www.cellecta.com(877) 938-3910

Pooled Lentiviral RNAi & CRISPR Libraries

Pooled lentiviral-based libraries allow you to assay the effects of many thousands of effector constructs simultaneously in one experiment. Producing a reliable and effective screening tool with such complexity requires considerable expertise. Cellecta has extensively optimized library construction protocols and has overcome a number of technical challenges to produce quality effector libraries. Over the last several years, we have built and tested over 400 shRNA, CRISPR, and other effector libraries.

Exceptional Oligonucleotides

• Synthesized using microarray-based oligonucleotide synthesis platform

• Minimal mutation rate (typically ~0.2%)

• Each oligo sequence in pool is fully defined without randomizations

• Solid support synthesis minimizes bias by providing similar yields of each individual oligonucleotide

Range of Well-Designed Vectors

• Lentiviral shRNA and sgRNA cloning vectors provide efficient delivery of complex libraries into a wide range of cell types

• Constitutive or tet-regulated promoters for expression of shRNA or sgRNA

• Single or dual selection marker (GFP, RFP, PuroR, BleoR, NeoR, HygroR, etc.) expressed from a single CMV, hEF1a, hUbiC, hPGK, or other promoter.

Markers:GFP, RFP, PuroR, BleoR, NeoR, HygroR, Hygro-HK, etc.

•

Marker promoter:hEF1αhPGKhUbiC

CMVCMV/Ferritin

Fully Customizable

Cellecta shRNA / sgRNA Expression Lentiviral Vector

BbsIBbsI

pRS(I/G)16-U6-(sh/sg)-UbiC-TagRFP-2A-Puro

~7.5 - 8.5 kb

AmpR

RSV 5’LTR

3’∆LTR

RRE

SV40 ORI

pUCORI

SV40 PolyA

cPPT

UbiC

TagRFPPuroR 2A

U6

Pooled LibraryScreening

Cells with SpecificPhenotype

Amplification ofshRNA/barcodes

or sgRNA

Packaged, pooledshRNA / sgRNA

Library

Treatment(e.g. drugs)(optional)

TransducedTarget Cells

Selection(e.g. growth,

FACS)

Treated Cells

U6H1U6-TetH1-Tet

shRNA/sgRNApromoter:

• Flexible array-based oligonucleotide synthesis enables rapid creation of complex libraries expressing any set of shRNAs or sgRNA to any targets

• Optimized shRNA / sgRNA expression vectors with various markers, selections and both inducible and constitutive promoters

• Lentiviral-based system ensures efficient delivery of high complexity, pooled libraries into a wide range of cell types

• Internal non-targeting negative controls and positive dropout controls ensure robust screening analysis

High-Quality Pooled RNAi& CRISPR Screening Libraries

Custom shRNA & CRISPR Libraries

You Give us Your List of Targets, and We Do the Rest!

• Target list may be gene identifiers or sequences, or shRNA / sgRNA sequences (other formats also acceptable)

• You choose the type of vector you would like us to use

• Cellecta provides the oligo design and synthesis and library construction

• All libraries are checked for shRNA / sgRNA representation by NGS

• A portion of individual clones are fully sequenced to assess mutation levels

• Library can be provided as ready-to-use viral particles or in plasmid form

• Turnaround time is approximately 3 months

Screen 100 to 10,000 Targets Simultaneously

• Library sizes from a few thousand to over 50,000 shRNAs or sgRNAs

• As many shRNA or sgRNA per target as desired--even "tiling" of targets

• Optional packaging of the libraries as ready-to-transduce pseudoviral particles

PCR & Cloning

LentivirusProduction

Oligo Detachment

Packaged,Pooled

shRNA / sgRNALibrary

PlasmidshRNA / sgRNA

Library

Oligo Pool

OligoSynthesis

shRNA/sgRNA

T6

barcode

Cellecta shRNA/sgRNA design

Any set of target genes

www.cellecta.com(877) 938-3910

Panel A plots the number of reads (out of 20 million) of each shRNA specific barcode present in DU145 cells compared with the corresponding number of barcodes.

Panel B is a “heat map” that depicts significance of depletion levels for each shRNA in each of 8 cell lines screened. Red or orange indicates no significant change in the shRNAs targeting the gene over time, whereas blue indicates genes with the most significantly depleted shRNAs.

Library Modules

• Module 1 targets signal transduction genes

• Members of major signal transduction pathways

• Top-ranking genes from the Cancer Genome Atlas

• Approved drug targets

• Many disease-associated genes

• Module 2 targets other disease-associated genes and known drug targets

• Module 3 targets cell surface markers, extracellular matrix genes, and DNA binding genes

Human Genome-Wide shRNA & CRISPR Libraries

• 3 Modules, together targeting nearly all protein coding genes

• Each gene is targeted by 8 shRNA or sgRNA for a total of 55,000 shRNA or sgRNA per module

• Modules can be combined for a single genome-wide screen

For Genome-Wide Knock-down or Knock-out Coverage in Human

www.cellecta.com(877) 938-3910

A.

9 di

visi

ons

Control (Infected Cells, Day 1)

- 4.8 +1.5

(-) Luc controls

-1.5

MedianFold Change

B.

P-value0 1

DU

145

PC

3LN

CaP

CW

R22

C42

HeL

aH

293

Hep

3B

Dropout viability RNAi screen to identify disease-specific essential genes

NEW!

Control Constructs for RNAi & CRISPR Libraries Cellecta provides predesigned control constructs for optimization of protocols and screening procedures. Constructs are available in plasmid form or as ready-to-transduce pseudoviral particles.

shRNA Controls

• Luciferase

• Non-targeting

• P53

• PSMA1

• PSMA6

• RPL30

• EIF3A

• KRAS

CRISPR sgRNA Controls

• CopGFP (variant)

• Non-targeting (empty)

• PCNA

• POLR2L

pRS(I/G)16-U6-(sh/sg)-UbiC-TagRFP-2A-Puro

8.02 kb

AmpR

RSV 5’LTR

3’∆LTRWPRE

RRE

SV40 ORI

pUC ORI

SV40 PolyA

cPPT

UbiC

TagRFP

PuroR2A

U6

Cellecta shRNA/ sgRNA Expression

Vector

www.cellecta.com(877) 938-3910

Next Generation Sequencing and Analysis ServicesFor laboratories needing support with sequencing and analysis, Cellecta can provide NGS and Barcode Enumeration of samples. You provide Cellecta with frozen cell pellets or DNA after screening, and Cellecta does the rest:

• Extracts genomic DNA

• Amplifies barcodes

• Performs NGS on the Illumina NextSeq or HiSeq

• Enumerates barcodes from raw sequencing data

• Additional bioinformatics and pathway analysis available

0500

100015002000250030003500400045005000

0 100 1000 10000

Freq

uenc

y (#

of s

hRN

A / s

gRN

A)

shRNA / sgRNA Representation(Bins / copy number)