CRISPR Screen In Vitro Steps with IT Capability Needed

LentiViral sgRNA Establish Cas9+ Cell Meta Data

Generate Pool Create Arms IT Capability Example Data Output
Library LInes (MVP)
sgRNA Library
START # of guides
Perturbation Sequences Registers sgRNA Library
Perturbation Type with ID to track

sgRNA library with

Register Vector
Vector backbone
Vector Map
Source Registers Vector
Antibiotic resistance with ID to track
CRISPR Function: KO, KD etc
Cas9 (Yes/No)

pDNA
sgRNA Library ID Registers pDNA with
Vector ID ID to track

sgRNA Library
Barcode Barcoded tubes with
Inventory
Amount (volume) location
Location

Vector
Barcode Barcoded tubes with
Amount (volume) location
Location

pDNA
Barcode Barcoded tubes with
Amount (volume) location
START
Location

Parent Cell line

Cell line Name Registered Cell Line
Make Virus with ID to track
Register

Virus
Cell line ID
pDNA ID
Seeding density
Doubling time
Infectability of cell line
Virus batch ID
Method used to establish line
Registered Virus
Pool or Clone
with ID to track
Clone info
Details for establishing line
Antibiotic type
Antibiotic Concentration
Cas9 Assessment method
% Expression
Cells Ready
%Activity
Activity Data

Cell Line
Barcode Barcoded tubes with
Inventory
Amount (volume) location
Location
Virus Batch
Barcode Barcoded tubes with
Amount (volume) location
Location

Titer of Virus Data Scale up Cell Mix calculations (per rep)

Cell Line ID Minimium wells all reps 107
Media used .+4 wells for no infection control and pipetting 111
Cells to infect /rep 3.33E+08
Virus Batch
Media (mL) without virus 216.45
Polybrene Conc:
Assess Virus Total volume with virus (mL) 222
Instrument Capture Other Infection Details Vicell Polybrene (ug) 888
Protocol used Manual Entry
Titer Assessment Scale up Infection Mix (per rep) calculations
Volume virus Wells to infect (min +1) 108
Expected Titer Functional Volume Media+Cells+PB Mix (mL) 210.6
Expected Titer Flow by Volume Total virus vol (mL) 5.4
vol to add to 12 wells / well (mL) 2

Cell Line Name

START Source: Vendor / inhouse
CAS9 source Registered Cell Line
Register QC mycoplasma Test with ID to track
QC Cell line Identity
Verify Cas9 Expression
Grow & Passage Cells
(with or without Cas9)
Barcode
Barcoded tubes with
Inventory Amount
location
Location

Verify Cas9 expression

(if built in house)
Instrument Capture Labware: Flasks, 96 well FACS Data
Cells Ready? (If plate multiple cell lines, 3
wells/line with reference
cells

Cell Line Name

Source: Vendor / inhouse
CAS9 source Registered Cell Line
YES QC mycoplasma Test with ID to track
QC Cell line Identity
Verify Cas9 Expression
Cell Line A375
Infection Format 12 well high density Total volume/well (uL) 2000
Infection Batch Registered Infection Cells/Well 3.00E+06 Max Viral Input (uL) 50
Polybrene (ug/mL) 4 Total Resuspended Cell/well (uL) 1950
Register Cell line ID Batch with ID to Total vol/well (mL) 2
Virus Batch ID track
Infection Stage Wells 120 Polybrene (ug) 960
Cells 3.60E+08 Polybrene stock (ug/uL) 10
Total Cells Cell Master Mix (mL) 234 Polybrene (uL) 96
Total yield Polybrene (uL) 96
Instrument Capture Date of experiment
Time virus added Cell Mix (mL) Virus (mL) Media (mL)
& Virus + cells (Master mix) 218.4 5.6 0
Assay Capture Time virus Removed
ViCell NIC 1.95 0 0.05
Labware- Flasks or Dish
CS JL
Polybrene concentration Infect cells and spin at 930x g
9/23/2019 14:45 9/23/2019 14:45
30C for 2hr
Titered cell Move to incubator 9/23/2019 16:45 9/23/2019 16:45
Polybrene Pool cells and plate in 5 stacks 9/24/2019 11:00 9/24/2019 12:00
Labware 12 well Passage into puro 9/25/2019 10:30 9/25/2019 10:30

9/13/2019
Total Cells count in & out of each RFP+ (SSC-A v. PE-A) Cell Counts
(+) Puro (-) Puro
9/13/2019 9/16/2019 9/13/2019 9/16/2019
passage NIC 0.33% 0.80% 0.92% 1.1% NIC 0.00358 0.390 0.92%
400uL 0.0751 0.354 21.21%
Media volume ViCell 400uL 90.71% 89.59% 21.21% 20.1%
200uL 0.149 0.312 47.76%
200uL 66.12% 67.97% 47.76% 28.5%

Instrument Capture Puro (+)(-) Reference

100uL 40.48% 44.68% 8.66% 0.0%
Reference
100uL 0.0227 0.262 8.66%
Antibiotic Selection Date of experiment Virus (sgGFP)
50uL 23.30% 27.49% 8.91% 1.8%
Virus (sgGFP)
50uL 0.0262 0.294 8.91%
Stage & 25uL 12.17% 14.21% 7.78% 0.5% 25uL 0.0238 0.306 7.78%

Assay Capture Time selection started 12.5uL 5.92% 8.13% 2.29% 0.2% 12.5uL 0.00954 0.416 2.29%
6.25uL 3.56% 5.54% 5.22% 1.2% 6.25uL 0.0167 0.320 5.22%
Passage times 400uL 89.72% 90.09% 52.53% 49.8%
400uL 0.249 0.474 52.53%
Time selection end 200uL
100uL
71.03%
48.86%
73.88%
51.11%
50.00%
20.41%
28.7%
9.5%
200uL 0.139 0.278 50.00%
80K_hGwKO 100uL 0.0596 0.292 20.41%
Labware: 6 well plate or Flask/dish _p0
50uL 29.21% 36.15% 16.12% 2.0% 80K_hGwKO
0.0382 0.237
Cytoflex 25uL 17.20% 21.04% 11.55% 1.5% _p0
50uL 16.12%
12.5uL 7.90% 10.76% 5.44% 0.2% 25uL 0.0298 0.258 11.55%
6.25uL 3.93% 3.83% 2.59% 0.6% 12.5uL 0.0131 0.241 5.44%
NIC 0.8% 6.25uL 0.00835 0.322 2.59%

Register each Arm

Selection Registered arm with
Register Small molecule Conc. Used ID to track
Conditions

Create & Maintain Cell Pellets of each Arm

Arm 1 to many (4) Inventory Barcode Inventory Pellet
Amount
location

Screen background:
Purpose Total Reseed
Representation
Since
Doublings
Through
Day Day of Week Screen action Rep Conditions Conc Volume Cells reseeded Pre-Puro Post-Puro Post-puro
Optimization 0 Monday Infection CS
cells
3.20E+08
Vol
3.20E+08 1200 4000
Infection Puro
0

Generate Unique screen id 0.9

1.9
Tuesday
Wednesday
Pool
Split to Puro
CS
CS
3.80E+05
9.69E+05
1000
500
3.8E+08
4.85E+08
1000
500
3.80E+08
4.85E+08
1425
1817
4750
6056
0.25
0.60 0.35
4 Friday Passage on Puro CS 1.51E+06 510 7.7E+08 130.5 1.97E+08 2888 9626 1.27 1.02
Cell Line ID 7.1 Monday Start Co-Cultures CS 1.75E+06 502 8.79E+08 82.3 1.44E+08 3294 10981 3.42 3.18

Reference Virus ID 9 Wednesday End Co-culture CS

IFNy
No_IFNy
T-cells
E:T_0 1.23E+06 502 6.17E+08 0.00E+00 2315 7718 5.52 5.28 2.10
Reference Titer ID 9 Wednesday End Co-culture CS Yes_IFNy E:T_0 9.12E+05 501 4.57E+08 0 1713 5711 5.09 4.84 1.67
Instrument Capture 9 Wednesday End Co-culture CS No_IFNy E:T_2 2.43E+05 503 1.22E+08 0 458 1528 3.19 2.94 -0.24
Per Arm 9 Wednesday End Co-culture CS Yes_IFNy E:T_2 1.87E+05 505 9.44E+07 0 354 1180 2.81 2.57 -0.61

& ViCell
Morphology change
Assay Capture
Time tracked for each passage time IFNy T-cells
No_IFNy E:T_0
Rep
CS
IFNy T-cells
Yes_IFNy E:T_0
Rep
CS
IFNy
No_IFNy
T-cells
E:T_2
Rep
CS
IFNy T-cells
Yes_IFNy E:T_2
Doublings Doublings Doublings Doublings
Time track for pellet harvest or Screen end From
Through Puro Post-Puro Day
From Through
Post-Puro Day From Infection
Through
Post-Puro Day
From Through
Infection Infection Puro Puro Infection Puro
Total Cells 0 0 0 0 0 0 0 0 0 0 0 0 0 0
0.247928 0 0 0.916667 0.247928 0 0 0.9166667 0.247927513 0 0 0.91666667 0.2479275 0
Total Media volume 0.598425 0.350497247
1.266974 1.019046378
0
0
1.916667
4
0.598425 0.350497
1.266974 1.019046
0
0
1.9166667
4
0.598424761 0.35049725
1.266973892 1.01904638
0
0
1.91666667
4
0.5984248 0.350497
1.2669739 1.019046
Viability 3.423418 3.175490308
5.52 5.28
0
2.10
7.083333
9
3.423418 3.17549
5.09 4.84
0
1.67
7.0833333
9
3.423417821 3.17549031
3.19 2.94
0
-0.24
7.08333333
9
3.4234178 3.17549
2.81 2.57

Day of Experiment
Labware: Flask /dish

Register DNA Pellet each arm Conc Post-Clean up Sample

Total Yield (ng) Representation (X) 11ug Aliquot
(ng/uL) Size
Cell Pellet ID Total Approx.
Cells of input Registered DNA with Sample
Representation
Nanodro
Qubit Volume
Nanodro
Qubit
Nanodro
Qubit
Nanodro
Qubit
By lowest
Register ID to track # p p p p quant
Preparation method used pre-clean up (mL)
1 1459.85 187 182 4 748000 728000 1416.667 1378.788 58.8 60.4 60.4
Modifications
2 1304.83 152.3 130 4.5 685350 585000 1298.011 1107.955 72.2 84.6 84.6
3 1360.23 192.1 146 3.6 691560 525600 1309.773 995.4545 57.3 75.3 75.3
DNA Pellet 4 1443.56 181.8 155 5 909000 775000 1721.591 1467.803 60.5 71.0 71.0
5 902.65 214.2 198 2 428400 396000 811.3636 750 51.4 55.6 55.6
DNA Pellet each arm 6 1654.26 184.5 154 4.5 830250 693000 1572.443 1312.5 59.6 71.4 71.4
Barcode Inventory Pellet
7 358.33 88.45 63 2.5 221125 157500 418.7973 298.2955 124.4 174.6 174.6
Inventory 8 1067.05 164.4 109 5 822000 545000 1556.818 1032.197 66.9 100.9 100.9
Amount
END location Sample gDNA
Rep T-cells IFNγ
# prep
1 CS No No 1,2 A+B
Data Capture each Pellet 2 JL No No 3,4 A+B
3 CS No Yes 5,6 A+B
DNA Pellet ID 4 JL No Yes 7,8 A+B
Assessment Method 5 CS Yes No 9 A+B
NanoDrop Concentration 6 JL Yes No 10,11 A+B
Instrument Capture Qubit Concentration Nanodrop 7 CS Yes Yes 12 A+B
8 JL Yes Yes 13,14 A+B
& Volume (manual)
Assay Capture Total yield
Total cells represented
Approximate library Qubit
representation
Labware: tube
CrispR Screen
Phase

INFy

Pick seq. guides LentiVirus

Sources
Vector map Other
(sgRNA 80K) sequence This is Human
Virus Optional step PBMC
Pick
Methodology Vector Cell Line Small
Vector Molecule
Data File Features Library And/OR
(txt)

START Activate TCells

Cell Line QC & CAS9 Compare retested

NO
Process

validation results vs original

Manufacture Assign Does viral

Pick Pick Vector Generate Infect cells Maintain
START
(Backbone) OR pDNA with vector Vectored
Lentivirus
Add Virus Titer Virus backbone have YES Validate Cas9 Cell line QC
with Library Selection Create ARMS
FEED ALL End Media
Create Pellets
Genomic DNA
Library & Library library Samples Genomics END
Cas9? ARMS Screen Removal Prep

NO

No YES
YES

Screening
Complete?
Selection
Complete?
Assay Types

In
DNA Functional Infection In
QC Selection Assay
Concentration Titer Screening

Media Viability Selection Method

Verify Cas9 Cell Cell Count Media Conc &
Growth Curve Cell Counts MycoPlasma Polybrene Infection Volume By Titer & Amount Cell Count
OR Expression Identity Conc
Total Cells Total Virus
Method Volume Viability Volume

Total Yield

NanoDrop Qubit
Provided by
Incucyte ViCell OR
manufacturer ViCell OR ViCell

Cas9
Volumetric or Combine cell, virus, &
Activity Fortess Cytoflex ViCell
Weighed on scale media to well (12 well
Instrument

GFP(NEW) Incucyte Fortess Cytoflex OR

plate)
Centrifuge
Remove & culture

NanoDrop

Qubit

Vector Features to track: Register Calculate & Graph

Register Data from ATCC Look up original QC
Antibiotic Resistance Virus Register in Excel or PRISM Record data Data from INDEXXX Record data
pDNA is source to data & compare
Function (KD, KO, activation) Virus in Excel or Charles River in Excel
compare results against new QC data Record data Inventory (-80)
Guide-only or All in one BioReg in Excel Record data Excel
Generation Associate information below in Excel
Vector map/features Prep method with registered cell line:
IT

Guides Volume generated % Survival

Concentration +/- selection by viral input
Selection used
Length of selection Record data
Infection method in Excel
 SAMPLE ASSAY
SAPIO ARIA STUDYLOG DATA LAKE DARE/IJC
REQUEST APPS

Electrophoresis – Spectrophotmeter
Fluorometer –
QC DNA, RNA – measure DNA,
quantify DNA
samples RNA concentration

Upload list of Samples

using an Excel Template
TapeStation Nano-drop Qubit

Fluorescence
Video imaging
Pull in any general Sequencing activated cell
Select Samples from the Perform any automated system to analyze
sorting
Any pre ARIA “Create Study” in ARIA - Enter Notebook # & information about the Trigger an Assay Add the list of Samples Send Samples to SAPIO, cells
New Experiment from Design Sample Execute Study, Collect “Sample Manifest” in Sample Manifest to validation of the Are the Assays handled in SAPIO? Is there any
Experiment workflow in in vivo Study or in vivo Experiment Name in Experiment, like title, Request from Sample to be analyzed to the Notify respective Study
Template in SAPIO Collection in ARIA Samples ARIA Sample Manager include in the Assay samples in the sample downstream workflow still in SAPIO?
SAPIO ARM in ARIA ARIA background, Manager Sample Assay Request Stakeholders
request list input
compounds, cell lines Quantification Assays
qPCR, Western Blot
MSD Genomics -
Vi-Cell Assay FACS Analysis
Flow Cytometry Illumina
NGS
CRISPR workflow
Design Sample
Collection and Record
Data in Studylog
Biomarker, targets Immunoblotting

MSD WES

Need a way to bring multiple

samples into system using a LC-MS
common ID Gyros
Barcode scanning
Cytokine Analysis
Collect Data files in common landing
-multiple samples for 1 project Nano string etc.
Send Samples to SARP, location DATA LAKE
-frost from freezer creates bad
scanning Bio Analysis Request
-tubes should have double ID to etc.
Ability to open a project page
help identify the sample. Other then
with drop down menu to
barcode Reported to DARE or
uploading files
-Location important to help identify
Easy to upload and store per IJC
the correct sample
assay type & instrument