TITLE: CRISPRi screen, MYH9 locus
AUTHORS: Brian D. Cosgrove, Lexi Bounds, Carson Key Taylor, Alan L. Su, Anthony J. Rizzo, Alejandro Barrera, Andrea R Daniel, Gregory E. Crawford, Brenton D. Hoffman, Charles A. Gersbach

[DESCRIPTION]
This protocol describes methods for a CRISPR interference screen to identify open chromatin regions that function as stiffness-responsive modulators of MYH9 expression.

[STEPS]
SECTION: Library design and cloning
1. Open chromatin regions within 440 kb of the MYH9 TSS are used to generate the oligo pool (identified from ATAC-seq of primary human fibroblasts cultured on soft and stiff ECM). For each ATAC-seq peak, any gRNA with a GuideScan specificity score of > 0.2 is included [which has previously been shown to increase the quality of non-coding screens (1)].  114 peaks are represented in the library, with an average of ~41 gRNA/peak.
SECTION: Library design and cloning
2. 500 non-targeting gRNA are included as negative controls (2). The combined gRNA
library of 5,192 gRNA is synthesized as an oligo pool by Twist Biosciences with common overhangs for cloning into our lentiviral backbone. 
SECTION: Library design and cloning
3. PCR amplify the oligo pool.
SECTION: Library design and cloning
4. A hU6-driven lentiviral gRNA vector (pBDC119) is then digested with Esp3I, gel purified, and ligated along with the amplified oligo pool by Gibson assembly.
SECTION: Library design and cloning
5. A 1x SPRI cleaning step is preformed, the Gibson assembly transformed into Endura competent cells (Lucigen) according to the manufacturer's protocol, and cultured overnight before maxi-prepping the gRNA-library plasmid.
SECTION: Library design and cloning
6. A PCR amplicon across the gRNA region of the resulting plasmid is sequenced to a depth of ~100k-1M read pairs on an Illumina miSeq in order to verify coverage across the entire gRNA library.
SECTION: Lentiviral generation and functional titering of MYH9 locus library
7. gRNA library plasmid is co-transfected into ~18M HEK293T cells along with two lentiviral packaging plasmids using Lipofectamine 3000 (ThermoFisher). 
SECTION: Lentiviral generation and functional titering of MYH9 locus library
8. 20 hours post-transfection, the transfection media is removed and fresh growth media added.
SECTION: Lentiviral generation and functional titering of MYH9 locus library
9. One day later, media containing viral particles is removed (at 48 hours post-transfection) and stored at 4C. Fresh media is added to the cultures and collected one day later for storage at 4C.
SECTION: Lentiviral generation and functional titering of MYH9 locus library
10. The media containing viral particles is combined and filtered using 0.45 μm low-protein binding filters. The filtered media is concentrated using Lenti-X Concentrator (Takara Bio) according to the manufacturer’s protocol.
SECTION: Lentiviral generation and functional titering of MYH9 locus library
11. Functional titering to determine MOI is performed by transducing HFF cells across a 50x-10,000x dilution range of the viral stock, and then subjecting the cells to FACS-based cell sorting to identify what percent of the population is mCherry+ for each viral stock dilution. 
SECTION: CRISPRi locus screen
12. A stable HFF (primary human neonatal foreskin fibroblasts) cell line created using a lentiviral
dCas9-KRAB construct [pLV-hUbC-dCas9-KRAB-2A-Blast (pJB289)]. The gRNA library is transduced at an MOI of ~0.33 followed by Puro selection for four days at 1 ug/mL. 
SECTION: CRISPRi locus screen
13. Cells are maintained for an additional four days, prior to trypsinization with 0.25% Trypsin-EDTA for 5 minutes at 37C. Trypsin is neutralized with 1X volumes of complete growth media, cells are centrifuged at 300g for 5 minutes, and the supernatant aspirated. The cells are rinsed once with 1X volume PBS followed by another centrifugation and aspiration leaving 200uL of PBS above the pellet.
SECTION: CRISPRi locus screen
14. The eBioScience ICC Fixation kit (ThermoFisher) is used to fix/permeabilize cells according to manufacturer’s instructions. Note, equilibrate reagents to room temp prior to usage. To fix cells, add 500uL eBioSciences Fix/Perm Buffer (ThermoFisher) to the 200uL PBS and pellet, and incubate at room temperature for 20 minutes.
SECTION: CRISPRi locus screen
15. Add 1X Permeabilization Buffer up to 8mL total volume, spin at 600g for 5 minutes, and add perm buffer rinse.
SECTION: CRISPRi locus screen
16. Count HFF cells and remove approximately 2 million to use as unsorted controls. Set aside another approximately 500,000 cells to use as control samples for single channel compensation controls.
SECTION: CRISPRi locus screen
17. Immunostaining is performed on the remaining cells using a AlexaFluor-488 conjugated Rabbit
monoclonal anti-NMMIIA antibody (clone EPR8965, Abcam, #ab204675) at a ratio of 0.5 uL antibody per 300k HFF cells per 100uL of Perm Buffer.
SECTION: CRISPRi locus screen
18. HFF cells are incubated for 30 minutes at room temperature in the dark on a nutating rocker, spun 600g for 5 minutes spin, followed by two repeats of 3mL 1X Perm Buffer rinse/spin cycles.
SECTION: CRISPRi locus screen
19. Cells were resuspended in FACS Buffer [1X PBS supplemented w/ 1% BSA (Sigma) and 0.5mM EDTA (Sigma)] at a density of ~9M cells/mL and sorted. A SH800 Cell Sorter (Sony Biotechnologies) is used to separate out the top/bottom-expressing MYH9 fractions following immunostaining.

SECTION: CRISPRi locus screen
20. Single channel expressing cell populations are used to set up compensation panels. These include untreated cells, antibody-only cells, mCherry-only cells. The top 10% and lower 10% of the MYH9 population is sorted and used for downstream gRNA-enrichment analysis and sequencing. 
SECTION: gDNA recovery and library preparation
21. Sorted cells are counted and DNA extracted form the fixed cells using the PicoPure DNA extraction kit (ThermoFisher) according to the manufacturer's protocol. 
SECTION: gDNA recovery and library preparation
22. Recovery digests are performed for 20 hours at 65C using up to 1.5M HFF cells per reaction volume. All gDNA is split between sample-indexed 100uL Q5 PCR reactions (up to ~340ng max input per 100uL reaction) to amplify out the gRNA protospacer from HFF cells.
SECTION: gDNA recovery and library preparation
23. gDNA PCR reaction conditions: 
98C for 30s / 25x: 98C for 10s, 60C for 30s, 72C for 15s / 72C for 2 min. 

Primers:

 
 
SECTION: gDNA recovery and library preparation
24. Individual PCRs are pooled together and subjected to a double-sided 0.65X/1X SPRI clean-up.
SECTION: gDNA recovery and library preparation
25. Quality control is performed by running the amplified libraries out on a High Sensitivity D1000 Tapestation (Agilent) to confirm expected size and Qubit dsDNA HS assays were performed to determine a final concentration.
SECTION: gDNA recovery and library preparation
26. All libraries are pooled to an effective concentration of 4 nM and combined in equal volumes prior to sequencing on an Illumina MiSeq, using a v2 50 cycle reagent kit with Read1 being 21 cycles (protospacer) and index read 1 being 6 reads (sample barcoding). 
SECTION: MYH9 locus library analysis
27. Resulting FASTQ files are aligned to a custom reference sequence corresponding to the given gRNA library using bowtie2 and all downstream analyses performed in R.
SECTION: MYH9 locus library analysis
28. All gRNA are verified to be represented in the baseline untreated library data Day 8 post-transduction, and counts+1 for each gRNA are taken (to normalize for samples that dropped out in one condition) and normalized by sequencing depth for each library before downstream analysis (in counts per million reads sequenced, ‘CPM’).
SECTION: MYH9 locus library analysis
29. Due to the highly-apparent strand bias in the positive-strand when targeting the MYH9 gene body  only include the non-interfering gRNA from the negative strand.
SECTION: MYH9 locus library analysis
30. Take a ratio of the CPM for each gRNA of the low MYH9 expression group to high MYH9 expression group to identify whether the gRNA perturbation led to increases in enrichment in either expression bin. 
SECTION: MYH9 locus library analysis
31. Next, for each screen replicate the Z-score is calculated for each gRNA relative to the control non-targeting gRNA population using similar methodologies as previously described in (3).
SECTION: MYH9 locus library analysis
32. First, each sample’s ratio is converted to a log2 fold-enrichment, and population statistics for the negative control non-targeting gRNAs (median, standard deviation, gRNA number) are calculated.
SECTION: MYH9 locus library analysis
33. For each individual gRNA, the median of the negative control fold-enrichment is subtracted from each individual gRNA’s log2 fold-enrichment value, and this value is further divided by the standard deviation of the negative control non-targeting gRNA population to get an individual Z-score relative to the negative control population. 
SECTION: MYH9 locus library analysis
34. Raw Z-score values from both replicates are pooled to calculate pRE-wide effects. Phenotype scores (t-score based) are calculated as: