More recently, electroporation of HSPCs with ribonucleoprotein
(RNP) complexes made from recombinant Cas9 protein and
synthetic chemically modified gRNAs has achieved high efficacy
across a number of targets.3-5 The RNP-based ArciTect™
CRISPR-Cas9 expression system includes custom synthetic
chemically modified gRNA (sgRNA or crRNA) and purified Cas9
protein to fully support genome editing of human HSPCs. In
addition to effective genome editing tools, manipulation of
HSPCs requires standardized culture conditions for optimal editing
efficiency and progenitor cell expansion. StemSpan™ media
supports standardized culture conditions for high-efficiency
genome editing, while allowing researchers the flexibility to
choose from serum-free, xeno-free, and animal component-free
formulations to establish specific conditions for their experiments.
The following protocol is for the preparation and subsequent
delivery of CRISPR-Cas9 RNP complexes into human CD34+ cells via electroporation using either the Neon® Transfection System or Lonza® 4D Nucleofector™ X Unit (Figure 1). It is important to first isolate CD34+ cells, as their frequency in cord blood (CB) or
other hematopoietic tissues is low, and cell quality and viability
can be variable between different primary samples. Despite the
small sample volume and relatively low frequency of CD34+ cells (typically 0.1 - 1% of nucleated cells), CB is a good source of HSPCs
for techniques such as gene editing. CD34+ cells can be isolated
from whole CB using EasySep™ Human Cord Blood CD34 Positive
Selection Kit II (Catalog #17896) in a simple, two-step procedure
(Document #DX20181). This protocol was validated using both
fresh and cryopreserved CB-derived CD34+ cells and may need to
be further optimized for CD34+ cells isolated from other sources.
Following the protocol, we present a comprehensive study of
pre- and post-editing culture conditions that led to identification
of optimal conditions for efficient CD34+ genome editing and
maintenance of the HSPC phenotype.
Delivery of CRISPR-Cas9 Ribonucleoprotein
Complexes into Human CD34++ HSPCs Using Electroporation
†ArciTect™ sgRNA is only available in Australia, Austria, Belgium, Canada,
China, Denmark, Finland, France, Germany, Iceland, Ireland, Luxembourg,
the Netherlands, New Zealand, Norway, Poland, Portugal, Singapore,
Spain, Sweden, Switzerland, the United Kingdom, and the United States.
*ArciTect™ Cas9-eGFP Nuclease (Catalog #76006) or ArciTect™ Cas9 Nickase (Catalog #76009) can also be used. All testing in CD34+ cells was performed using ArciTect™ Cas9 Nuclease.
Case Study: Evaluation of Optimal Culture Methods for High-Efficiency Editing in CD34+ Human Hematopoietic Stem and Progenitor Cells (HSPCs)
To demonstrate genetic knockout with the ArciTect™ CRISPR-Cas9 system, we targeted two genes: CD45 and β2 Microglobulin (B2M) in CD34+ HSPCs. Loss of function due to CRISPR-Cas9-mediated INDEL generation at either locus is readily identifiable by the lack of CD45
or MHC-I expression on the cell surface. Using these experimental systems, we also tested multiple pre- and post-editing media to identify
optimal conditions for efficient genome editing and maintenance of the HSPC phenotype.
We first optimized electroporation conditions by targeting CD45, as previously described.4
cells were isolated from human
CB using EasySep™ Human Cord Blood CD34 Positive Selection Kit II. On
day 0, approximately 80 - 95% of the cells were CD34+
(data not shown). The
cells were cultured in StemSpan™ SFEM II supplemented with StemSpan™ CD34+ Expansion Supplement for 2 days prior to CRISPR-Cas9
RNP delivery, as post-isolation culture with cytokine exposure for 48 hours prior to RNP delivery has been shown to increase transfection
efficiency.4 After 2 days, cells were electroporated with RNP complexes containing crRNA:tracrRNA duplexes or sgRNA targeting the CD45 gene, using either the Neon® Transfection System or the Lonza® 4D-Nucleofector™ X Unit. The electroporation settings are described in the
protocol section of this document (Section F). Four days after electroporation, cell viability and expression of CD34 and CD45 were assessed
using flow cytometry (Figure 2). RNP-electroporated samples exhibited loss of CD45 expression on the cell surface (Figure 2A, B; > 50%
average knockout efficiency across all tested donors), while the percentage of CD34-expressing cells were unaffected by RNP delivery (Figure
Figure 2. High-Efficiency Genome Editing in CD34+ Cells Using the ArciTect™ CRISPR-Cas9 System
(A) Representative histogram of CD45 flow cytometry data from CD34+ cells cultured in StemSpan™ SFEM II plus StemSpan™ CD34+ Expansion Supplement, 4 days after
delivery of CRISPR-Cas9 RNP complexes containing either crRNA (dark grey bars) or sgRNA (orange bars) targeting CD45. Non-electroporated (No EP; light grey bars)
CD34+ cells served as a control. (B) CD45 editing efficiency and (C) the percentage of CD34+ cells were monitored by flow cytometry using a fluorophore-conjugated CD45
antibody and a fluorophore-conjugated CD34 antibody, respectively, 4 days after electroporation with CRISPR-Cas9 RNP complexes containing Cas9 Nuclease and either
crRNA:tracrRNA duplexes or sgRNA using either the Neon® Transfection system or Lonza® 4D-Nucleofector™ X Unit. (D) Post-electroporation cell recovery was measured
by dividing the total number of cells collected 4 days post electroporation by the number of electroporated cells. Each data point represents an individual donor; n = 4 - 7
donors, **P < 0.01, ***P < 0.001. Error bars represent standard deviation. (E, F) Genome editing (cleavage) efficiency was assessed 4 days post electroporation with
CRISPR-Cas9 RNP complexes containing Cas9 Nuclease and crRNA:tracrRNA duplexes using the Neon® Transfection system (E) or Lonza® 4D-Nucleofector™ system (F) in
CD34+ cells using ArciTect™ T7 Endonuclease I Kit. No electroporation: - RNP; RNP electroporated: + RNP.
Post-electroporation cell recovery, as measured by the total number of cells present after 4 days of culture divided by the total number of
cells electroporated per condition, was negatively impacted by the delivery of RNP complexes containing crRNA:tracrRNA duplexes or sgRNA
compared to non-electroporated (no EP) samples (Figure 2D). This is consistent with previous reports of delayed HSPC proliferation in response
to genome editing with programmable nucleases including CRISPR-Cas9.6 INDEL generation was confirmed by the T7 Endonuclease I Assay (Figure 2E, F).
Next, we targeted a second locus in CD34+
cells to validate the optimized protocol and evaluate the functional impact of genome editing
on HSPC lineage commitment using the colony-forming unit (CFU) assay. We chose to target B2M, which encodes the accessory chain of
major histocompatibility complex (MHC) class I molecules, using a single ArciTect™ crRNA sequence targeting exon 1 of B2M, as previously described.7 This strategy enabled monitoring of editing efficiency by flow cytometry with a fluorophore-conjugated MHC-I antibody, since
expression of B2M
is required for surface expression of MHC-I.8,9 CD34+ cells were
electroporated with RNP complexes using the Neon®
Transfection System. Four days after electroporation, cell viability and expression of CD34 and MHC-I were assessed using flow cytometry
(Figure 3). RNP-electroporated samples exhibited loss of MHC-I expression on the cell surface (Figure 3A, B; > 50% knockout efficiency
across 10 donors). The percentage of CD34-expressing cells and cell viability were unaffected by RNP delivery (Figure 3C and data not
shown, respectively). Both RNP-electroporated (RNP) and cells electroporated with Cas9 only (Cas9 Only) conditions exhibited reduced postelectroporation
cell recovery relative to non-electroporated (no EP) controls, suggesting that electroporation, rather than loss of target gene
expression, is the primary contributor to reduced CD34+ cell recovery/growth in the genome editing workflow (Figure 3D).
Four days after electroporation, a portion of the cells were plated in methylcellulose medium (MethoCult™ H4435 Enriched; Catalog #04435) and a 14-day CFU assay was performed (Figure 3E, F). RNP-electroporated cells showed a similar composition of erythroid (BFU-E), granulocyte/macrophage (CFU-GM), and multilineage (CFU-GEMM) progenitor cells as compared to non-electroporated and Cas9-only conditions (Figure 3E), and plating efficiency was similar between conditions (Figure 3F). Together, this suggests that genome editing of B2M with the ArciTect™ system has minimal impact on the function of myeloid, erythroid, and granulocytic progenitor cells.
Figure 3. Genome-Edited CD34+ Cells Retain Colony-Forming Potential
(A) Representative histogram of MHC-I flow cytometry data from RNP-electroporated (RNP, orange) and non-electroporated (no EP; dark grey) CD34+ cells cultured in
StemSpan™ CD34+ Expansion Supplement, as described in section A of the protocol, 4 days after delivery of the CRISPR-Cas9 RNP complexes containing containing gRNA
targeting B2M.(B) B2M editing efficiency (% MHC-I- viable cells) and (C) the percentage of CD34+ cells were monitored by flow cytometry using a fluorophore-conjugated
MHC-I antibody and a fluorophore-conjugated CD34 antibody, respectively, in non-electroporated (no EP, dark grey), Cas9 only (cells electroporated with Cas9 only without
gRNA, light grey), and RNP-electroporated (RNP) cells. (D) Post-electroporation cell recovery was measured by dividing the total number of cells collected 4 days post
electroporation by the number of electroporated cells. (E-F) 4 days post-electroporation, cells were plated in MethoCult™ H4435 Enriched (Catalog #04435) and cultured for
an additional 14 days, before counting. Distribution of CFU colony sub-types (E) or the number of colonies per input cell (F) were comparable between edited and non-edited
controls. Each data point per condition represents an individual donor; (A-D) n = 10 donors; (E,F) n = 7 donors. Error bars represent standard deviation.
StemSpan™ media promote similar or greater expansion of CD34+ cells compared with alternative commercial media products, providing an optimal media system to support CRISPR-Cas9 genome editing in CD34+
cells (Figure 4). Using the ArciTect™
CRISPR-Cas9 system, B2M editing efficiency
(Figure 4A) and cell viability (data not shown) was comparable across medium conditions. However, StemSpan™ media best support maintenance of
CD34 expression (Figure 4B) and expansion of more primitive subsets of HSPCs defined by CD34+CD90+CD45RA-
phenotype after genome
editing (Figure 4C).
Figure 4. StemSpan™ Media Support Better CD34+ and Primitive CD34+CD90+CD45RA- HSPC Expansion for Genome Editing Applications Compared with Alternative Commercial Media
Cells were cultured in the indicated medium supplemented with StemSpan™ CD34+ Expansion Supplement plus 175nM UM171* for 2 days, electroporated with CRISPR-Cas9 RNP complexes containing crRNA:tracrRNA targeting B2M. Non-electroporated (no EP) and cells electroporated with Cas9 only without gRNA (Cas9 Only) conditions were cultured in StemSpanTM SFEM II supplemented with StemSpanTM CD34+ Expansion Supplement plus 175 nM UM171*. (A) B2M knockout efficiency (% MHC-I- viable cells) was monitored by flow cytometry using a fluorophore-conjugated MHC-I antibody. (B) The percentage of CD34+ cells and (C) CD34+CD90+CD45RA- cells were quantified by flow cytometry 4 days post-electroporation. Each data point per condition represents an individual donor; n = 4 donors, **P < 0.01. Error bars represent standard deviation. Xeno-free Commercial Alternative 1 - 5 included, in random order, CTS™ StemPro™ HSC (Thermo), SCGM (Cellgenix), X-VIVO™ 15 (Lonza), Stemline™ II (Sigma), and StemPro™-34 (Thermo)
Note: Data for StemSpan™-AOF shown were generated with the original phenol red-containing version StemSpan™-ACF (Catalog #09855). However internal testing showed that the performance of the new phenol red-free, cGMP-manufactured version, StemSpan™-AOF (Catalog #100-0130) was comparable.
*The small molecule UM171 was used to generate data in Figure 4. UM171 is no longer licensed for sale by STEMCELL Technologies, however similar results are expected when using UM729 (Catalog #72332) prepared to a final concentration of 1 μM (data not shown). Further titration may be necessary to optimize cell fold expansion in specific conditions. For more information including data comparing UM171 and UM729, see Fares et al.10
Here, we describe an optimized protocol for high efficiency genome
editing of CD34+ cells using the ArciTect™ CRISPR-Cas9 system and
StemSpan™ media for pre- and post-editing culture. We found that
sgRNA exhibits higher editing efficiency compared to two-part
crRNA:tracrRNA duplexes. While pre- and post-editing culture in all
StemSpan™ media and supplements were compatible with the genome
editing workflow, we found that StemSpan™ SFEM II supplemented
with StemSpan™ CD34+ Expansion Supplement best supports
maintenance of CD34 expression and expansion of primitive HSPC
subsets after genome editing.
The genome editing strategy outlined in this document is typical for
most genetic knockout applications. Application-specific protocol
modifications, not presented here, might include: use of ArciTect™
Cas9-eGFP Nuclease (Catalog #76006) for visualization of positive
transfectants; use of the single-strand endonuclease ArciTect™ Cas9
Nickase (Catalog #76009) with two flanking gRNAs; or the addition of
a DNA donor template for homology-directed repair (HDR)-mediated