TITLE: Multiplexed CRISPR-based target-enriched next-generation sequencing for detecting antibiotic resistance genes in environmental samples
AUTHORS: Yuqing Mao, Thanh H Nguyen

[DESCRIPTION]
High-throughput detection of antibiotic resistance genes (ARGs) in complex environmental samples is challenging for two reasons: 1) ARGs account for less than 0.1% of total DNA in an environmental sample, and 2) it is difficult to detect thousands of ARGs in one reaction. Conventional methods, including metagenomic sequencing and quantitative polymerase chain reaction (qPCR), have their limitations with sensitivity and target range, respectively. Here, we propose a multiplexed CRISPR-Cas9-based target-enriched next-generation sequencing (NGS) method to detect thousands of ARGs in complex environmental samples, using sewage as a testbed. This protocol includes guide RNA design, guide RNA synthesis, DNA sample preparation, CRISPR-NGS library preparation, and data processing steps. With this protocol, ARGs in low abundances can be detected with increased read depth and higher sensitivity than regular metagenomic NGS methods. This protocol is also applicable for detecting other low-abundance genetic markers, for example, bacterial virulence factors, in environmental samples.

[BEFORE_START]
It is highly recommended to use DNA Away and RNase Away to clean all surfaces and equipment before wet lab experiments.

[STEPS]
SECTION: Guide RNA preparation
12. Mix the DNA template for either crRNA or tracrRNA, forward and corresponding
reverse primers, Phusion High-Fidelity PCR Master Mix, and molecular biology
grade water in a nuclease-free PCR tube following the volumes listed in the
table below. Pipette up and down 10 times or until well mixed. 
     Reagent      Volume

SECTION: Guide RNA preparation
13. Amplify the DNA templates for crRNA and tracrRNA in a thermal cycler for PCR. The steps in the thermal cycle are listed below.
     Step      Temperature
  (℃)      Time (s)      Cycles        Initial denaturation      98 ℃      10      1x        Denaturation      98 ℃      5      12x        Annealing      55 ℃      15        Final
  extension      72 ℃      60      1x        Hold      4 ℃      ∞      1x   

 

SECTION: Guide RNA preparation
14. Mix ATP, UTP, GTP, and CTP provided in TranscriptAid T7 High Yield Transcription Kit in 1:1:1:1 ratio in a nuclease-free microcentrifuge tube. Pipette up and down for 10 times or until well mixed.
SECTION: Guide RNA preparation
15. For the transcription reaction of crRNA and tracrRNA, mix the reagents
from TranscriptAid T7 High Yield Transcription Kit and the PCR-amplified DNA
templates in nuclease-free PCR tubes following the volumes provided in the
table below. Pipette up and down 10 times or until well mixed. A 50-μL
PCR-amplified DNA template can be divided into 4 transcription reactions in
this step.
 
     Reagent      Volume
  (μL)        Mixed
  NTP      16        PCR-amplified
  DNA template      12        5X
  TranscriptAid Reaction Buffer      8        TranscriptAid
  Enzyme Mix      4        Total      40   

 

 

SECTION: Guide RNA preparation
16. Incubate the RNA transcription samples at 37 ℃ for 4 hours.
 

SECTION: Guide RNA preparation
17. Add 5 μL of DNase I and 5 μL of DNA Digestion Buffer provided in RNA Clean & Concentrator-5 (DNase Included) to each 40-μL RNA transcription reaction. Pipette up and down 10 times or until well mixed.
SECTION: Guide RNA preparation
18. Incubate at room temperature for 15 min.
SECTION: Guide RNA preparation
19. Follow the “Total RNA Clean-up” instructions in the user manual of RNA Clean & Concentrator-5. Use 15 μL of DNase/RNase-Free Water to elute the purified crRNA or tracrRNA product. Incubate for 5 min before the final centrifugation to ensure maximum yield.

 

SECTION: Guide RNA preparation
20. Pipette each purified crRNA or tracrRNA product up and down for 10 times or until well mixed. Take 1 μL from each purified RNA sample, dilute 100-fold in 99 μL of molecular biology grade water. Pipette up and down for 10 times or until well mixed. Quantify each 100-fold-diluted purified crRNA or tracrRNA using Qubit‱RNA Broad Range (BR) Assay Kit by adding 10 μL diluted sample to 190 μL of master mix.
the
 

SECTION: Guide RNA preparation
21. Aliquot the crRNA and tracrRNA samples and store at -80 ℃ before use.
SECTION: Guide RNA preparation
22. Right before making CRISPR-NGS library, mix crRNA and tracrRNA in
anequi-molar ratio (see Equation 1), then add Nuclease-Free Duplex Buffer to
reach a final guide RNA concentration of 1500 ng/μL (see Equation 2). Pipette
up and down 10 times or until well mixed.

 
 

SECTION: Guide RNA preparation
23. Incubate the mixture in a thermal cycler at 94 ℃ for 2 min, then slowly cool down to room temperature. The guide RNA is ready to use.
SECTION: DNA sample preparation (Sewage sample as an example)
24. Shake the sewage sample until well mixed, and filter 50 mL of the sewage sample through
0.45 μm pore size membrane filter. 
 

SECTION: DNA sample preparation (Sewage sample as an example)
25. Store the membrane filter at -80 ℃ until DNA extraction.
 

SECTION: DNA sample preparation (Sewage sample as an example)
26. Extract DNA from the membrane filter using FastDNA‱ SPIN Kit for Soil following the user’s manual. Elute the DNA samples using 100 μL of DES provided in the kit.
SECTION: DNA sample preparation (Sewage sample as an example)
27. Purify the DNA samples using OneStep PCR Inhibitor Removal Kit following the user’s manual.
SECTION: DNA sample preparation (Sewage sample as an example)
28. Determine the concentrations of the DNA samples using Qubit‱ 1X dsDNA High Sensitivity (HS) Assay Kit by adding 2 μL diluted sample to 198 μL of master mix.
 

SECTION: DNA sample preparation (Sewage sample as an example)
29. Aliquot and store the DNA samples at -20 ℃ or -80 ℃ until library preparation.

SECTION: CRISPR-NGS library preparation
30. Right before library preparation, dilute the “NH8B” external standard 100-fold using
molecular biology grade water.
SECTION: CRISPR-NGS library preparation
31. Determine the concentration of the diluted “NH8B” external standard using Qubit‱ 1X dsDNA High Sensitivity (HS) Assay Kit by adding 2 μL diluted external standard to 198 μL of master mix.
SECTION: CRISPR-NGS library preparation
32. Mix the diluted Cas9 protein, the duplexed guide RNA, and NEBuffer‱ r3.1
in nuclease-free PCR tubes with the volumes listed in the table below. Pipette
up and down 10 times or until well mixed.
     Reagent      Volume
  (μL)        Cas9      2        Guide
  RNA      10        NEBuffer‱  r3.1      3        Molecular
  biology grade water      4        Total      19   

 

 

SECTION: CRISPR-NGS library preparation
33. Incubate the above mixture at room temperature for at least 15 min to bind guide RNA to Cas9.
SECTION: CRISPR-NGS library preparation
34. Block the DNA samples by removing 5’ phosphate group using rAPid Alkaline Phosphatase with the volumes listed in the table below.
     Reagent      Amount        rAPid
  Alkaline Phosphatase Buffer 10x concentrated      2
  μL        rAPid
  Alkaline Phosphatase 1 U/μl      1
  μL        DNA
  sample      ~
  200 ng        Molecular
  biology grade water      Fill
  up the volume to 20 μL   

 

 

 

SECTION: CRISPR-NGS library preparation
35. Incubate the DNA-blocking reaction mixture in a thermal cycler with the following thermal conditions.
     Step      Temperature
  (℃)      Time
  (min)        Incubation      37      10        Phosphatase
  inactivation      75      2        Hold      4      ∞   

SECTION: CRISPR-NGS library preparation
36. Mix the blocked DNA samples, the mixture of Cas9 and guide RNA, and the “NH8B” external standard with the volumes listed in the table below. Pipette up and down 10 times or until well mixed. 
     Reagent      Volume
  (μL)        Mixture
  of Cas9 and guide RNA      19        Blocked
  DNA      10        100-fold
  diluted “NH8B” external standard      1        Total      30   

SECTION: CRISPR-NGS library preparation
37. Incubate the above mixture at 37 ℃ for 16 hours.
SECTION: CRISPR-NGS library preparation
38. Add 5 μL of RNase T1 to the mixture, pipette up and down for 10 times, and incubate at 37 ℃ for 15 min to remove guide RNA.
SECTION: CRISPR-NGS library preparation
39. Prepare the master mix for dA-tailing, using the reagents and volumes listed in the table below. Pipette up and down 10 times or until well mixed.
     Reagent      Volume
  (μL)        dATP
  (100 mM)      2        Taq
  DNA Polymerase      5        ThermoPol‱  Reaction Buffer      80        Molecular
  biology grade water      13        Total      100   

 

SECTION: CRISPR-NGS library preparation
40. Add 5 μL of the dA-tailing master mix to each 35-μL mixture after RNase T1 treatment to reach a 40 μL total volume. Pipette up and down 10 times or until well mixed.
SECTION: CRISPR-NGS library preparation
41. Incubate the mixture at 72 ℃ for 20 min for dA-tailing and Cas9 inactivation.
SECTION: CRISPR-NGS library preparation
42. Dilute adapters using molecular biology grade water or xGen‱ Adapter Buffer based on the total DNA input, according to the table below.
     Total
  DNA input (ng)      Adapter
  dilution ratio        >100      100x        <100      200x   

SECTION: CRISPR-NGS library preparation
43. Ligate adapters to targeted DNA fragments using the reagents and volumes listed in the table below.
     Reagent      Volume
  (μL)        dA-tailed
  DNA sample      35        Diluted
  adapter      2.5        NEBNext‱  Ligation Enhancer      1        NEBNext‱  Ultra‱ II Ligation Master Mix      30        Total      68.5   

 

SECTION: CRISPR-NGS library preparation
44. Incubate the ligation mixture at room temperature for 15 min.
SECTION: CRISPR-NGS library preparation
45. Dilute 1x TE buffer 10-fold to make 0.1x TE buffer.
SECTION: CRISPR-NGS library preparation
46. Purify the adapter-ligated DNA samples using AMPure XP SPRI beads with beads:DNA ratio of 0.8:1 (57 μL of SPRI beads for 68.5 μL of adapter-ligated DNA sample). The detailed SPRI beads cleaning steps are listed in the table below.
     Step      On/Off
  the magnetic rack      Time
  (min)        Bind
  DNA sample to the beads      off      5        Separate
  the beads from the liquid phase      on      5        Discard
  the supernatant      on      /        1st  wash by 80% ethanol      on      2        Discard
  the supernatant      on      /        2nd  wash by 80% ethanol      on      2        Discard
  the supernatant      on      /        Air
  dry the beads with lid open      on      3-5        Resuspend
  the beads by 17 μL of 0.1x TE buffer      off      /        Release
  DNA from the beads to the liquid phase      off      10        Separate
  the beads from the liquid phase      on      5        Transfer
  15 μL of the supernatant to a clean PCR tube      on      /   

SECTION: CRISPR-NGS library preparation
47. Dilute the xGen‱ Library Amplification Primer Mix 2-fold by adding an equal volume of molecular biology grade water.
SECTION: CRISPR-NGS library preparation
48. Mix the beads-purified DNA sample, diluted primer mix, and NEBNext‱Ultra‱ II Q5‱ Master Mix in a nuclease-free PCR tube according to the table below. Pipette up and down 10 times or until well mixed.
     Reagent      Volume
  (μL)        Beads-purified
  DNA sample      15        Diluted
  primer mix      10        NEBNext‱  Ultra‱ II Q5‱ Master Mix      25        Total      50   

SECTION: CRISPR-NGS library preparation
49. Incubate the mixture above in a thermal cycler using the thermal cycle listed in the table below.
     Step      Temperature
  (℃)      Time      Cycles        Initial
  denaturation      98 ℃      30 s      1x        Denaturation      98 ℃      10 s      22x for
  >100 ng DNA input;  30 x for
  <100 ng DNA input        Annealing      65 ℃      75 s        Final
  extension      65 ℃      5 min      1x        Hold      4 ℃      ∞      1x   

SECTION: CRISPR-NGS library preparation
50. Purify the PCR product using AMPure XP SPRI beads with beads:DNA ratio
of 0.9:1 (45 μL of SPRI beads for 50 μL of PCR product). The detailed SPRI
beads cleaning steps are listed in the table below. 
     Step      On/Off
  the magnetic rack      Time
  (min)        Bind
  the PCR product to the beads      off      5        Separate
  the beads from the liquid phase      on      5        Discard
  the supernatant      on      /        1st  wash by 80% ethanol      on      2        Discard
  the supernatant      on      /        2nd  wash by 80% ethanol      on      2        Discard
  the supernatant      on      /        Air
  dry the beads with lid open      on      3-5        Resuspend
  the beads by 33 μL of 0.1x TE buffer      off      /        Release
  DNA from the beads to the liquid phase      off      10        Separate
  the beads from the liquid phase      on      5        Transfer
  30 μL of the supernatant to a clean tube      on      /   

SECTION: CRISPR-NGS library preparation
51. Determine the DNA concentration of the library using Qubit‱ 1X dsDNA High Sensitivity (HS) Assay Kit by adding 2 μL diluted external standard to 198 μL of master mix.
 

SECTION: CRISPR-NGS library preparation
52. According to the DNA concentration, take 1-2 μL of the library and dilute to ~1 ng/μL for the fragment analyzer.
 

SECTION: CRISPR-NGS library preparation
53. Store the libraries at -20 ℃ or -80 ℃ until the sequencing run.
SECTION: Multiplex crRNA design (using FLASHit as an example)
1. Create a Linux environment. It can be set up in MobaXterm (https://mobaxterm.mobatek.net/) or other preferred terminal software. Using MobaXterm as an example, the Linux environment can be created by “Sessions” -> “New session” -> WSL. Then, select “Ubuntu” for “Distribution”. Click on “OK”, and the session will be created and saved.
SECTION: Multiplex crRNA design (using FLASHit as an example)
2. Install FLASHit (https://github.com/czbiohub-sf/flash) in the Linux environment according to the instructions in “Prerequisite” on its GitHub webpage.
 

SECTION: Multiplex crRNA design (using FLASHit as an example)
3. Collect all target genes from databases. Here, as an example, all available sequences for antibiotic resistance genes (ARGs) from The Comprehensive Antibiotic Resistance Database (CARD) (https://card.mcmaster.ca/) were downloaded using the link: https://card.mcmaster.ca/latest/data.
SECTION: Multiplex crRNA design (using FLASHit as an example)
4. For an ARG detection project, among all downloaded “.fasta” files, “nucleotide_fasta_protein_homolog_model.fasta” was used as the input, because the ARGs that have raised high concerns such as the CTX-M gene families and the mcr gene families are included in the protein homolog model.
SECTION: Multiplex crRNA design (using FLASHit as an example)
5. Trim the “nucleotide_fasta_protein_homolog_model.fasta” file using the Python code below to keep only the antibiotic resistance ontology (ARO) of the ARGs in the titles, because special characters cannot be processed by FLASHit.
 

 


SECTION: Multiplex crRNA design (using FLASHit as an example)
6. In Linux terminal, activate the conda environment, then activate the environment for running FLASHit.
SECTION: Multiplex crRNA design (using FLASHit as an example)
7. [optional] By default, FLASHit excludes the off-target sites from human genomes and the E. coli BL21 genome. If an environmental sample is expected to include undesired genomes other than these two, for example, swine genomes, users can modify the files in /flash/generated_files/ accordingly.
SECTION: Multiplex crRNA design (using FLASHit as an example)
7.1. Search the reference genome of the undesired off-targets from NCBI Genome database (https://www.ncbi.nlm.nih.gov/datasets/genome/) by typing the species name in the search box.
SECTION: Multiplex crRNA design (using FLASHit as an example)
7.2. From the search results, click on the genome with theNCBI RefSeq label.
SECTION: Multiplex crRNA design (using FLASHit as an example)
7.3. Download the genome sequence by choosing “RefSeq only” and “Genome
sequences (FASTA)”.
SECTION: Multiplex crRNA design (using FLASHit as an example)
7.4. Use each “.fasta” file as an input to FLASHit following the guidance in “Creating
your own library” in the“Workflow” section on the GitHub page of FLASHit.
SECTION: Multiplex crRNA design (using FLASHit as an example)
7.5. After “Will discard xxx targets in amibiguous_targets.txt affecting xxx not necessarily unique genes.” is shown on the screen, break the current FLASHit run by hitting Ctrl+C. 
SECTION: Multiplex crRNA design (using FLASHit as an example)
7.6. Go to the directory /flash/generated_files/target_index/, copy “all_targets.txt” to a customized directory, and rename it by the input “.fasta” file name.
SECTION: Multiplex crRNA design (using FLASHit as an example)
7.7. After collecting and renaming all “all_targets.txt” files to the customized directory, split files larger than 10 Mb to separated 10 Mb files into a new directory using the command below.
 

 

SECTION: Multiplex crRNA design (using FLASHit as an example)
7.8. In the new directory containing all split files, add “.txt” suffix to all files using the command below.
 

 

SECTION: Multiplex crRNA design (using FLASHit as an example)
7.9. Copy and paste the other files smaller than 10 Mb to the directory containing all split files.
SECTION: Multiplex crRNA design (using FLASHit as an example)
7.10. Run the following python code to remove replicated off-targets and organize all off-targets to the same file.

 


 

SECTION: Multiplex crRNA design (using FLASHit as an example)
7.11. If there are multiple off-target genomes, place all output “.txt” files generated by the above code into the same directory. The list for human genome off-targets is already provided by FLASHit with the file path "/flash/generated_files/human_guides_38.txt". Combine all off-targets into the same “all_offtargets.txt” file using the following Python code.
 

 


SECTION: Multiplex crRNA design (using FLASHit as an example)
7.12. Replace the “all_offtargets.txt” file in “/flash/generated_files/” with the new “.txt” file generated by the code above. Make sure to rename the newly generated “.txt” file to “all_offtargets.txt”.
SECTION: Multiplex crRNA design (using FLASHit as an example)
7.13. Rename “human_guides_38.txt” and “ecoli_bl21_de3_offtargets.txt” in “/flash/generated_files/” to “human_guides_38.txt1” and “ecoli_bl21_de3_offtargets.txt1” to avoid those two files to be identified by FLASHit by default.
SECTION: Multiplex crRNA design (using FLASHit as an example)
8. Follow the guidance in “Creating your own library” in “Workflow” section on the GitHub page of FLASHit to generate a list for the multiplexed 20-nt target regions for the template of crRNA.
SECTION: Multiplex crRNA design (using FLASHit as an example)
9. Follow the guidance in “Creating a bed file of the guides” on the GitHub page of FLASHit to generate a file showing the cleavage sites of the crRNA on the target genes.
SECTION: Multiplex crRNA design (using FLASHit as an example)
10. Assemble the full crRNA templates by replacing the XXXXXXXXXXXXXXXXXXXX in 5’-TAATACGACTCACTATAGXXXXXXXXXXXXXXXXXXXXGTTTTAGAGCTATGCTGTTTTG-3’ by the 20-nt sequences generated by FLASHit.
SECTION: Multiplex crRNA design (using FLASHit as an example)
11. The assembled nucleotide sequences can be used for purchasing DNA oligo pools.
SECTION: NGS read mapping
54. After sequencing, download all raw sequencing data files. 
SECTION: NGS read mapping
55. Unzip the files to get ".fastq" files for each library.
SECTION: NGS read mapping
56. Download PRICE from https://derisilab.ucsf.edu/software/price/index.html to the local Linux environment. Install by navigating to the PRICE directory and typing "make" in the command line tool.
 

 

SECTION: NGS read mapping
57. Clone the KMA repository from https://bitbucket.org/genomicepidemiology/kma/src/master/ using the command below to the local Linux environment. Install by navigating to the KMA directory and typing "make" in the command line tool.
 

 

 

SECTION: NGS read mapping
58. Screen the low-quality sequencing reads using PriceSeqFilter with 85% of nucleotides in a read must be in high quality, the minimum allowed probability of a nucleotide being correct is 98%, and 90% of nucleotides in a read that must be called. An example of the command for paired sequences is shown below.
 

 

 

SECTION: NGS read mapping
59. Make a copy of the ".fasta" file used for generating guide RNA in Step 8. Add the sequence of the "NH8B" external standard to the end of the copied file. This ".fasta" file will be used as a reference gene list.

 

 

SECTION: NGS read mapping
60. Index the ".fasta" reference gene list file with KMA using the command below.
 

 

SECTION: NGS read mapping
61. Map the filtered ".fastq" files to the indexed reference gene list with KMA using the command below.

 

 


SECTION: NGS read mapping
62. The read mapping results for the target genes are available in the ".res" file generated by KMA.