CG000197 RevATECHNICAL NOTE

Guide RNA Specifications Compatible
with Feature Barcoding technology
for CRISPR ScreeningIntroduction
The Single Cell Gene Expression Solution coupled with Feature Barcoding technology enables profiling of gene
expression in conjunction with additional cellular features from the same single cell. Specifically, the Single
Cell Gene Expression and CRISPR Screening Solution provides a high-throughput and scalable approach toobtain gene expression profiles along with CRISPR-mediated perturbation phenotypes in transduced cells via
direct capture of poly-adenylated mRNAs and single-guide RNAs (sgRNAs) (Figure 1). This document provides
specifications for engineering Feature Barcoding technology compatible sgRNAs for use with the Chromium
Single Cell 3ʹ workflow (Figure 2) for CRISPR screening.

Chromium Single Cell 3ʹ v3 Gel Beads

Capture Sequence Integration in sgRNAIn addition to a poly(dT) primer sequence that enables
the production of barcoded, full-length cDNA from polyadenylated mRNA, the Single Cell 3’ v3 Gel Beads (Figure1) also include two additional primer sequences (CaptureSequence 1 and Capture Sequence 2) for direct capture andpriming of Feature Barcoding technology compatible sgRNAs
present in a cell inside a Gel-bead-in-emulsion (GEM).

To enable direct capture, each sgRNA should beengineered to contain either Capture Sequence 1 orCapture Sequence 2, along with a protospacer (FeatureBarcode), designed to target gene/s of interest. Two
possible locations for integrating the capture sequence inthe sgRNA include (1) within the sgRNA hairpin structure,or (2) immediately before the sgRNA termination signal,
elongating the 3’ end of the sgRNA (Table 1). However,alternate sgRNA integration locations for either of thetwo capture sequences may be possible depending on thespecific application, type of construct used etc.

TruSeq Read 1
Nextera Read 1(Read 1N)

Single Cell 3ʹ
v3 Gel Bead

Nextera Read 1(Read 1N)

10x UMIBC

Poly(dT)VN

10x UMIBC

Capture Sequence

10x UMIBC

Capture Sequence 2

Figure1. Schematic of a Chromium Single Cell 3’ v3 Gel Bead, including theCapture Sequence 1 and the Capture Sequence 2 for direct capture of sgRNA
molecules from a cell inside a GEM.

Transduced Cells

Cells

Performing sgRNA QC by qPCR, NGS or other methods
is recommended before proceeding with 10x Genomics
Single Cell Solutions. The Chromium Single Cell GeneExpression and CRIPSR Screening Solution has beenshown to be compatible with sgRNA constructs in several
CRISPR applications, including interference (CRISPRi),
activation (CRISPRa), and cutting.
Cell Selection

Library Preparation

Sequencing

Pooled sgRNA

Figure 2. Transduced cells with integrated sgRNA for the Chromium Single Cell 3ʹ workflow for CRISPR screening.
10xGenomics.com

Guide RNA Specifications Compatible with Feature Barcoding technology for CRISPR Screening | Rev ACapture Sequence 1
Capture Sequence 1 on Gel Bead: 5ʹ-TTGCTAGGACCGGCCTTAAAGC-3ʹ
Capture Sequence 1
integrated in sgRNA hairpin

Capture Sequence 1
Hairpin 1

Upper Stem
Nexus

Lower Stem

Hairpin 2

Protospacer

Termination Signal-3ʹ

5ʹ-5ʹ-NNNNNNNNNNNNNNNNNNNNGTTTAAGAGCTAAGCTGGAAACAGCATAGCAAGTTTAAATAAGGCTAGTCCGTTATCAACTTggccGCTTTAAGGCCGGTCCTAGCAAggccAAGTGGCACCGAGTCGGTGCTTTTTTT-3ʹ

Capture Sequence 1
integrated in sgRNA 3ʹ-end

Upper Stem

Hairpin 1
Nexus

Hairpin 2

Lower Stem

CaptureSequence 1

Protospacer

Termination Signal-3ʹ

5ʹ-5ʹ-NNNNNNNNNNNNNNNNNNNNGTTTAAGAGCTAAGCTGGAAACAGCATAGCAAGTTTAAATAAGGCTAGTCCGTTATCAACTTgaaaAAGTGGCACCGAGTCGGTGCGCTTTAAGGCCGGTCCTAGCAATTTTTTT-3ʹ

Capture Sequence 2
Capture Sequence 2 on Gel Bead: 5ʹ-CCTTAGCCGCTAATAGGTGAGC-3ʹ
Capture Sequence 2
integrated in sgRNA hairpin

Capture Sequence 2
Upper Stem

Hairpin 1
Nexus

Lower Stem
Protospacer

Hairpin 2

Termination Signal-3ʹ

5ʹ-5ʹ- NNNNNNNNNNNNNNNNNNNNGTTTAAGAGCTAAGCTGGAAACAGCATAGCAAGTTTAAATAAGGCTAGTCCGTTATCAACTTggccGCTCACCTATTAGCGGCTAAGGggccAAGTGGCACCGAGTCGGTGCTTTTTTT -3ʹ

Capture Sequence 2
integrated in sgRNA 3ʹ-end

Upper Stem

5ʹ-

Hairpin 1
Nexus

Hairpin 2

Lower Stem
Protospacer

CaptureSequence 2

Termination Signal-3ʹ

5ʹ- NNNNNNNNNNNNNNNNNNNNGTTTAAGAGCTAAGCTGGAAACAGCATAGCAAGTTTAAATAAGGCTAGTCCGTTATCAACTTgaaaAAGTGGCACCGAGTCGGTGCGCTCACCTATTAGCGGCTAAGGTTTTTTT -3ʹ

Table 1. Integration of Capture Sequence 1 and Capture Sequence 2 in sgRNA.
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Guide RNA Specifications Compatible with Feature Barcoding technology for CRISPR Screening | Rev A2