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Design of Hybrid RNA Polymerase III Promoters for Efficient CRISPR-Cas9 Function
设计合成RNA聚合酶III启动子以高效启动CRISPR-Cas9功能   

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ACS Synthetic Biology
Apr 2016

Abstract

The discovery of the CRISPR-Cas9 system from Streptococcus pyogenes has allowed the development of genome engineering tools in a variety of organisms. A frequent limitation in CRISPR-Cas9 function is adequate expression levels of sgRNA. This protocol provides a strategy to construct hybrid RNA polymerase III (Pol III) promoters that facilitate high expression of sgRNA and improved CRISPR-Cas9 function. We provide selection criteria of Pol III promoters, efficient promoter construction methods, and a sample screening technique to test the efficiency of the hybrid promoters. A hybrid promoter system developed for Yarrowia lipolytica will serve as a model.

Keywords: Synthetic biology (合成生物学), CRISPR-Cas9 (CRISPR-Cas9), RNA polymerase III promoters (RNA聚合酶III启动子), Hybrid promoters (杂交启动子), sgRNA (sgRNA), Yarrowia lipolytica (解脂耶罗威亚酵母)

Background

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) is a collection of DNA sequences found in bacteria that contain snippets of viral DNA from previous exposures (Marraffini and Sontheimer, 2010). The snippets are referred to as spacer DNA, and they are flanked by short, repetitive palindromic sequences. Bacteria use these stored spacer sequences as a template to express RNA to recognize and attack specific viruses if they are exposed again. When combined with CRISPR-associated (Cas) proteins, CRISPR-Cas systems can recognize and cut foreign DNA or RNA, destroying the virus and protecting the host from repeated infections (Barrangou, 2013).

A specific CRISPR system, the type II CRISPR-Cas9 from Streptococcus pyogenes, has been modified into a simpler system for use in genomic editing. With this system, researchers are able to design specific single-guide RNA (sgRNA) sequences that are complementary to a 20 bp sequence of a gene of interest that has an upstream protospacer adjacent motif (PAM; ‘NGG’) (Jinek et al., 2012). When the designed sgRNA complexes with the Cas9 protein, the assembled ribonucleoprotein binds to and introduces a double strand break (DSB) in the target DNA sequence. In genome editing applications, this DSB is then repaired by a cell’s native repair mechanisms. In the absence of an introduced repair template, the nonhomologous end-joining DNA repair pathway is normally used to repair the break in most eukaryotes (Moore and Haber, 1996). Repair via nonhomologous end-joining frequently results in an indel mutation that causes a frameshift mutation and disrupts the gene’s function. The simple programmability of the sgRNA sequences allows for unprecedented precision in genomic edits. In addition, the portability of the CRISPR-Cas9 system has allowed precise genome editing and other applications in organisms where it was previously tedious or impossible (Mali et al., 2013; Wang et al., 2013; Lobs et al., 2017b; Schwartz et al., 2017b and 2017c). The efficiency of the CRISPR-Cas9 system has been shown to correlate with sgRNA expression (Hsu et al., 2013; Ryan et al., 2014; Yuen et al., 2017). Because of this, a range of strategies for sgRNA expression have been developed. RNA polymerase II promoters, which primarily serve to drive expression of mRNA, have been used because they are widely studied and offer a high degree of control over expression (Deaner et al., 2017). More commonly for CRISPR systems, RNA polymerase III (Pol III) promoters have been used to drive sgRNA expression. Pol III promoters natively drive expression of smaller RNAs, most notably tRNAs, and yield higher transcript levels (Schwartz et al., 2016). To increase functional sgRNA expression levels even higher, Pol III promoters concatenated with tRNAs have been used (Xie et al., 2015; Schwartz et al., 2016; Lobs et al., 2017a). Implementation of synthetic hybrid Pol III promoter systems can improve CRISPR-Cas9 mediated genome editing for efficient gene disruption.

Materials and Reagents

  1. 10 µl pipette tips (Fisher Scientific, FisherbrandTM, catalog number: 02-707-438 )
  2. 200 µl pipette tips (Fisher Scientific, FisherbrandTM, catalog number: 02-707-417 )
  3. 1,000 µl pipette tips (Fisher Scientific, FisherbrandTM, catalog number: 02-707-403 )
  4. 1.5 ml microcentrifuge tubes (Fisher Scientific, FisherbrandTM, catalog number: 05-408-129 )
  5. 0.2 ml PCR tubes (Fisher Scientific, FisherbrandTM, catalog number: 14-230-215 )
  6. 100 x 15 mm Petri dishes (Fisher Scientific, FisherbrandTM, catalog number: FB0875712 )
  7. 14 ml culture tubes (Corning, Falcon®, catalog number: 352057 )
  8. Competent DH5α Escherichia coli (New England Biolabs, catalog number: C2987I )
  9. Yarrowia lipolytica strain Po1f (ATCC, catalog number: MYA-2613 )
  10. pCRISPRyl (Addgene, catalog number: 70007 ) or Episomal Cas9 plasmid (see Notes)
  11. Q5 HF polymerase (New England Biolabs, catalog number: M0491L )
  12. YeaStarTM Genomic DNA Kit (Zymo Research, catalog number: D2002 )
  13. DNA Clean & ConcentratorTM (Zymo Research, catalog number: D4004 )
  14. Gibson Assembly Master mix (New England Biolabs, catalog number: E2611L )
  15. ZyppyTM Plasmid Miniprep Kit (Zymo Research, catalog number: D4037 )
  16. CutSmart Buffer (New England Biolabs, catalog number: B7204S )
  17. AvrII restriction enzyme (New England Biolabs, catalog number: R0174S )
  18. Taq DNA polymerase (New England Biolabs, catalog number: M0273L )
  19. Yeast extract (BD, DifcoTM, catalog number: 212750 )
  20. Peptone (BD, DifcoTM, catalog number: 211677 )
  21. Glucose (Fisher Scientific, FisherbrandTM, catalog number: D16-10 )
  22. Agar (Sigma-Aldrich, catalog number: A7002-1KG )
  23. Yeast nitrogen base without amino acids (BD, DifcoTM, catalog number: 291940 )
  24. Complete Supplemental Mixture without Leucine (CSM-leu) (Sunrise Science, catalog number: 1005-010 )
  25. Complete Supplemental Mixture (CSM) (SunriseScience, catalog number: 1001-010 )
  26. Oleic acid (MP Biomedicals, catalog number: 0215178125 )
  27. Tween 20 (Sigma-Aldrich, catalog number: P9416-50ML )
  28. LB broth (Sigma-Aldrich, catalog number: L3022-1KG )
  29. Ampicillin (Sigma-Aldrich, catalog number: A0166 )
  30. YPD media/agar (see Recipes)
  31. SD-leu media/agar (see Recipes)
  32. SD oleic acid agar (see Recipes)
  33. LB agar (see Recipes)

Equipment

  1. Pipettes (Gilson, model: PIPETMANTM Variable Volume, catalog number: F167370 )
  2. Benchtop microcentrifuge (Eppendorf, model: 5424 , catalog number: 022620401)
  3. PCR thermocycler (Bio-Rad Laboratories, model: T100TM, catalog number: 1861096 )
  4. Incubation shaker (Infors, model: Multitron Standard )
  5. Incubator (Thermo Fisher Scientific, Thermo ScientificTM, model: HerathermTM IGS60, catalog number: 51028063 )
  6. Gel electrophoresis tank (Bio-Rad Laboratories, model: Wide Mini-Sub®, catalog number: 1704468 )
  7. Gel electrophoresis power supply (Bio-Rad Laboratories, model: PowerPacTM Basic, catalog number: 1645050 )
  8. Gel imager (Bio-Rad Laboratories, model: Gel DocTM XR+, catalog number: 1708195 )

Procedure

  1. Promoter selection criteria
    Our lab has previously developed CRISPR-Cas9 systems for the yeasts Y. lipolytica and Kluyveromyces marxianus (Schwartz et al., 2016; Lobs et al., 2017a). In these works, we compared CRISPR-Cas9 activity with sgRNA expression from native Pol III promoters, tRNAs, and hybrid Pol III promoters combining native Pol III promoters with a tRNA. In both organisms, hybrid Pol III promoters outperformed native Pol III promoters and tRNAs; however, the native Pol III promoter used in the best hybrid promoter was different in each organism. To date, only class II RNA Pol III promoters (those that are tRNA-like) have been demonstrated in hybrid promoters as described in this protocol. The class II promoters which can be tried include SNR52, SNR6, RPR1, and SCR1 (Marck et al., 2006). These can be identified in annotated genomes of an organism, or found via BLAST search using a closely related organism as input. In plant and mammalian systems, the Pol III U3 and U6 promoters have been extensively used for sgRNA expression, and so may be adaptable to a hybrid promoter approach (Cong et al., 2013; Mali et al., 2013; Shan et al., 2013).

  2. Hybrid promoter construction
    The hybrid promoter system combines the selected Pol III promoter and a tRNA sequence as shown in Figure 1. Sequences of tRNA from an organism can be identified via computational methods (Marck and Grosjean, 2002) or from a public database (http://gtrnadb.ucsc.edu/). Most often, such databases provide the mature tRNA sequence, which can be used to identify the full-length sequence in the genome via a BLAST search or a similar local alignment tool. In cases where the mature sequence is predicted or not know, experimental validation may be required. The addition of a tRNA allows the sgRNA to mature and be excised from the primary transcript. In addition, tRNAs are self-contained RNA Pol III promoters, which may result in improved sgRNA expression. The hybrid promoter consisting of the chosen RNA Pol III promoter and tRNA is placed upstream of the sgRNA encoding sequence with a polyT sequence for termination. This forms the complete hybrid promoter construct.


    Figure 1. Synthetic hybrid promoter construction. A. Schematic of the assembly of Pol III promoter, tRNA, and AvrII-containing sgRNA via Gibson assembly. Similar colored boxes denote overlap sequences and the AvrII site. B. Schematic of cloning specific 20 bp sgRNA target sequence into digested AvrII site.

    1. The desired Pol III promoter is amplified from genomic DNA in a PCR reaction with Q5 DNA polymerase. Genomic DNA can be extracted using an organism specific kit (for Y. lipolytica and K. marxianus we have used the YeaStarTM kit). Primers should be designed to bind ~200-300 bp upstream the ‘A-box’ and approximately 25 bp downstream from the ‘B-box’ of the Pol III promoter. The ‘B-box’ can be identified both by its putative consensus sequence (from Saccharomyces cerevisiae) ‘GWTCRAnnC’ and by its position downstream of an ‘A-box’ (consensus sequence ‘TRGYnnAnnnG’) (Marck et al., 2006). An example of a Pol III promoter amplified from genomic DNA is shown in Figure 2A. The primers used in the PCR reactions contain ~20-30 bp overlap sequences to join the truncated Pol III to the Cas9 plasmid backbone and the tRNA sequence via Gibson Assembly. The backbone homology sequence should contain a selected restriction site overhang. Example primers are shown below. The underlined sequences correspond to the promoter sequence of interest while the non-underlined sequences are the backbone and tRNA overlaps.
      Forward primer:
      CACATTTCCCCGAAAAGTGCCACCTGACGTCCCCAGTTGCAAAAGTTGACAC
      Reverse primer:
      AAACCATCGGCGCATTAGAGGTATTTTTCAAAGTAGCCCAGTGCAGAGTCC


      Figure 2. Pol III promoter and tRNA design. A. Schematic of a Pol III promoter amplified from genomic DNA. The italicized dimensions are recommendations but will differ between organisms and between promoters. B. Schematic of a tRNA sequence amplified from genomic DNA. Similar to A, the italicized dimensions will differ between different tRNAs. The sequences that flank upstream the A-box and downstream the B-box are essential for Rnase P and Rnase Z binding and excision of the tRNA.

    2. The tRNA sequence is similarly amplified from genomic DNA with Q5 DNA polymerase. Primers are designed ~20-30 bp upstream the A-box and ~20 bp downstream the B-box to allow for RNase P and RNase Z binding, respectively. Conserving these flanking sequences ensures excision of the matured tRNA. An example is seen in Figure 2B. The forward primer contains overlaps with the truncated promoter sequence. The reverse primer contains an overhang with an AvrII restriction site followed by an overlap of the sgRNA sequence. The AvrII restriction digestion site allows for easy addition of an N20 sgRNA targeting sequence. An example set of primers are shown below. The underlined sequence contains the tRNA sequence and the non-underlined sequences on the forward and reverse primer correspond to the truncated promoter and the AvrII (lower-cased)-sgRNA overlaps, respectively.
      Forward primer:
      CGAGTTCTGGACTCTGCACTGGGCTACTTTGAAAAATACCTCTAATGCGCCG
      Reverse primer:
      AACTTGCTATTTCTAGCTCTAAAAcctaggTCAACCTGCGCCGACCC
    3. The final fragment containing the sgRNA sequence is amplified from plasmid pCRISPRyl (Addgene #70007) using primers containing AvrII- tRNA and Cas9 backbone overlaps. The underlined sequence contains the sgRNA sequence while the non-underlined sequences contain the AvrII(lower-cased)- tRNA overlap and plasmid backbone overlap with a restriction site overhang.
      Forward primer:
      CCGGTTCGATTCCGGGTCGGCGCAGGTTGAcctaggTTTTAGAGCTAGAAATAGCAAG
      Reverse primer:
      GTCATGATAATAATGGTTTCTTAGACGTAAAAAAAAGCACCGACTCGGTG
    4. Once the Pol III, tRNA, and sgRNA sequences have been amplified and isolated using a PCR clean-up kit, like DNA Clean & ConcentratorTM, they are combined with the backbone plasmid in a single Gibson Assembly reaction as described below at a 1:3 ratio of backbone to insert. The backbone plasmid is cut with a restriction enzyme at a site distal to the Cas9 sequence. In pCRISPRyl, the AatII site is used.
      1. Gibson Assembly Conditions (10μl)
        Gibson Master mix
        5 μl
        Backbone plasmid
        0.5 pmol
        Insert 1: Pol III fragment
        1.5 pmol
        Insert 2: tRNA fragment
        1.5 pmol
        Insert 3: sgRNA fragment
        1.5 pmol
        H2O up to
        10 μl
        Incubate at 50 °C for 1 h.
    5. The product can then be directly transformed into DH5α competent cells for replication and extracted using ZyppyTM Plasmid Miniprep Kit.

  3. sgRNA target sequence design
    Selected sgRNA sequences can be designed into primers for cloning following the format below. The underlined segment is the desired N20 sgRNA sequence, the lowercase sequence is homologous to the AvrII restriction site while the rest of the sequence is overlap with the tRNA sequence and sgRNA. The overlaps allow sgRNA target sequence to be combined via Gibson Assembly into the AvrII digested CRISPR plasmid, as shown in Figure 1.
    Forward primer:
    GGGTCGGCGCAGGTTgacgtNNNNNNNNNNNNNNNNNNNNGTTTTAGAGCTAGAAATAGC
    Reverse primer:
    GCTATTTCTAGCTCTAAAACNNNNNNNNNNNNNNNNNNNNacgtcAACCTGCGCCGACCC
    1. The forward and reverse primers are annealed using the following protocol in a thermocycler to obtain a linear fragment.
      1. Annealing conditions (25 μl)
        CutSmart buffer
        2.5 μl
        Forward primer (10 μM)
        5 μl
        Reverse primer (10 μM)
        5 μl
        H2O
        12.5 μl
      2. Thermocycler protocol (7 min)
        95 °C                         
        3 min
        90 °C
        30 sec
        85 °C
        30 sec
        Continue decreasing in increments of 5 °C until the final temperature of 60 °C.
    2. The backbone plasmid digested with AvrII as described below:
      1. Restriction digest conditions (50 μl)
        CutSmart buffer                            
        5 μl
        AvrII
        1 μl
        Backbone plasmid
        1 μg
        H2O
        up to 50 μl  
      2. Incubate at 37 °C for at least 1 h.
      3. Purify using DNA clean up kit.
    3. Finally, the annealed fragment is combined with the backbone digested with AvrII via Gibson Assembly as described above at a ratio of 1:3 backbone to insert.

  4. Measuring gene disruption efficiency
    1. Here, we describe an efficient screening method for verifying gene disruption in transformed colonies using PCR. This method can be used to simultaneously screen dozens of colonies.
      1. Isolate genomic DNA from random colonies after transformation using the YeaStar Genomic DNA Kit.
      2. Design primers that are 200 base pairs upstream and downstream of the sgRNA target sequence that will be used for amplification.
      3. Amplify the region flanking target site via PCR with Taq polymerase and purify.
      4. Sanger sequence the purified fragment and align with corresponding wild-type sequence to identify any indel or frameshift mutations.
    2. Alternatively, quantification of gene disruption can be done with an easily selectable growth-associated phenotype. Here we use Y. lipolytica as an example. In Y. lipolytica, disruption of the gene PEX10 prevents peroxisome biogenesis and results in an inability to use long-chain fatty acids as an energy source (Blazeck et al., 2014; Schwartz et al., 2017a). This means that colonies with this phenotype are unable to grow on minimal media with oleic acid as the sole carbon source. Therefore, a CRISPR plasmid containing a sgRNA target sequence that targets PEX10 can be used to screen for genetic disruption, allowing simultaneous screening of hundreds of colonies.
      1. The wild-type strain of Y. lipolytica, PO1f, is transformed with the designed CRISPR plasmid at stationary phase (Schwartz et al., 2016). Successful transformants are selected for via outgrowth in either liquid SD-leu media or on SD-leu agar plates. Selection in SD-leu media ensures that all tested colonies contain the CRISPR plasmid. Outgrowth allows for expression of the CRISPR system and gene disruption before screening.
      2. Transformed colonies are randomly selected and streaked on both YPD and SD oleic acid agar plates and incubated at 30 °C.
      3. Colonies that grow on YPD media but not SD oleic acid plates demonstrate genetic disruption of PEX10 as seen in Figure 3.


        Figure 3. Phenotype of PEX10 disruptants. Example plates screening of PEX10 disrupted phenotypes on YPD and SD oleic acid media. PO1f is shown as a control, light blue indicates RPR1’-tRNAgly, orange indicates SNR52’-tRNAgly, and SCR1’-tRNAgly is shown in dark blue. Adapted with permission from Schwartz et al., 2016. Copyright © American Chemical Society 2015.

Data analysis

  1. Determining disruptions from sequence alignment
    Alignment of amplified genomic sequences to the wild type may be done using a multiple sequence alignment program such as MUSCLE (https://www.ebi.ac.uk/Tools/msa/muscle/). This particular program allows for simultaneous alignment of up to 500 sequences. Aligning at least 50 bp sequences that span the target site allows for identification of indels. Indels of 1, 2, 4, and 5 bp (or any number not divisible by 3) indicate a frameshift mutation and a successful gene disruption.

  2. Disruption efficiency
    Disruption efficiency is measured in triplicate with each sample consisting of 30 randomly selected colonies. The percentage of disrupted colonies in each sample is calculated, and the mean disruption efficiency and standard deviation are determined and reported. For example, after 2 days of outgrowth, the synthetic hybrid promoter SCR1’-tRNAgly disrupted 15/30, 14/30, and 20/30 of colonies which gives a disruption efficiency of 54 ± 11% (Schwartz et al., 2016).

Notes

  1. Selection and design of an episomal Cas9 backbone plasmid is highly dependent on the organism. For example, pCRISPRyl used in our lab contains a Cas9 sequence that has been codon optimized for Y. lipolytica, a Y. lipolytica CEN sequence, and a Leucine selective marker. The plasmid also contains an ampicillin resistance cassette and origin of replication for propagation in E. coli. An analogous vector is needed for the organism of interest.
  2. The efficacy of selected tRNAs can differ between organisms, and so multiple different tRNA sequences may need to be tested in each case. Selection criteria include high native abundance and short length. In Y. lipolytica, tRNAgly was selected based on its high native abundance according to codon usage in the Y. lipolytica genome. Our particular tRNA sequence was the shortest tRNAgly.
  3. The PEX10 gene in Y. lipolytica allowed for an easily screened phenotype. However, while not all organisms can grow with oleic acid as a sole carbon source, other organisms may have similar growth-associated genes that allow for straight-forward screening methods. For example, the ADE2 gene in S. cerevisiae causes cells to appear red in the absence of adenine (Jones and Fink, 1982), while disruption of the XYL2 gene in K. marxianus eliminates its ability to grow on xylitol (Lobs et al., 2017a).

Recipes

  1. YPD media/agar
    10 g/L yeast extract
    20 g/L peptone
    20 g/L glucose
    For agar plates, add 20 g/L agar
    Note: Glucose must be added after autoclaving and cooling to 50 °C.
  2. SD-leu media/agar
    7 g/L yeast nitrogen base without amino acids
    0.69 g/L CSM-leu
    20 g/L glucose
    For agar plates, add 20 g/L agar
    Note: Glucose must be added after autoclaving and cooling to 50 °C.
  3. SD oleic acid agar
    7 g/L yeast nitrogen base without amino acids
    0.69 g/L CSM
    20 g/L agar
    0.3% oleic acid
    0.2% Tween 20
    Note: Oleic acid and Tween 20 must be added after autoclaving and cooling to 50 °C.
  4. LB agar
    20 g/L LB broth
    15 g/L agar
    Note: For selective plates add 50 μg/ml ampicillin after autoclaving and cooling to 50 °C.

Acknowledgments

This protocol was adapted from a previously published work (Schwartz et al., 2016) and supported by NSF CBET-1403264 and -1403099 and the University of California, Riverside Chancellor’s Research Fellowship. The authors declare no conflict of interest or competing interest.

References

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简介

来自化脓性链球菌的CRISPR-Cas9系统的发现使得在各种生物体中开发基因组工程工具成为可能。 CRISPR-Cas9功能的频繁限制是足够的sgRNA表达水平。 该协议提供了构建杂合RNA聚合酶III(Pol III)启动子的策略,其促进sgRNA的高表达和改善的CRISPR-Cas9功能。 我们提供Pol III启动子的选择标准,有效的启动子构建方法以及样品筛选技术来检测杂合启动子的效率。 为解脂耶氏酵母开发的杂交启动子系统将用作模型。

【背景】CRISPR(成簇定期间隔短回文重复序列)是细菌中发现的DNA序列的集合,其中含有先前曝光的病毒DNA片段(Marraffini和Sontheimer,2010)。片段被称为间隔区DNA,并且它们侧面是短的,重复的回文序列。细菌使用这些存储的间隔序列作为模板来表达RNA,以识别和攻击再次暴露的特定病毒。当与CRISPR相关(Cas)蛋白结合时,CRISPR-Cas系统可以识别和切割外源DNA或RNA,破坏病毒并保护宿主免受重复感染(Barrangou,2013)。

一种特定的CRISPR系统,来自化脓链球菌的II型CRISPR-Cas9已经被修改成用于基因组编辑的更简单的系统。利用这个系统,研究人员能够设计特定的单引导RNA(sgRNA)序列,它与具有上游原型间隔区相邻基序(PAM;'NGG')的目的基因的20bp序列互补(Jinek et al。,2012)。当设计的sgRNA与Cas9蛋白复合时,组装的核糖核蛋白结合并在靶DNA序列中引入双链断裂(DSB)。在基因组编辑应用程序中,此DSB然后通过单元的本机修复机制进行修复。在缺乏引入的修复模板的情况下,非同源末端连接DNA修复途径通常用于修复大多数真核生物的破坏(Moore和Haber,1996)。通过非同源末端连接修复通常会导致indel突变,导致移码突变并破坏基因的功能。 sgRNA序列的简单可编程性允许在基因组编辑中实现前所未有的精确度。此外,CRISPR-Cas9系统的便携性允许精确的基因组编辑和其他先前单调乏味或不可能的生物体的应用(Mali等人,2013; Wang等人, ,2013; Lobs et al。,2017b; Schwartz et al。,2017b和2017c)。已显示CRISPR-Cas9系统的效率与sgRNA表达相关(Hsu等人,2013; Ryan等人,2014; Yuen等人,2017)。正因为如此,已经开发了一系列针对sgRNA表达的策略。主要用于驱动mRNA表达的RNA聚合酶II启动子已被使用,因为它们被广泛研究并提供对表达的高度控制(Deaner等人,2017)。对于CRISPR系统更常见的是,RNA聚合酶III(Pol III)启动子已被用于驱动sgRNA表达。 Pol III启动子天然驱动较小RNA的表达,最显着的是tRNA,并产生较高的转录水平(Schwartz等人,2016)。为了增加功能性sgRNA表达水平甚至更高,已经使用与tRNA连接的Pol III启动子(Xie等人,2015; Schwartz等人,2016; Lobs 等。,2017a)。合成杂种Pol III启动子系统的实施可以改进CRISPR-Cas9介导的基因组编辑以实现有效的基因破坏。

关键字:合成生物学, CRISPR-Cas9, RNA聚合酶III启动子, 杂交启动子, sgRNA, 解脂耶罗威亚酵母

材料和试剂

  1. 10μl移液器吸头(Fisher Scientific,Fisherbrand TM TM,目录号:02-707-438)
  2. 200μl移液器吸头(Fisher Scientific,Fisherbrand TM,产品目录号:02-707-417)
  3. 1,000μl移液器吸头(Fisher Scientific,Fisherbrand TM,产品目录号:02-707-403)
  4. 1.5ml微量离心管(Fisher Scientific,Fisherbrand TM,目录号:05-408-129)
  5. 0.2ml PCR管(Fisher Scientific,Fisherbrand TM,目录号:14-230-215)
  6. 100×15mm培养皿(Fisher Scientific,Fisherbrand TM,目录号:FB0875712)

  7. 14毫升培养管(Corning,Falcon ,产品目录号:352057)
  8. 主要的DH5α大肠杆菌(New England Biolabs,目录号:C2987I)
  9. 解脂耶氏酵母属菌株Po1f(ATCC,目录号:MYA-2613)
  10. pCRISPRyl(Addgene,目录号:70007)或Episomal Cas9质粒(见注)
  11. Q5 HF聚合酶(New England Biolabs,目录号:M0491L)
  12. YeaStar TM基因组DNA试剂盒(Zymo Research,目录号:D2002)
  13. DNA Clean&浓缩器TM(Zymo Research,目录号:D4004)
  14. Gibson Assembly Master mix(新英格兰生物实验室,目录号:E2611L)
  15. Zyppy TM质粒小量制备试剂盒(Zymo Research,目录号:D4037)
  16. CutSmart缓冲液(新英格兰生物实验室,目录号:B7204S)
  17. Avr II限制酶(New England Biolabs,目录号:R0174S)
  18. Taq DNA聚合酶(New England Biolabs,目录号:M0273L)
  19. 酵母提取物(BD,Difco TM,目录号:212750)
  20. 蛋白胨(BD,Difco TM,产品目录号:211677)
  21. 葡萄糖(Fisher Scientific,Fisherbrand TM,目录号:D16-10)
  22. 琼脂(Sigma-Aldrich,目录号:A7002-1KG)
  23. 不含氨基酸的酵母氮碱(BD,Difco TM,目录号:291940)
  24. 完全没有亮氨酸的补充混合物(CSM-leu)(Sunrise Science,目录号:1005-010)
  25. 完全补充混合物(CSM)(SunriseScience,目录号:1001-010)
  26. 油酸(MP Biomedicals,目录号:0215178125)
  27. 吐温20(Sigma-Aldrich,目录号:P9416-50ML)
  28. LB肉汤(Sigma-Aldrich,目录号:L3022-1KG)
  29. 氨苄青霉素(Sigma-Aldrich,目录号:A0166)
  30. YPD媒体/琼脂(见食谱)
  31. SD-leu媒体/琼脂(见食谱)
  32. SD油酸琼脂(见食谱)
  33. LB琼脂(见食谱)

设备

  1. 移液器(Gilson,型号:PIPETMAN TM可变容量,目录号:F167370)
  2. 台式微量离心机(Eppendorf,型号:5424,目录号:022620401)
  3. PCR热循环仪(Bio-Rad Laboratories,型号:T100 TM,目录号:1861096)。
  4. 培养摇床(Infors,型号:Multitron标准)
  5. 培养箱(Thermo Fisher Scientific,Thermo Scientific TM,型号:Heratherm TM TM IGS60,目录号:51028063)
  6. 凝胶电泳槽(Bio-Rad Laboratories,型号:Wide Mini-Sub,目录号:1704468)
  7. 凝胶电泳电源(Bio-Rad Laboratories,型号:PowerPac TM Basic,目录号:1645050)
  8. 凝胶成像仪(Bio-Rad Laboratories,型号:Gel Doc TM TM XR +,目录号:1708195)

程序

  1. 促销员选择标准
    我们的实验室先前开发了用于酵母菌Y的CRISPR-Cas9系统。 (Kluyveromyces marxianus)(Schwartz等人,2016; Lobs等人,2017a)。在这些作品中,我们将CRISPR-Cas9活性与来自天然Pol III启动子,tRNAs和将天然Pol III启动子与tRNA组合的杂种Pol III启动子的sgRNA表达进行比较。在这两种生物体中,杂种Pol III启动子优于天然Pol III启动子和tRNA;然而,用于最佳杂交启动子的天然Pol III启动子在每种生物体中都不同。迄今为止,只有II类RNA Pol III启动子(tRNA样启动子)已在本协议中描述的杂合启动子中得到证实。可以尝试的II类启动子包括:SNR52,SNR6,RPR1和SCR1(Marck et al。,2006)。这些可以在生物体的注释基因组中被鉴定,或者通过使用密切相关的生物体作为输入的BLAST搜索来发现。在植物和哺乳动物系统中,Pol III U3和U6启动子已广泛用于sgRNA表达,因此可适用于杂合启动子方法(Cong等人,2013; Mali等人, ,2013; Shan et。,2013)。

  2. 杂交启动子结构
    杂合启动子系统将选择的Pol III启动子和tRNA序列结合起来,如图1所示。可以通过计算方法(Marck and Grosjean,2002)或从公共数据库( http://gtrnadb.ucsc.edu/ )。通常,这样的数据库提供了成熟的tRNA序列,其可以通过BLAST搜索或类似的局部比对工具用于鉴定基因组中的全长序列。在成熟序列被预测或不知道的情况下,可能需要实验验证。 tRNA的添加允许sgRNA成熟并从初级转录物中切除。另外,tRNA是自我包含的RNA Pol III启动子,其可以导致改善的sgRNA表达。由选择的RNA Pol III启动子和tRNA组成的杂合启动子位于具有polyT序列的sgRNA编码序列的上游用于终止。这形成了完整的杂合启动子构建体。


    图1.合成的杂合启动子构建A.通过Gibson装配组装Pol III启动子,tRNA和含有Avr II的sgRNA的示意图。类似的彩色框表示重叠序列和Avr II网站。 B.将特定的20bp sgRNA靶序列克隆到消化的AvrII位点的示意图。

    1. 所需的Pol III启动子在与Q5 DNA聚合酶的PCR反应中从基因组DNA扩增。基因组DNA可以使用有机体特异性试剂盒(对于解脂耶氏酵母和马克斯克鲁维酵母我们已经使用YeaStar TM试剂盒)进行提取。引物应设计为与'A-box'上游约200-300bp和Pol III启动子的'B-box'下游约25bp结合。 'B盒'可以通过其推定的共有序列(来自酿酒酵母)'GWTCRAnnC'和通过其在'A盒'(共有序列'TRGYnnAnnnG')下游的位置来识别( Marck等人,2006)。图2A显示了从基因组DNA扩增的Pol III启动子的一个例子。用于PCR反应的引物含有〜20-30bp重叠序列,以通过Gibson Assembly将截短的Pol III连接到Cas9质粒骨架和tRNA序列。骨架同源序列应包含选定的限制性位点突出。示例引物如下所示。加下划线的序列对应于感兴趣的启动子序列,而非加下划线的序列是骨架和tRNA重叠。
      正向引物:
      CACATTTCCCCGAAAAGTGCCACCTGACGT CCCCAGTTGCAAAAGTTGACAC
      反向引物:
      AAACCATCGGCGCATTAGAGGTATTTTTC AAAGTAGCCCAGTGCAGAGTCC


      图2. Pol III启动子和tRNA设计A.从基因组DNA扩增的Pol III启动子的示意图。斜体大小是推荐值,但在生物体和启动子之间会有所不同。 B.从基因组DNA扩增的tRNA序列的示意图。与A相似,不同tRNA之间的斜体大小会有所不同。 A框上游侧和B框下游侧的序列对于RNA酶P和RNA酶Z的结合和tRNA的切除是必不可少的。

    2. 用Q5 DNA聚合酶从基因组DNA中类似地扩增tRNA序列。引物设计为A盒上游约20-30bp和B盒下游约20bp,以分别允许RNase P和RNase Z结合。保存这些侧翼序列确保切除成熟的tRNA。图2B显示了一个例子。正向引物含有与截短的启动子序列重叠。反向引物含有具有AvrII限制性位点的突出端,随后是sgRNA序列的重叠。 Avr II限制性消化位点允许容易地添加N sub 20 sgRNA靶向序列。下面显示了一组示例性引物。加下划线的序列含有tRNA序列,正向和反向引物上的非下划线序列分别对应截短的启动子和AvrII(下壳)-sgRNA重叠。
      正向引物:
      CGAGTTCTGGACTCTGCACTGGGCTACTT TGAAAAATACCTCTAATGCGCCG
      反向引物:
      AACTTGCTATTTCTAGCTCTAAAAcctaggTC AACCTGCGCCGACCC
    3. 使用含有Avr II-tRNA和Cas9骨架重叠的引物从质粒pCRISPRyl(Addgene#70007)扩增含有sgRNA序列的最终片段。加下划线的序列含有sgRNA序列,而未加下划线的序列含有AvrII(下套)-tRNA重叠,质粒骨架与限制性位点突出重叠。
      正向引物:
      CCGGTTCGATTCCGGGTCGGCGCAGGTTGAcctagg TTTTAGAGCTAGAAATAGCAAG
      反向引物:
      GTCATGATAATAATGGTTTCTTAGACGT AAAAAAAAGCACCGACTCGGTG
    4. 一旦Pol III,tRNA和sgRNA序列已经使用PCR清洁试剂盒如DNA Clean&如下所述,它们与骨架质粒在单一吉布森组装反应中以1:3的骨架与插入的比例结合。骨架质粒在Cas9序列的远端位点用限制性酶切割。在pCRISPRyl中,使用了 Aat II网站。
      1. 吉布森装配条件(10μl)
        吉布森大师混音
        5微升
        骨干质粒
        0.5 pmol
        插入1:Pol III片段
        1.5 pmol
        插入2:tRNA片段
        1.5 pmol
        插入3:sgRNA片段
        1.5 pmol
        H 2 0直至
        10微升
        在50°C孵育1小时。
      2. 然后可以将产物直接转化到DH5α感受态细胞中进行复制,并使用Zyppy TM Plasmid Miniprep Kit进行提取。

      3. sgRNA靶序列设计
        可以将选定的sgRNA序列设计到用于克隆的引物中,按照以下格式。加下划线的片段是所需的N sub 20 sgRNA序列,小写序列与Avr II限制性位点同源,而该序列的其余部分与tRNA序列和sgRNA重叠。重叠使得sgRNA靶序列能够通过Gibson组合进入AvrII II消化的CRISPR质粒中,如图1所示。
        正向引物:
        GGGTCGGCGCAGGTTgacgt NNNNNNNNNNNNNNNNNNNN GTTTTAGAGCTAGAAATAGC
        反向引物:
        GCTATTTCTAGCTCTAAAAC NNNNNNNNNNNNNNNNNNNN acgtcAACCTGCGCCGACCC
        1. 正向和反向引物使用以下方案在热循环仪中退火以获得线性片段。
          1. 退火条件(25微升)
            CutSmart缓冲区
            2.5微升
            正向引物(10μM)
            5微升
            反向引物(10μM)
            5微升
            H 2 O
            12.5微升
          2. 热循环仪方案(7分钟)
            95°C                 
            3分钟
            90°C
            30秒
            85°C
            30秒
            继续以5°C的增量降低,直到最终温度达到60°C。
          3. 如下所述用Avr II消化的骨架质粒:
            1. 限制消化条件(50μl)
              CutSmart缓冲区           &nbsp ;   
              5微升
              Avr II
              1微升
              骨干质粒
              1微克
              H 2 O
              多达50μl  

            2. 在37°C孵育至少1小时
            3. 使用DNA清理试剂盒进行纯化。
            4. 最后,将退火的片段与通过如上所述的Gibson组件消化的AvrII以1:3的骨架与插入物的比率结合的骨架组合。

            5. 测量基因中断效率
              1. 在这里,我们描述了一种使用PCR验证转化菌落中基因破坏的有效筛选方法。该方法可用于同时筛选数十个菌落。

                1. 使用YeaStar Genomic DNA Kit从转化后的随机菌落中分离基因组DNA
                2. 设计将用于扩增的sgRNA靶序列上游和下游200个碱基对的引物。

                3. 用Taq聚合酶通过PCR扩增区域侧翼目标位点并纯化。
                4. Sanger对纯化的片段进行测序并与相应的野生型序列进行比对以鉴定任何indel或移码突变。
              2. 或者,可以用易于选择的生长相关表型进行基因破坏的定量。在这里,我们使用解脂耶氏酵母作为一个例子。在 Y中。解脂基因PEX10阻止过氧化物酶体的生物合成并导致不能使用长链脂肪酸作为能源(Blazeck等人, 2014; Schwartz et al。,2017a)。这意味着具有这种表型的菌落不能在以油酸作为唯一碳源的基本培养基上生长。因此,含有靶向PEX10的sgRNA靶序列的CRISPR质粒可用于筛选遗传中断,允许同时筛选数百个菌落。
                1. Y的野生型菌株。在设计的CRISPR质粒在固定相中转化解脂耶氏酵母PO1f(Schwartz等人,2016)。在液体SD-leu培养基或SD-leu琼脂平板上选择成功的转化体通过生长。 SD-leu培养基中的选择可确保所有测试菌落含有CRISPR质粒。生长可以在筛选之前表达CRISPR系统和基因中断。
                2. 随机选择转化的菌落并在YPD和SD油酸琼脂平板上划线并在30℃温育。
                3. 在YPD培养基但不是SD油酸平板上生长的菌落证明了PEX10的基因破坏,如图3所示。


                  图3. PEX10中断体的表型在YPD和SD油酸培养基上筛选PEX10 的实例平板破坏了表型。 PO1f表示为对照,浅蓝色表示RPR1'-tRNA gly,橙色表示SNR52'-tRNA gly,SCR1'-tRNA gly >以深蓝色显示。根据Schwartz等人的许可修改2016年版权所有©美国化学学会2015年。
            6. 数据分析

              1. 确定序列对齐中断
                扩增的基因组序列与野生型的比对可以使用多序列比对程序来完成,例如MUSCLE( https://www.ebi.ac.uk/Tools/msa/muscle/ )。这个特定的程序允许同时对齐多达500个序列。对齐跨越目标站点的至少50bp序列可以识别插入。 1,2,4和5 bp的indels(或任何不能被3整除的数字)表示移码突变和成功的基因中断。

              2. 中断效率
                每个样品由30个随机选择的菌落组成,一式三份测量破坏效率。计算每个样品中破坏菌落的百分比,并确定并报告平均破坏效率和标准偏差。例如,在生长2天后,合成的杂合启动子SCR1'-tRNA ^ gly破坏了15 / 30,14 / 30和20/30个菌落,其破坏效率为54±11% (Schwartz等人,2016年)。

              笔记

              1. 附加型Cas9主链质粒的选择和设计高度依赖于生物体。例如,我们实验室中使用的pCRISPRyl含有已经针对解脂耶罗维亚酵母Y进行密码子优化的Cas9序列。解脂亚种CEN序列和亮氨酸选择标记。该质粒还含有氨苄青霉素抗性盒和用于在E中繁殖的复制起点。大肠杆菌。感兴趣的生物体需要类似的载体。
              2. 选定的tRNA的功效可以在生物体之间有所不同,因此可能需要在每种情况下测试多种不同的tRNA序列。选择标准包括天然丰度高和长度短。在解脂耶氏酵母中,根据Y中的密码子选择,基于其高天然丰度选择tRNA gly。解脂耶氏酵母基因组。我们特定的tRNA序列是最短的tRNA gly 。
              3. 中的 PEX10 基因。解脂耶氏酵母允许容易筛选的表型。然而,尽管不是所有的生物体都可以与油酸一起作为唯一碳源生长,但其他生物体可能具有相似的生长相关基因,允许直接筛选方法。例如,酿酒酵母中的ADE2基因导致细胞在没有腺嘌呤的情况下显示为红色(Jones和Fink,1982),而破坏XYL2 中的基因。 marxianus 消除了它在木糖醇上生长的能力(Lobs et al。,2017a)。

              食谱

              1. YPD媒体/琼脂
                10克/升酵母提取物
                20克/升蛋白胨
                20克/升葡萄糖
                对于琼脂平板,加入20克/升琼脂
                注意:葡萄糖必须在高压灭菌并冷却至50°C后加入。
              2. SD-leu媒体/琼脂
                不含氨基酸的酵母氮碱7克/升
                0.69克/升CSM-leu
                20克/升葡萄糖
                对于琼脂平板,加入20克/升琼脂
                注意:葡萄糖必须在高压灭菌并冷却至50°C后加入。
              3. SD油酸琼脂
                不含氨基酸的酵母氮碱7克/升
                0.69克/升CSM
                20克/升琼脂
                0.3%油酸
                0.2%吐温20
                注意:必须在高压灭菌并冷却至50℃后加入油酸和吐温20。
              4. LB琼脂
                20克/升LB肉汤

                15克/升琼脂 注意:对于选择性平板,在高压灭菌并冷却至50℃后加入50μg/ ml氨苄青霉素。

              致谢

              该协议改编自以前发表的作品(Schwartz等人,2016年),并得到NSF CBET-1403264和-1403099以及加州大学河滨校长研究奖学金的支持。作者声明不存在利益冲突或利益冲突。

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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
引用:Misa, J., Schwartz, C. and Wheeldon, I. (2018). Design of Hybrid RNA Polymerase III Promoters for Efficient CRISPR-Cas9 Function. Bio-protocol 8(6): e2779. DOI: 10.21769/BioProtoc.2779.
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