乙醇(AmericanBio,目录号:AB00515-00500)
  15. 70%乙醇(AmericanBio,目录号:AB04010-00500)
  16. 1kb加DNA标准品(Thermo Fisher Scientific,Invitrogen TM,目录号:10787018)
  17. 琼脂糖,低熔点(AmericanBio,目录号:AB00981)
  18. 10 bp DNA标准品(Thermo Fisher Scientific,Invitrogen TM,目录号:10821015)
  19. NEBNext End Repair Module(New England Biolabs,目录号:E6050S)
  20. 10,000x SYBR安全DNA凝胶染色剂(Thermo Fisher Scientific,Invitrogen TM,目录号:S33102)
  21. QIAEX II凝胶提取试剂盒(QIAGEN,目录号:20021)
  22. QIAquick凝胶提取试剂盒(QIAGEN,目录号:28704)
  23. p15I-接头寡核苷酸对:有义的aaaactcgag cagcag tggatccG和反义/5phos/Cggatcca ctgctg ctcgag(Integrated DNA Technologies; 25nmole DNA小规模;标准脱盐净化)。反义寡核苷酸具有5'-磷酸酶修饰物
  24. 100 bp DNA标准品(New England Biolabs,目录号:N3231S)
  25. P P15I酶(New England Biolabs,目录号:R0646L)
  26. PCI:苯酚:氯仿:异戊醇25:24:1,用TE(10mM Tris,pH8.0,1mM EDTA)饱和(Sigma-Aldrich,目录号:P2069-100ML)
  27. 用Tris饱和的酚,pH7.5(Thermo Fisher Scientific,Thermo Scientific TM,目录号:17914)
  28. 氯仿(AmericanBio,目录号:AB00350-00500)
  29. PCI(苯酚:氯仿:异戊醇25:24:1),Tris饱和(Roche Diagnostics,目录号:03117944001)
  30. 3'sgRNA骨架衔接子寡核苷酸对:sense/5phos/nngttttagagctagaaatagcaagttaaaataaggctagtccgttatcaacttgaaaaagtggcaccgagtcggtgc-ttttttatactctatat和anti-sense ataaagcttaaaaaaagcaccgactcggtgccactttttcaagttgataac-ggactagccttattttaacttgctatttctagctctaaac(Integrated DNA Technologies,100nmole DNA oligo scale,Standard desalting purification)。敏感寡核苷酸具有5'-磷酸酶修饰
  31. HI-HF酶(New England Biolabs,目录号:R3136S)
  32. dIII-HF酶(New England Biolabs,目录号:R3104S)
  33. MiniElute凝胶提取试剂盒(QIAGEN,目录号:28604)
  34. LB培养基(Thermo Fisher Scientific,Invitrogen TM,目录号:12795027)
  35. QIAprep Spin Miniprep Kit(QIAGEN,目录号:27104)
  36. 用于克隆sgRNA的Sanger测序中的寡核苷酸:ctccctttatccagccctca(Intergatred DNA Technologies,25nmole DNA oligo scale,Standard desalting purification)
  37. 氨苄青霉素(AmericanBio,目录号:AB00115-00100)
  38. Tris碱(Sigma-Aldrich,目录号:T6066)
  39. 乙二胺四乙酸(EDTA)(Sigma-Aldrich,目录号:EDS-100G)
  40. 冰醋酸(Sigma-Aldrich,目录号:695092)
  41. 琼脂(AmericanBio,目录号:AB01185-00500)
  42. 50x TAE凝胶运行缓冲液(见配方)
    1. 材料和试剂

      1. 移液器提示(更喜欢使用过滤器以最大限度地减少污染,例如来自Denville Scientific)
      2. 管(Denville Scientific,目录号:C2170)
      3. 培养皿(Corning,Falcon ®,目录号:351029)
      4. NEB 5-alpha Electrocompetent E.大肠杆菌(New England Biolabs,目录号:C2989K)
      5. 逆转录病毒载体pSUPER-CRISPR含有嘌呤霉素选择标记和U6启动子,以驱动在HindⅢ位点上克隆的sgRNA的表达(详见参见Cheng等人,2016b)。 注意:请写信请求本材料。
      6. T4 DNA连接酶,2000,000 U/ml(New England Biolabs,目录号:M0202T)。用于需要T4 DNA连接酶在小体积中过量的连接,如步骤2中所述的
      7. T4 DNA连接酶在40万U/ml(New England Biolabs,目录号:M0202S)
      8. T4多核苷酸激酶(New England Biolabs,目录号:M0201S)
      9. 蒸馏水(AmericanBio,目录号:AB02123-00500)
      10. QIAquick PCR纯化试剂盒(QIAGEN,目录号:28104)
      11. 琼脂糖(AmericanBio,目录号:AB00972)
      12. 溴化乙锭,10mg/ml(Sigma-Aldrich,目录号:E1510)
      13. 3 M醋酸钠,pH 5.2
      14. 100%乙醇(AmericanBio,目录号:AB00515-00500)
      15. 70%乙醇(AmericanBio,目录号:AB04010-00500)
      16. 1kb加DNA标准品(Thermo Fisher Scientific,Invitrogen TM,目录号:10787018)
      17. 琼脂糖,低熔点(AmericanBio,目录号:AB00981)
      18. 10 bp DNA标准品(Thermo Fisher Scientific,Invitrogen TM,目录号:10821015)
      19. NEBNext End Repair Module(New England Biolabs,目录号:E6050S)
      20. 10,000x SYBR安全DNA凝胶染色剂(Thermo Fisher Scientific,Invitrogen TM,目录号:S33102)
      21. QIAEX II凝胶提取试剂盒(QIAGEN,目录号:20021)
      22. QIAquick凝胶提取试剂盒(QIAGEN,目录号:28704)
      23. p15I-接头寡核苷酸对:有义的aaaactcgag cagcag tggatccG和反义/5phos/Cggatcca ctgctg ctcgag(Integrated DNA Technologies; 25nmole DNA小规模;标准脱盐净化)反义寡核苷酸具有5'-磷酸酶修饰物
      24. 100 bp DNA标准品(New England Biolabs,目录号:N3231S)
      25. P


        P15I酶(New England Biolabs,目录号:R0646L)
      26. PCI:苯酚:氯仿:异戊醇25:24:1,用TE(10mM Tris,pH8.0,1mM EDTA)饱和(Sigma-Aldrich,目录号:P2069-100ML)
      27. 用Tris饱和的酚,pH7.5(Thermo Fisher Scientific,Thermo Scientific TM,目录号:17914)
      28. 氯仿(AmericanBio,目录号:AB00350-00500)
      29. PCI(苯酚:氯仿:异戊醇25:24:1),Tris饱和(Roche Diagnostics,目录号:03117944001)
      30. 3'sgRNA骨架衔接子寡核苷酸对:感测/5phos/nngttttagagctagaaatagcaagttaaaataaggctagtccgttatcaacttgaaaaagtggcaccgagtcggtgc-ttttttatactctatat和反义ataaagcttaaaaaaagcaccgactcggtgccactttttcaagttgataac-ggactagccttattttaacttgctatttctagctctaaac(集成DNA技术,DNA 100nmole寡规模,标准脱盐纯化)敏感寡核苷酸具有5'-磷酸酶修饰
      31. HI-HF酶(New England Biolabs,目录号:R3136S)
      32. dIII-HF酶(New England Biolabs,目录号:R3104S)
      33. MiniElute凝胶提取试剂盒(QIAGEN,目录号:28604)
      34. LB培养基(Thermo Fisher Scientific,Invitrogen TM,目录号:12795027)
      35. QIAprep Spin Miniprep Kit(QIAGEN,目录号:27104)
      36. 用于克隆sgRNA的Sanger测试中的寡核苷酸:ctccctttatccagccctca(Intergatred DNA Technologies,25nmole DNA oligo scale,Standard desalting purification)
      37. 氨苄青霉素(AmericanBio,目录号:AB00115-00100)
      38. Tris碱(Sigma-Aldrich,目录号:T6066)
      39. 乙二胺四乙酸(EDTA)(Sigma-Aldrich,目录号:EDS-100G)
      40. 冰醋酸(Sigma-Aldrich,目录号:695092)
      41. 琼脂(AmericanBio,目录号:AB01185-00500)
      42. 50x TAE凝胶运行缓冲液(见配方)
        1. 程序

          1. 对于大的DNA片段,例如BAC克隆,从步骤6开始。如果输入DNA很小并且由多个片段组成,例如单独的PCR产物,则通过NanoDrop 2000定量纯化的DNA片段并以相等的摩尔比合并。 br />
          2. 如果使用多片DNA的输入池,则随机连接较大的产物,然后使用T4 DNA连接酶过量产生。具体地说,对于每μgDNA,将DNA库与4,000U T4 DNA连接酶(New England Biolabs)在50μl反应中在37℃下连接3小时。我们从约5μg磷酸基团的总DNA约20μg开始。
            注意:添加此步骤以避免偏离输入DNA片段末端附近的区域(因为我们在此片段化步骤之后进行大小选择,并且接近末端的序列将在碎片后由非常小的片段表示) ,因此在最终图书馆中的代表性不足)。
          3. 如果直接使用PCR产物,则在使用T4多核苷酸激酶(TPK)进行连接之前进行激酶反应。具体来说,我们遵循制造NEB的方案,该反应由300 pmol的PCR DNA,5μl的10x缓冲液,5μl的10mM ATP和1μl(10U)的T4 PNK组成,并补充蒸馏水至总计50将反应物在37℃下孵育30分钟,然后在65℃灭活20分钟。磷酸化反应中的DNA然后通过QIAGEN PCR DNA纯化试剂盒纯化,并在连接前在50μl蒸馏水中洗脱。
          4. 在1x TAE缓冲液中,用0.5μg/ml溴化乙锭在1%琼脂糖凝胶上运行10μl或200ng连接产物,以检查连接产物的大小(平均通常> 10kb)。
          5. 通过乙醇沉淀纯化连接的大量DNA。加入10%体积的3M乙酸钠,pH 5.2和2体积的100%乙醇;混合,然后在-20℃下沉淀1小时;使用预冷微量离心机旋转10分钟,用70%乙醇洗涤;空气干燥1分钟;并在150μl水中重新悬浮。
          6. 为了产生随机DNA片段,在S220聚焦 - 超声波仪中超声处理约120微升输入DNA(如果从多个片段连接起来),在120μl水中超声处理90秒以产生400-450bp的片段峰值功率= 140 V,占空因数= 5,循环/突发= 200,平均功率= 7)。使用1 kb Plus DNA标准品(Invitrogen)在2%琼脂糖凝胶上使用溴化乙锭至少运行2-4μl(250-500 ng)DNA,以检查碎片DNA的峰值大小(图1A)。


            图1.分子芯片方法中关键步骤的DNA凝胶实例 A.步骤6中的超声处理的DNA(2-4μl或250-500ng)在2%琼脂糖凝胶上显现,借助于250 ng 1 kb Plus DNA标准品,显示400-450 bp的峰片段大小。 B.在具有10bp DNA标准的4%LMP琼脂糖凝胶上观察到在步骤9中连接到衔接子的5μl(〜200ng)DNA片段,显示了更高分子量的衔接子二聚体和三聚体和连接的DNA片段的形成。 C.步骤9中的总连接装载到1%琼脂糖凝胶上,以显现并回收200-1,000bp线性DNA范围内的连接的DNA片段,如步骤10所详述。D.将适配器连接的DNA片段用 Eco P15I以释放38bp的DNA末端并加载到4%的LMP琼脂糖凝胶上以纯化38bp的DNA库,如步骤11中所详述的.E。全长sgRNA库用<将Hind III和Hind III载于4%LMP琼脂糖凝胶上以观察和纯化115bp的sgRNA DNA池,如步骤15所详述。注意,〜115-由于背景较高,bp DNA条带显得微弱
          7. 超声处理的片段在150μl末端修复反应中用15μlNEBnextEnd Repairing Enzyme Mix,30μlNEBNextEnd修复反应10x缓冲液修复,补充有蒸馏水至总共300μl,并在25℃下反应30分钟,然后用400-450bp的DNA片段进行1%的琼脂糖凝胶纯化(用1x-SYBR安全在蓝色凝胶底座如Maestrogen Illuminator凝胶中显现,以避免在UV光下DNA损伤,通过使用QIAEX II凝胶提取试剂盒,通常在实验室可以使用溴化乙锭来观察DNA)
          8. 为了从随机DNA片段的两端获得片段末端,首先通过退火两种寡核苷酸aaaactcgag cagcag tggatccG和/5phos/Cggatcca 来制备 ctgctg ctcgag(IDT)在1x连接缓冲液(NEB)中以10μM的比例加热至95℃,然后在PCR机中逐渐降温至1℃至25℃,通过在开水中逐渐冷却至室温。退火的DNA适配器含有EcoIp15I位点(粗体),然后是总共8-bp的间隔区,包括Bam HI位点(下划线)和G(大写)最终用于后续的sgRNA克隆作为U6启动子的转录起始位点
          9. 将〜12μg的DNA片段以〜1:10的摩尔比与6.0μg的上述退火的Eco-P15I-衔接子与20,000U T4 DNA连接酶(New England Biolabs)在300μl反应3 h,37°C。具有10-bp DNA标准(Invitrogen)的4%低熔点(LMP)凝胶上行5μl连接,以通过可视化形成44-bp适配器二聚体(我们也看到三聚体形成)并且存在较高MW DNA(图1B)
          10. 通过将其连接到具有NEB 100bp DNA梯的1%琼脂糖凝胶(图1C)上,并通过使用具有200-1,000bp线性标准范围的衔接物连接的DNA片段,从衔接子单体和其它非特异性条带纯化QIAEX II凝胶提取试剂盒1%凝胶对于最小化凝胶体积以增加产量为重要的。
          11. 将约5μg的EcoRⅠ连接的凝胶纯化的DNA用于100U的EcoRI P15I酶(New England Biolabs)在300μl与指定的缓冲液和ATP浓度在37°C正好1小时。为了检查步骤9中适配器连接的消化和效率的完整性,在4%低熔点琼脂糖凝胶上用溴化乙锭进行15μlDNA标准品(Invitrogen)的15μl消化,以显示38 bp(期望〜25 -50ng的38bp DNA片段;图1D)
          12. 消化后,通过等体积连续的苯酚/PCI /氯仿提取物清洗P15I消化反应,并在每次提取后以18,400×g离心5分钟,以除去潜在的与DNA结合的P15I蛋白反应提取时总是收集上层相。通过加入1/10体积的3M乙酸钠,pH 5.7和2体积的100%乙醇,用乙醇沉淀最终的上清液以沉淀DNA,以18,400×g离心10分钟并洗涤用70%乙醇的DNA沉淀,空气干燥,最后将DNA沉淀重新悬浮于50μl蒸馏水中。
          13. 沉淀的消化产物被凝胶纯化(在4%低熔点琼脂糖凝胶上),以获得约38bp的DNA片段(EcoRI/P15I-衔接子+来自随机末端的19/17碱基)DNA片段)通过用2倍体积的1×TAE缓冲液稀释凝胶切片,将加热块上70℃的凝胶切片溶解,用苯酚/PCI /氯仿萃取,并将DNA溶于20μl水中。
          14. 为了连接到其余的sgRNA主链,将280ng纯化的38-bp DNA库与40,000U(New England Biolabs,M0202T)T4 DNA连接酶在37℃下以50μl反应连接3小时,以1:5摩尔比至2.75微克的sgRNA-主链转接头。sgRNA-主链 - 衔接子含有两个Ns,用于结合来自EcoRI P15I消化产物的悬垂,剩余的sgRNA序列(无目标识别结构域),聚合酶III转录终止的polyT拉伸和 dIII克隆位点。通过使用QIAEX II凝胶提取试剂盒,通过退火两种寡核苷酸,随后进行4%低熔点琼脂糖凝胶纯化来制备该sgRNA-主链衔接子,以消除不合适的退火产物 两个寡核苷酸:/5phos/nngttttagagctagaaatagcaagttaaaataaggctagtccgttatcaacttgaaaaagtggcaccgagtcggtgc-tttttttaagctttat
            ataaagcttaaaaaaagcaccgactcggtgccactttttcaagttgataacggactagccttattttaacttgctatttctctctaaac(IDT)。
            有关退火两个寡核苷酸以准备sgRNA-主链转基因DNA的详细信息,请参见步骤8
          15. 连接的sgRNA DNA池通过QIAquick PCR Purification Kit进行清洗,在50μl中用20μL每个BamHⅠ和HindⅢ(New England Biolabs)消化,37℃过夜通过在4%低熔点琼脂糖(图1E)上运行10bp DNA标准品(Invitrogen)并通过使用MiniElute凝胶提取试剂盒获得〜115bp的sgRNA库来凝胶纯化。当运行大量连接的DNA量时,凝胶上的115-bp DNA带通常显得微弱,因为高凝胶背景
          16. 通过SYBR Safe Gel Stain(Invitrogen)在荧光计上定量该sgRNA库(总共〜10ng sgRNA库DNA,由步骤9中规定的原料产生),并以40:1的T4 DNA连接酶以3:1的摩尔比连接( New England Biolabs),每100μl总DNA在室温下每10μl反应过夜进入含有U6启动子的逆转录病毒载体pSUPER-CRISPR的BamHⅠ-HindⅢ位点和嘌呤霉素选择标记(Cheng等人,2016b)。
          17. 连接的DNA是乙醇沉淀的(详见步骤4),并溶于10μl水中
          18. 使用GenePulser Xcell通过电穿孔将连接产物以30μl等分试样转化为NEB5α感受态细胞。然后将来自10次电转化的转化细胞合并在一起,在10ml LB培养基中培养1小时。进行几个小部分转化以估计总转化克隆。为了估计%克隆效率,使用QIAprep Spin Miniprep试剂盒将10-20个菌落生长至微量制备DNA,通过使用寡核苷酸ctccctttatccagccctca的Sanger测序证实克隆的sgRNA插入片段。我们发现约80%的克隆克隆了19-20个mer sgRNA。其他20%的克隆克隆了更短的sgRNA,这可能是由于EcoRI酶P15I的非特异性切割活性。
          19. 转化培养物在100ml含有100μg/ml氨苄青霉素的LB培养基中生长过夜,用于质粒DNA制备。正确准备,一个> 100万克隆可以轻松实现。
          20. 对于逆转录病毒制备,功能性报告筛选的例子,sgRNA富集数据分析以鉴定功能性非编码基因组区域,以及机械研究的实例,参见我们以前的出版物(Cheng等人,2016b)。
            1. 数据分析

              1. 使用ApE程序中的DNA对齐特征分析Sanger DNA测序数据,可以在 http://biologylabs.utah.edu/jorgensen/wayned/ape/。要做到这一点,打开ApE中的两个序列,在程序中的"工具"下选择"对齐两个序列",在新窗口的下拉菜单中指定两个序列文件,然后选择"确定"计算并生成新的对齐文件。 br />
              2. sgRNA文库复杂度计算为总计E。大肠杆菌菌落数在平板时间稀释因子上
                1. 笔记

                  1. 为了更好地了解sgRNA库的构建的不同阶段,请参阅我们以前的出版物(Cheng等人,2016b)中的整个技术过程的流程图以及Molecular chipper的一般概念流程图与传统的阵列合成的sgRNA文库生成相比,sgRNA文库产生(Cheng等人,2016a)。
                  2. 指定的优化结扎和消化时间很重要。
                  3. 从大量的输入DNA开始至关重要。我们从原始报告(Cheng等人,2016b)中以最少20μg的输入DNA开始,并且通常在任何中间步骤的情况下制备至少40-60μg的起始DNA材料在程序中
                  4. 1:5摩尔比与衔接子的连接对于将DNA连接到DNA片段的两端是有效的连接。更多的适配器可能有助于提高连接效率,但会导致凝胶纯化的背景更高,因此难以使连接的DNA产物可视化。
                  5. 获得数百纳克的38bp片段用于连接到sgRNA骨架是前进的必要条件。
                  6. 通过转变足够的高素质能力的细胞来实现所需的复杂性/覆盖范围。我们比较了可用的商业细胞,发现NEB 5-αElectrocompetent E。大肠杆菌是最高的能力之一
                  7. 如果在第一次生态P15I消化后不存在38bp的条带,则1)检查适配器是否退火良好:只连接适配器,这将导致存在单体的主要带, 4%低熔点凝胶上的次要非特异性条带; 2)检查超声处理的DNA是否被良好地修复,如果连接良好:在72℃下在dNTP存在下,通过Taq DNA聚合酶处理小部分修复的DNA 5分钟,随后进行TA克隆(Invitrogen):如果终端修复工作良好,并且克隆插入片段的测序应显示克隆片段两端的衔接子,则应出现许多白色菌落; 3)通过在3-4%LMP凝胶上运行,通过连接的DNA凝胶纯化来减少背景物质的杂质。
                  8. 如果在转化最终连接后没有足够的菌落/覆盖率,1)鉴定具有高转化效率的感受态细胞的商业来源,例如来自NEB的电穿孔感受态细胞; 2)或者,使您自己的电感细胞效率更高; 3)进行测试转化,计算整个菌落数/复杂度/覆盖率,然后再转换其余的连接以制作整个DNA文库。
                  9. 如果克隆效率不高,1)用BamHⅠ和HindⅢ全长连接的sgRNA片段消化6小时,而不是过夜以使星形活性降至最低; 2)通过克隆一个模拟蛋白质HI-HindⅢ片段测试载体质量,如果需要,首先可以克隆填充物以有效制备双酶消化的载体。
                    1. 食谱

                      1. 50升TAE凝胶运行缓冲液每升
                        242克Tris碱
                        18.61g EDTA
                        57.1毫升冰醋酸
                        溶解于蒸馏水中,最终体积为1升
                        1. 致谢

                          这项研究部分得到NIH授予R01CA149109(至J.L.)和R01GM099811(Y.D.和J.L.)的支持。该协议是从以前的工作(Cheng等人,2016a和2016b)进行了改编的。作者声明没有可能影响本协议的设计和实施的利益冲突或竞争利益。

                          参考

                          1. Arakawa,H。(2016)。  转换方法mRNA转入用于任何生物体的CRISPR/Cas9编辑的gRNA文库。 2(8):e1600699。
                          2. Canver,MC,Smith,EC,Sher,F.,Pinello,L.,Sanjana,NE,Shalem,O.,Chen,DD,Schupp,PG,Vinjamur,DS,Garcia,SP,Luc,S.,Kurita, R.,Nakamura,Y.,Fujiwara,Y.,Maeda,T.,Yuan,GC,Zhang,F.,Orkin,SH和Bauer,DE(2015)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/26375006"target ="_ blank">通过Cas9介导的原位饱和诱变的BCL11A增强子解剖自然 527(7577):192-197。
                          3. Cheng,J.,Pan,W.,Roden,C.,Chen,Z.,and Lu,J.(2016a)。  Molecular Chipper:Functional Mapping of the使用CRISPR的非编码基因组。 下一代生成Sequenc&申请 3:132.
                          4. Cheng,J.,Roden,CA,Pan,W.,Zhu,S.,Baccei,A.,Pan,X.,Jiang,T.,Kluger,Y.,Weissman,SM,Guo,S.,Flavell, RA,Ding,Y.和Lu,J.(2016b)。用于CRISPR sgRNA文库生成的分子切片技术和非编码区域的功能定位。 Nat Commun 7:11178.
                          5. Koike-Yusa,H.,Li,Y.,Tan,EP,Velasco-Herrera Mdel,C.and Yusa,K。(2014)。  使用慢病毒CRISPR引导RNA文库的哺乳动物细胞中的全基因组隐性遗传筛选 Nat Biotechnol 32(3):267-273。
                          6. Korkmaz,G.,Lopes,R.,Ugalde,AP,Nevedomskaya,E.,Han,R.,Myacheva,K.,Zwart,W.,Elkon,R。和Agami,R。(2016)使用CRISPR-Cas9的人类基因组中增强子元件的功能遗传筛选"class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/26751173"target ="_ blank">。 生物技术 34(2):192-198。
                          7. Lane,AB,Strzelecka,M.,Ettinger,A.,Grenfell,AW,Wittmann,T。和Heald,R。(2015)。  用于基因组标记和筛选的酶促产生的CRISPR文库。 Dev Cell 34(3):373-378 。
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引用:Cheng, J., Pan, W. and Lu, J. (2017). Dense sgRNA Library Construction Using a Molecular Chipper Approach. Bio-protocol 7(12): e2373. DOI: 10.21769/BioProtoc.2373.
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