Case Study

Synthetic sgRNAs
Enable Researchers
to Study Viral
Infection in Resting
Human CD4+ T Cells

ICONS FROM THE ORION SE

T

Featuring
Dr. Manuel Albanese
Postdoctoral Researcher, Max von Pettenkofer
Institute and Gene Center, Virology, National
Reference Center for Retroviruses, LMU München

APPLICATIONRESEARCH AREA

PRODUCT

Bioengineering

Infectious Disease

Synthetic sgRNAAt A Glance
Executive Summary
Dr. Manuel Albanese and his colleagues used Synthego’s synthetic
single guide RNA (sgRNA) and Lonza’s 4D-Nucleofector® System tooptimize Nucleofection® Conditions in resting human CD4+ T cells.
Dr. Albanese conducted this study as a postdoctoral researcher
together with Adrian Ruhle in Dr. Oliver Keppler’s lab at the Max
von Pettenkofer Institute and Gene Center in Munich, Germany.
The study was published in Nature Methods in January 2022, titled,“Rapid, efficient and activation-neutral gene editing of polyclonal
primary human resting CD4+ T cells allows complex functional
analyses”.

Case Study | Synthetic sgRNADr. Manuel Albanese
Postdoctoral Researcher
Max von Pettenkofer Instituteand Gene Center, Virology,
National Reference Center for
Retroviruses, LMU München

We initiallystartedexperimenting
with guides from someother companies.
However, Synthego’s
modified single guide
RNAs always worked
best in our hands, so we
decided to move ahead
entirely with Synthego.
We always use acombination of
two guide RNAs forbest efficiency.

Introduction
Resting CD4+ T cells are highly resistant to humanimmunodeficiency virus-1 (HIV-1) infection, present multiple
blocks to active HIV-1 replication, and often act as reservoirs oflatent HIV-1 infection. As such, it is important to study the biologyof these cells and mimic the viral interaction and replication
steps in naïve, resting CD4+ T cells. The authors targeted multiple
genes related to viral infectivity (up to six genes were knocked
out simultaneously at rates greater than 98% while maintaining
high viability), knocked in large reporter gene segments, andperformed downstream functional assays to characterize theirvarious gene edits.

Challenge
Previous attempts at gene editing of resting CD4+ T cells
have been met with minimal success and low cell viability.
Conventionally, it is challenging to maintain the resting statesin CD4+ T cells subsequent to gene knockout, thereby limiting
the analysis of pathways and factors that trigger CD4+ T cell
activation. This is particularly challenging within the context
of HIV-1 infection as it restricts the study of pro- and anti-viral
infection factors in host immune cells.

Solution
Through a combination of optimized culturing conditions suchas interleukin supplementation, Synthego’s sgRNA, and Lonza’s
4D-Nucleofector® System, the authors were able to optimize theediting conditions for resting human CD4+ T cells and maintainhigh cell viability for several weeks post Nucleofection®. Theseedited CD4+ T cells presented a robust system to evaluate theimpact of these individual genes on HIV infectivity in resting T cells

2through a series of downstream functional assays such as T cell
migration and fluorescence-activated cell sorting for fluorescenceexpressing HIV vectors.

Introduction
Resting human CD4+ T cells are highly resistant to active HIV-1 replication andact as reservoirs of latent infection. As such, it is critically important to study the
biology of these cells in naïve, resting CD4+ T cells. The systemic interrogationof the role of canonical genes in viral infection has been challenging as thesecells are not compatible with standard transfection protocols and often resultin low cell viability following gene editing. Unlike activated CD4+ T cells, resting
CD4+ T cells do not proliferate, thus necessitating a highly efficient protocol
for generating knockout CD4+ T cells. Previous studies in the field used
activated human CD4+ T cells for editing and allowed them to return to resting
physiological states for analysis. However, a caveat of using post-activatedresting CD4+ T cells is that they do not completely recapitulate the naïve T cell
physiology. This has greatly limited scientists from studying the different factors
and pathways involved with viral entry and replication in resting CD4+ T cells.
The present case study describes Dr. Albanese’s optimized methods to transfectCase Study | Synthetic sgRNA

resting human CD4+ T cells using Synthego’s single guide RNAs and Lonza’s
4D-Nucleofector® System. Using the optimized transfection conditions, Dr.
Albanese could knock out up to six genes (CD46, CXCR4, PSGL-1, CD4, TRIM5a,and CPSF6) simultaneously in resting CD4+ T cells with high editing efficiencies
and maintained high cellular survivability for several weeks following gene
editing. This allowed the authors to interrogate the functionality of these genes
in resting CD4+ T cells in a series of downstream assays. Furthermore, theauthors utilized this optimized protocol to knock in a green fluorescent protein(GFP) to key genes that regulate viral infection to study alternative pathways forHIV-1 infectivity through simple assays such as fluorescent-activated cell sorting.
Overall, the authors conclude that the impact of this study opens new avenues
to investigate virus and human CD4+ T cell interactions and viral latency inphysiologically relevant resting CD4+ T cells.

Pipeline Schematic to Establish Polyclonal Knockouts in Human Resting CD4+ T Cells*The above and following figures were adapted from the original article from Albanese et al,
Nat Methods 19, 81–89 (2022) and is licensed under the Creative Commons Attribution License 4.0
which permits the use, sharing, distribution, and reproduction in any format so long as appropriate
credit is given to the original authors.

3

Methods
Dr. Albanese chose Synthego’s synthetic sgRNAs and Lonza’s 4D Nucleofector® System forhis experiments, in combination with optimal cell culture media conditions. This enabled
the team to obtain the following:• Achieve high and consistent knockout efficiencies in resting primary humanCD4+ T cells• Consistent single gene knockout efficiency enabled experiments studying
multi-gene knockouts• High viability in resting human CD4+ T cells following transfection (unlike priorstudies that were limited to 72 hours)• Ability to perform downstream functional studies due to sustained high viability

Case Study | Synthetic sgRNA

Synthego’s synthetic sgRNA addressed one of the major challenges for Dr. Albanese’s
study, by providing consistently high knockout efficiencies (>98%) in resting humanCD4+ T cells. This was also critical to the downstream functional characterization studies.
Dr. Albanese used a combination of two synthetic sgRNAs for each target gene knockoutin order to achieve the best editing efficiency. For delivery into resting CD4+ T cells
Dr. Albanese used the 16-well strip of the Lonza 4D-Nucleofector® X Unit. In a total volumeof 20 µl, he transfected 2x106 cells with the pre-complexed RNPs. The nuanced pulsing of
the 4D-Nucleofector® allowed him to gain high editing efficiencies while maintaining ahigh viability.
Please refer to the detailed protocol on how to edit resting CD4+ T cells.

Design Your Own Synthetic sgRNA
Synthego’s highly pure synthetic sgRNAs ensure high experimental reproducibility. Input
your own gRNA sequence or use Synthego’s CRISPR Design Tool to start your CRISPR
experiment today.

Synthego.com/products/crispr-kits/synthetic-sgrna

4

Results
Dr. Albanese’s work describes a robust approach to editing primary humanresting CD4+ T cells through a combination of Synthego’s highly efficientsynthetic sgRNAs to ensure successful editing, Lonza’s 4D-Nucleofector®, andoptimal culturing conditions to ensure high cell survival following
Nucleofection®. This optimized approach allows for a greater number ofavailable edited cells for the study of both previously evaluated and novel
mechanisms of infection in resting CD4+ T cells.

CD4+ T Cells Retain High Viability Following
Single and Multi-gene Gene Knockouts
Dr. Albanese and his team demonstrated robust single and multi-gene
knockout of various genes integral for viral infection (CD46, CXCR4, PSGL-1,
CD4, TRIM5a, and CPSF6) in resting CD4+ T cells (Figs 1a-b). Importantly, cell
viability remained relatively high following single and multi-gene knockout(50-80% for 4 weeks following infection, Fig 1c). Upon activation, edited
T cells continued to express canonical activated T cell markers CD25, CD39
CD69, and HLA-DR (Fig 1d).

c.

b.

Case Study | Synthetic sgRNAa.

d.

Figure 1. Highly Efficient Multi-gene KO in Primary Human Resting CD4+ T Cells
Simultaneous, polyclonal six-gene knockout following a single RNP nucleofection with
Synthego’s sgRNAs resulted in the depletion of 4 cell surface markers (CD46, CXCR4,
PSGL-1, and CD4) within 2 weeks via fluorescent-activated cell sorting (a) and depletionof cytoplasmic proteins (TRIM5a and CPSF6) within 25 days via immunoblotting (b).
Viability of both single gene and multi-gene (4- and 6-gene) knockout demonstrated highcell survival 4 weeks following nucleofection as compared to wild-type (WT) controls (c).
Multi-gene-edited CD4+ T cells demonstrated a typical activated CD4+ T cell profile
following stimulation (T-Activator CD3/CD28 beads and IL-2 medium) as assessed by canonical active CD4+ T cell markers CD25, CD69, CD38, and HLA-DR via fluorescent activatedcell sorting 2 weeks following nucleofection (d). (Figure adapted from Albanese et al, Nat
Methods 19, 81–89 (2022).

5

Functional Characterization of Single Gene Knockouts
Single gene KOs were systematically evaluated for their role in HIV-1 infectivity. Cell surface
marker CXCR4 is well-known for its role in T cell chemotaxis and HIV-1 entry and infection.
The knockout of CXCR4 gene resulted in both a reduction in the ability of CD4+ T cells to
migrate and also for its ability to be infected by a GFP-expressing HIV-1 (Figs 2a-b).
Dr. Albanese and his team further evaluated transcription factors implicated in HIV-1
infection (MX2, CPSF6, PSGL-1) that had been studied in other cell systems but not resting
CD4+ T cells. Their work showed that CPSF6 did not mediate pre- and post-entry of HIV-1
into host CD4+ cells. MX2 and CPSF6 are both noted for their role in interacting with theHIV-1 capsid during nuclear entry of the pre-integration complex and proviral integration.
Interestingly, the depletion of CPSF6, but not MX2, resulted in resting CD4+ T cells beingrefractory toward HIV-1 infection (Fig 2c). These are novel findings in the field of resting
human CD4+ infectivity by HIV-1.

Case Study | Synthetic sgRNA

a.

b.

c.

Figure 2. Single Gene Knockout Allows for
Phenotypic Characterization of Cell Migration andCanonical/Non-Canonical Viral Infection in Primary
Human Resting CD4+ T Cells
The impact of CXCR4 knockout in SDF-1a (CXCL12)-driven
chemotaxis of resting CD4+ T cells was assessed 1 week
after nucleofection (a). CXCR4 knockout in resting
CD4+ T cells demonstrated reduced efficacy of GFPexpressing HIV-1 infection as compared to the NTC
group and assessed by fluorescent activated cell sorting(b). CPSF6 knockout in resting CD4+ T cells demonstrate
a reduced ability for HIV-1 infection, however, MX2
knockout does not 4 weeks after nucleofection (c).
(Figure adapted from Albanese et al, Nat Methods 19,
81–89 (2022).

6

eGFP Knock-In Upstream of SAMHD1 Enables Studying Alternative
Pathways for HIV-1 Infection
Dr. Albanese created a functional reporter system to study the mechanism of SAMHD1
in regulating viral infection. SAMHD1 is a human anti-viral protein that prevents viral
replication of HIV-1. The specific knockout of the SAMHD1 gene has been studied in otherdiseases, and loss-of-function mutations in SAMHD1 have resulted in a greater vulnerability
to viral infection.
In order to study the role of SAMHD1 in resting CD4+ T cells, Dr. Albanese and teamknocked in an N-terminal eGFP protein into the SAMHD1 gene by using one sgRNA from
Synthego and a double-stranded plasmid DNA donor template with approximately 550
base pair homology arms (Fig 3a). This resulted in a 2.3% knock-in efficiency of GFP into
T cells that were then subsequently enriched for downstream assays via FACS. ThisGFP-SAMHD1 CD4+ T cell line was then used to study alternative infectivity pathways withhigh resolution by the researchers. The team used a reporter HIV-1-BFP/SAMHD1-GFP
CD4+ T cell system where BFP and GFP were used to demonstrate shifts in the status ofT cells following infection.

a.

Case Study | Synthetic sgRNAGFP-SAMHD1 resting CD4+T cells were largely refractory to infection by a BFP-expressing
HIV-1 vector unless a known degrader for SAMHD1, Vpx, was introduced which resulted
in a decrease in the number of SAMHD1-GFP+ cells and a concomitant increase in HIV-1BFP+ cells (Fig 3b, left 4 panels). To further explore additional pathways of HIV-1
infectivity, Dr. Albanese and colleagues introduced EFV, a reverse transcriptase inhibitor,
to Vpx-treated GFP-SAMHD1 CD4+ T cells resulting in an abrogation of BFP+ cells whilestill maintaining a depletion of GFP+ T cells (Fig 3b, right 2 panels) thus allowing forhigh-resolution analysis of alternative pathways for viral infection.
b.

Figure 3. Large Knock-in of eGFP into anti-viral protein SAMHD1 Loci Allows for Study of HIV-1
Infection in Primary Human Resting CD4+ T Cells
SAMHD1, an innate human protein that blocks viral replication of HIV-1 in dendritic cells, macrophages,
monocytes, and resting CD4+ lymphocytes, was studied for its role in CD4+ T cell infectivity by HIV-1. eGFP
was knocked in upstream of the SAMHD1 loci utilizing a double-stranded plasmid DNA donor template with
550 bp homology arms for exon 1 of SAMHD1 to generate GFP-SAMHD1 expressing resting CD4+ T cells(a). Infection of the GFP-SAMHD1 CD4+ T cells with BFP-expressing HIV-1 virions is inhibited except whenVpx protein is introduced which degrades SAMHD1 protein (b, middle column). The inclusion of EFV,a reverse transcriptase inhibitor, resulted in an inhibition of BFP-HIV-1 infection despite SAMHD1
depletion (b, right column). (Figure adapted from Albanese et al, Nat Methods 19, 81–89 (2022).

7

Synthego’s Synthetic Single Guide RNAs Enable High-Efficiency
Knockouts in Resting CD4+ T Cells
When Dr. Albanese and his team compared Synthego’s synthetic sgRNAs (freshly preparedor frozen and then thawed) against another major vendor’s synthetic sgRNAs, Synthego’s
sgRNAs resulted in consistently greater (more than 2x) KO efficiency than the other vendor’s(Fig 4).

Case Study | Synthetic sgRNAThis efficient gene editing in resting CD4+ T cells is important as these assays require theuse of valuable donor cells from humans. A gene-editing protocol that results in high cell
viability and high gene editing efficiency is critical in enabling downstream experiments.
Furthermore, high editing efficiency is crucial for future downstream clinical applications.
A large amount of successfully edited donor cells will impact how many cells are available
for patient use. To ensure enough viable cells for the downstream functional assays,
Dr. Albanese and the team started out with 2.5x more cells than typically required forthese assays.

Figure 4. Synthego’s Synthetic Guides Outperform Another Vendor’s in Editing Resting CD4+ T Cells
The editing efficiency of both frozen and freshly prepared Synthego synthetic sgRNAs were directlycompared against another vendor’s sgRNA and crRNA:tracrRNA in editing the CD46 loci in resting CD4+ T
cells. Synthego’s synthetic sgRNAs demonstrated consistently high (60%+) knockout efficiency as assessedby TIDE analysis regardless of preparation condition. (Unpublished data, Albanese et al.)Pre-Designed Multi-Guide sgRNA for Every Human Gene
Choose your gene of interest, and we’ll deliver our algorithmically designed multi-guide
Gene Knockout Kit v2 kit to you. If you don’t achieve knockout at the protein level, youwill get your money back (non-essential human genes only).

Synthego.com/products/crispr-kits/gene-knockout-kit

8Conclusions
Previous viral interaction studies have utilized CRISPR
editing approaches in post-activated CD4+ T cells as
they can be readily expanded following gene editing,whereas maintaining viability in resting CD4+ T cells
post-gene editing is a persistent problem that has
challenged the field. Also, such studies have largely

About Synthego

only targeted single cellular factors and are often

Synthego is a genome engineering
company that enables theacceleration of life science researchand development in the pursuitof improved human health. With
its foundations in engineering,the company leverages machinelearning, automation, and gene
editing to build platforms toadvance both basic research andtherapeutic development programs.

extremely time-consuming. The few studies that havelooked at CRISPR-mediated genetic manipulation in
resting T cells are greatly limited due to cytotoxicity.
In many cases, the cells do not survive beyond threedays after transfection.
In this study, Dr. Albanese and his colleagues
utilized optimized media conditions and the Lonza
4D-Nucleofector® System, which resulted in highLonza Cologne GmbH – 50829
Cologne Germany
For research use only. Not for use indiagnostic procedures.
The Nucleofector® Technology is covered bypatent and/or patent pending rights ownedby the Lonza Group Ltd or its affiliates. All
trademarks belong to Lonza, registered inUSA, EU or CH or to third party owners andused only for informational purposes. All
third party copyrights have been reproducedwith permission or belong to Lonza. The
information contained herein is believed to becorrect and corresponds to the latest state ofscientific and technical knowledge. However,no warranty is made, either expressed or
implied, regarding its accuracy or the results tobe obtained from the use of such informationand no warranty is expressed or implied
concerning the use of these products. The
buyer assumes all risks of use and/or handling.
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any third party’s intellectual property rights.
The user bears the sole responsibility for
determining the existence of any such third
party rights, as well as obtaining any
necessary licenses.

This optimized protocol, combined with the efficacy
of Synthego’s sgRNAs in generating highly efficient
knockouts for both single and multiple genes, enabled
the successful analysis of several genes that mediateHIV-1 infectivity in resting CD4+ T cells. Learnings
from this study could be extended beyond virushost cell interactions, including areas such as
immuno-oncology and T cell biology.
To learn more about Synthego’s CRISPRevolution
products, go to Synthego.com/products/crispr-kits
To learn more about Lonza’s 4D-Nucleofector® System,
go to Bioscience.lonza.com/nucleofector-technology

Our work is vital to the larger T cell community
because now scientists can knock out genes in
resting T cells and continue to maintain theirresting states. This will allow scientists tostudy T cell activation factors. Our multiple
gene knockout approach allows researchers to
study T cell signaling pathways with redundant

For more details: www.lonza.com/legal.

protein functions© 2023 Lonza. CD-MN053 03/23.

- Dr. Manuel Albanese

9Case Study | Synthetic sgRNAcell survival of edited resting human CD4+ T cells.

PROTOCOLCRISPR Editing Human Primary Resting CD4+
T Cells with RNPs using Nucleofector®
Technology
Developed by researchers at the University of Munich in collaboration with Synthego and Lonza

Introduction
This protocol describes how to deliver ribonucleoprotein (RNP) complexes that consist of
purified SpCas9 nuclease complexed with Synthego’s chemically modified synthetic single guide
RNA (sgRNA) to human primary resting CD4+ T cells. RNP delivery is accomplished using the
Lonza 4D-Nucleofector® System.
Chemically modified sgRNAs are designed to resist degradation by exonucleases and prevent
innate intracellular immune cascades that can lead to cell death. The present protocol byAlbanese et al. may be used for the generation of single or multiple gene knockouts in humanprimary resting CD4+ T cells1. This protocol was successfully used for the knock-in of GFP into
different loci and may also be used for the knock-in of other genes of interest.
To learn more about the success of this protocol, please read the associated case study.

Abbreviations:
CRISPR: clustered regularly interspaced short palindromic repeats
SpCas9: Streptococcus pyogenes CRISPR-associated protein 9
sgRNA: single guide RNA
RNP: ribonucleoprotein
PCR: polymerase chain reaction
ICE: Inference of CRISPR Edits
TE: Tris-EDTA
PBS: phosphate-buffered saline
GFP: green fluorescent protein
FBS: fetal bovine serum
HDR: Homology-directed repair

CRISPR Editing Human Primary Resting CD4+ T cells with RNPs using
Nucleofector® Technology© 2023

1Synthego.com/contact(888) 611-6883
20201009

PROTOCOL

Materials Required
Material

Ordering Information

sgRNA Synthetic sgRNA Kit (Synthego Standard Modifications)(Synthego), available here

PCR & sequencing primers

Multiple vendors

Nuclease-free 1X TE buffer

Multiple vendors (e.g., Thermo Fisher Scientific, Catalog #AM9849)

Nuclease-free water

Multiple vendors (e.g, Thermo Fisher Scientific)

1X PBS, cell culture grade

Multiple vendors (e.g, Thermo Fisher Scientific)

4D-Nucleofector® Core Unit and X Unit

Lonza, 4D-Nucleofector® Core Unit | Lonza (AAF-1003B)
Lonza, 4D-Nucleofector® X Unit | Lonza (AAF-1003X)

P3 Primary Cell 4D-Nucleofector® X Kit S (for 20 µl transfection
volume)(Lonza, P3 Primary Cell 4D-Nucleofector® X Kit S | Lonza (V4XP3032) for 16x 20 µl transfection volume)

RPMI 1640 with GlutaMAX Supplement

Gibco RPMI 1640 Medium, GlutaMAX™ Supplement (thermofisher.
com)

10% (v/v) FBS(Sigma)Penicillin/streptomycin (100 IU/ ml)(Thermo Fisher Scientific)

EasySep™ Human CD4+ T Cell Enrichment Kit(Stem Cell; 19052)

IL-7 and IL-15 (2 ng/ ml each)(Peprotech)

SpCas9 Nuclease

Multiple vendors (IDT; 1081059)

Genomic Isolation Kit

Multiple vendors (e.g, Thermo Fisher Scientific)

CaCl2 (1mM)

Multiple vendors (e.g, Thermo Fisher Scientific)

MgCl2 (3mM)

Multiple vendors (e.g, Thermo Fisher Scientific)

EDTA (1mM)

Multiple vendors (e.g, Thermo Fisher Scientific)

Triton (X 100)

Multiple vendors (e.g, Thermo Fisher Scientific)

Tris (10 mM, pH 7.5)

Multiple vendors (e.g, Thermo Fisher Scientific)

Proteinase K

Multiple vendors (e.g, Thermo Fisher Scientific)

96-well flat-bottom plates

Multiple vendors (tissue culture grade)

48-well plates

Multiple vendors (tissue culture grade)

dsDNA HDR Template (optional)

Multiple vendors (e.g. Twist Bioscience)

Note: All protocols outlined have been validated using materials mentioned in this manual. Materials other than the ones outlined
in our manual may require additional optimization by the user.
CRISPR Editing Human Primary Resting CD4+ T cells with RNPs using
Nucleofector® Technology© 20232

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PROTOCOL

General Guidelines• Wearing gloves and using nuclease-free tubes and reagents is recommended to avoid RNase contamination.
• Always maintain sterile technique, and use sterile filter pipette tips.
• All Synthego and Lonza reagents should be stored according to the manufacturer’s recommendations.
• Synthetic sgRNA should be dissolved in TE buffer and diluted to a working concentration using
nuclease-free water.
• RNP complexes are stable at room temperature for up to 1 hour.
• This protocol uses 16-well Nucleocuvette® Strips for the 4D-Nucleofector® X Unit. Please note that
Nucleofection® Conditions are transferable between different formats.
For further information regarding the above-described guidelines please check the Tips & Tricks section at
the end of this protocol.

Workflow Graphic

The above figure was adapted from the original article from Albanese et al, Nat Methods 19, 81–89 (2022) and is licensed under
the Creative Commons Attribution License 4.0 which permits the use, sharing, distribution, and reproduction in any format solong as appropriate credit is given to the original authors.

CRISPR Editing Human Primary Resting CD4+ T cells with RNPs using
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PROTOCOL

Protocol
1. Isolation of Primary T cells• Heparinized blood retained in leukocyte reduction chambers from healthy donors was used to isolate CD4+
T cells.
• Blood cells were diluted with non-filter sterilized PBS; isolation of CD4+ T cells was done using the EasySepTM
Rosette Human CD4+ T cell enrichment kits according to the manufacturer’s protocols.
• For further information please check the Tips & Tricks section at the end of this protocol.

2. Pre Nucleofection®
A. Please make sure that the Nucleofector® Solution supplement is added to the P3 Nucleofector® Solution(16.4 µl P3 Solution + 3.6 µl supplement per sample).
B. Start 4D-Nucleofector® System and create experimental parameter file (for details see device manual).
C. Select the Nucleofector® Program EH-100. For other program recommendations, e.g., to achieve higher efficiency or higher viability, please see the Tips & Tricks at the end of this protocol.
D. Pre-warm an aliquot of culture medium RPMI 1640 GlutaMAX (without supplements) to 37°C (100 µl per
sample); see Step 4 for recovery post Nucleofection®.
E. Prepare cell culture plates by filling the appropriate number of wells with the desired volume ofrecommended media.

3. Nucleofection® Process
3.1. Dissolve and Dilute your sgRNAA. Briefly centrifuge your tubes containing the sgRNA to ensure that the dried RNA pellet is collected at thebottom.
B. If you are working with a 1.5 nmol sgRNA, rehydrate sgRNA in 15 μl nuclease-free buffer (1X TE buffer)and pulse vortex for 30 seconds to ensure complete mixing. This will make a stock solution of 100 μM(100 pmol/μl) of sgRNA.
Note: If sgRNA is not fully dissolved, let it sit overnight and up to 72 hours at 4°C. If the sgRNA is not
being used immediately, dissolved sgRNA should be aliquoted into 6 μl per tube and stored at -20°C.
Under these conditions, the sgRNA is stable for up to 3 years if not repeatedly thawed. For quantification
guidelines and further information, please check the Tips & Tricks section at the end of this protocol.
C. The sgRNA 100 μM stock solution is ready to use. If you wish to dilute your sgRNA to a different working
concentration, please use nuclease-free water, pulse vortex the sgRNA/nuclease-free water mix for 30
seconds and incubate at room temperature for 5 minutes to dissolve the sgRNA.

CRISPR Editing Human Primary Resting CD4+ T cells with RNPs using
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4

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PROTOCOL

Option A: Knockout Generation in Resting Human CD4+ T Cells
This protocol describes amounts for one sample; multiple samples can be processed simultaneously if desired.

3.2. Assemble RNP Complex (2.5:1 sgRNA to SpCas9 ratio) and Transfect Cells
A. Prepare the RNP by incubating synthetic sgRNAs together with SpCas9 at a ratio of 2.5:1 to form the RNP
complex as recommended in the table below.
Note: SpCas9 protein is provided in solution and can be used directly. Please use sterile filtered(0.22 µm) PBS to dilute the RNP to a final concentration of 20 µM.
RNP Preparation: Components, Concentration & Volume
Component

Quantity (pmol)

Concentration (µM)

Volume (µl)

sgRNA

250

100

2.5

SpCas9*

100

62

1.6

PBS (filter sterilized)

N/A

N/A

0.9

Total Volume

N/A

N/A

5

*Please adjust volumes if a different SpCas9 concentration is used. The original protocol uses SpCas9 from IDT, alternatively,
Synthego’s 2NLS SpCas9 can also be used, available here.

B. Incubate RNP mix at room temperature for 10 minutes.
C. Count an aliquot of the cells and determine cell density.
D. Wash cells twice with non-filtered sterilized PBS, centrifuge 2 x 106 cells at 200 x g for 10 minutes atroom temperature. Remove supernatant completely and resuspend the pellet carefully in 20 µl room
temperature P3 Nucleofector® Solution.
E. Mix the RNP solution with the cell suspension as described in the table below depending on yourediting method.
Nucleofection® Sample Preparation for Different Editing Methods
MethodVolume of RNP Solution

Cell Resuspension Volume in P3
Nucleofector® Solution

Single sgRNA editing

5 µl of RNP

20 µl of cells

Multi-guide sgRNA editing

2 µl of each RNP complexes for each sgRNA

20 µl of cells

Co-editing of genes

0.5 µl of each RNP complexes for each sgRNA

20 µl of cells

F.

Transfer 20 µl of the cells mixed with RNPs into a well of the 16-well Nucleocuvette® Strip.
Note: For example, for single sgRNA editing, mix 5 µl + 20 µl very well and only use 20 µlof sample for transfection.

G. Transfect cells using Nucleofector® Program EH-100.
H. Recover the cells according to Step 4.

CRISPR Editing Human Primary Resting CD4+ T cells with RNPs using
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PROTOCOL

Option B: Knock­­–in Generation in Resting Human CD4+ T Cells
This protocol describes amounts for one sample: multiple samples can be processed simultaneously if desired.
For the knock-in of GFP or any other gene of interest, a double-stranded DNA HDR donor template with around 550
bp in front 5’ and 550bp after 3’ the sgRNA cutting site is recommended.

3.3. In vitro digestion of knock-in HDR DNA templates to confirm no cleavage of the HDR templateby the RNP (Optional)
A. Mix the RNP complex (1µM), the HDR DNA template (1µg) and single-cutter restriction enzyme for the
specific plasmid (positive control of DNA cleavage).
B. Incubate samples at 37°C for 2h.
C. Add 1 µl of proteinase K (20 mg/ ml).
D. Incubate at 56°C for 10 minutes.
E. Visualize sample on a 1% agarose gel.

3.4. Assemble RNP Complex (2.5:1 sgRNA to SpCas9 Ratio) and Transfect Cells
A. Prepare the RNP by incubating synthetic sgRNAs together with SpCas9 at a ratio of 2.5:1 to form the RNP
complex as recommended in the table below.
Note: SpCas9 protein is provided in solution and can be used directly. Please use sterile filtered(0.22 µm) PBS to dilute the RNP to a final concentration of 20 µM.

RNP and HDR Donor Preparation: Components, Concentration & Volume
Component

Quantity (pmol)

Concentration (µM)

Volume (µl)

sgRNA

250

100

2.5

SpCas91*

100

62

1.6

PBS (filter sterilized)

N/A

N/A

0.9

Total Volume

N/A

N/A

51*

DNA HDR donor
template

0.8-1.12 pmol2* To be added after preparation of cell suspension

Please adjust volumes if a different SpCas9 concentration is used. The original protocol uses SpCas9 from IDT, alternatively,

1*

Synthego’s 2NLS SpCas9 can also be used, available here.
2*
The pmol will vary with the DNA HDR donor template used. Usual templates span from 1900 bp to 1350 bp which correspondto the pmol above.

B. Count an aliquot of the cells and determine cell density.
C. Wash cells twice with non-filter sterilized PBS, centrifuge 2 x 106 cells at 200 x g for 10 minutes at room
temperature. Remove supernatant completely and resuspend the pellet carefully in 20 µl room temperature P3 Nucleofector® Solution.
D. Mix 5 µl of the RNP template solution with 20 µl of the cell suspension.

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E. Add donor DNA template (0.8-1.12 pmol).
F.

Mix well and transfer 20 µl of the sample into a well of the 16-well Nucleocuvette® Strip.

G. Transfect cells using Nucleofector® Program EH-100.
H. Recover the cells according to Step 4.

4. Post Nucleofection®
A. Add 100 µl of pre-warmed RPMI 1640 GlutaMAX (without supplements) to each well in the 48-well plate.
B. Transfer cells to 48-well plates and allow them to recover for 10 min at 37 °C.
C. After recovery, cultivate and continue regular media changes until analysis as described in Step 5 below.

5. Cultivation of Primary Human CD4+ T Cells
A. Cultivate cells in 96-well flat-bottom plates at a cell density of 1 × 106 cells/ ml.
B. Add complete culture medium; RPMI 1640 GlutaMAX supplemented with 10% (v/v) FBS, penicillin/streptomycin (100 IU/ ml) and IL-7 and IL-15 (2 ng/ ml each) supplements.
C. Replace complete medium (including FBS, antibiotics and IL-7, IL-15 supplements) every 3 days.

6. Genotyping Analysis
Editing efficiencies can be assessed via Sanger sequencing through a variety of analysis tools. Synthego's ICE
analysis tool can be used to assess both CRISPR knockout and knock-in edits.
To assess editing efficiency with ICE:
A. Design PCR primers compatible with Inference of CRISPR Edits (ICE) analysis.
B. 1 week post Nucleofection®, collect cells (5 × 104) and lyse in lysis buffer (20 μl) (1 mM CaCl2, 3 mM MgCl2,
1 mM EDTA, 1% Triton X 100 and 10 mM Tris, pH 7.5) with the addition of proteinase K (20 μg/ml; Thermo
Fisher Scientific). Incubate the cell lysate at 65 °C for 20 min, followed by 95 °C for 15 min and store at −20°C
until the PCR specific for the CRISPR/Cas9 target sites is performed.
C. Use 1 μl of cell lysate as a PCR template and Sanger-sequence the PCR amplicons.
D. Alternatively, you can follow the Genotyping guidelines provided in our Genotyping protocol for DNA
isolation, PCR and Sanger sequencing.
E. Conduct Inference of CRISPR Edits (ICE) analysis on Sanger sequences to determine editing efficiency. See
our ICE Knockout Analysis protocol or ICE Knock-in Analysis protocol for instructions on how to use ICE andinterpret results.
Note: ICE should be able to handle any deletion sizes and work with any donor template size. However, Synthegohas only validated ICE analysis of deletions up to 40 bases for sgRNA and up to 150 bases for multi-guideexperiments. For knock-in experiments, we have tested insertions of up to 270 bases long.

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The larger the deletion, the shorter the available Sanger sequence that will be used for the alignment with thecontrol, hence the higher chance that ICE will display warning messages which are indicated when the analysis
displays “Succeeded” in orange with the warning message below it. The Sanger sequence quality/ length will dictate
if the analysis is successful.
Alternatively, if ICE analysis is not successful in genotyping your large knock-in edit please refer to loci-specific
Sanger sequencing or functional assays to determine large knock-in success.

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Tips & Tricks• Cell preparation/ cell source
Alternatively to isolating primary T cells, cryopreserved PBMCs can be used to isolate CD4+ cells or cryopreserved
CD4+ T cells can be used:
PBMCs (different fill sizes), cryopreserved

CC-2702 (50M), CC-2703 (100M), CC-2704 (10M), CC-2705 (25M)

CD4+ T cells, cryopreserved

2W-200• Nucleofector® Program
The above protocol describes the use of pulse code EH-100 to transfect resting T-cells with Nucleofector®
Technology;
Depending on your desired outcome (e.g. editing efficiency, number of viable transfected cells) you might want totest additional pulses. Please test (some of) the following pulse conditions along with a no pulse control:

The P3 Primary Cell 4D-Nucleofector® X Kit S (Lonza) is supplied with the pmaxGFP™ Vector as a positive control for the Nucleofection® Process. We recommend using 0.4 µg pmaxGFP™ Vector for a transfection volume of 20 µl.

• Viability
The centrifugation step prior to Nucleofection® Process highly influences the viability of transfected cells. Reducing
centrifugation speed to 90 x g can improve cell viability.
To achieve maximum viability, we highly recommend adding pre-warmed media to each well of the Nucleocuvette®
Strip directly after Nucleofection® and incubate for 10 minutes at room temperature. After that, carefully transfer
the cells to a prepared culture dish in appropriate seeding density.
Medium change 6 hours post Nucleofection® may lead to increased viability after transfection. Spin the culture
dishes for 8 minutes at 140 x g in a tissue culture centrifuge. Carefully remove the medium and add fresh pre-equilibrated culture medium as described in the protocol.

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Nucleofection® Conditions are transferable between different formats. The 4D-Nucleofector® X Unit offers thepossibility to transfect in 20 µl format (described in this protocol) or in 100 µl format. For upscaling to 100 µl pleaseadjust cell number and amount of RNP as shown in the table below:
20 µl Volume

100 µl Volume

Kit #P3 Primary Cell 4D-Nucleofector® X Kit S(V4XP-3032 for 16 x 20 µl reaction)

P3 Primary Cell 4D-Nucleofector® X Kit L(V4XP-3012 for 12 x 100 µl reaction; V4XP3024 for 24 x 100 µl reaction)

Cell number

1-2 x 106

0.5-1 x 107

Nucleofector® Solution

20 µl (P3 + supplement)

100 µl (P3 + supplement)

SpCas9100 pmol

500 pmolsgRNAs

40 pmol

200 pmol

HDR template0.8-1.12 pmol

4 - 5.6 pmol

In addition, it is possible to perform transfections in 20 µl volume in 96-well format using the 4D-Nucleofector® 96-well Unit or in
384-well format using the 384-well Nucleofector® System.

• Genome Editing in Other Cell Types
As a starting point, this protocol may also be used for activated T cells.
If you want to perform CRISPR Cas9-mediated genome editing or use a different editing system in other cell types(primary cells or cell lines), please check Lonza’s Knowledge Center for detailed protocols and pulse recommendations or contact our Scientific Support Team.
Additionally, Synthego has other optimized RNP protocols available using synthetic sgRNAs for other cell types suchas the Immortalized Cell Nucleofection Protocol and the iPS Cell Nucleofection Protocol.
• sgRNA and RNP
A. sgRNA Design
For knockouts, it is generally recommended to try 3-5 sgRNAs with unique target sequences (evaluated independently) per target that cut near (<10 bp) the edit site and check their indel percentage by ICE first. Whenusing two sgRNAs together to create a knockout, the distance between the cutting sites must not be multiple of
3 nt. Otherwise, you remove only few amino acids and the protein can still be functional.
For knock-ins, the higher the indel efficiency of a given sgRNA, the higher the knock-in efficiency. Therefore, it isgenerally recommended to try 3-5 sgRNAs with unique target sequences (evaluated independently) per target
that cut near (<10 bp) the edit site and check their indel percentage by ICE first.
B. sgRNA Rehydration, Storage, and Quantification
Synthetic sgRNA can also be dissolved in water if desired, however, we recommend using TE buffer to help stabilize pH when complexing with the nuclease. Please refer to our sgRNA Quick Start Guide and Gene Knockout
Kit v2 Quick Start Guide for Rehydration, Storage, and Quantification guidelines.

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C. RNP
It is important to work as quickly as possible as RNP complexes begin to become unstable after 1 hour at room
temperature. Additionally, leaving cells in Nucleofector® Solution for extended periods of time may lead to reduced transfection efficiency and viability.
RNP complexes may also be stored at 4°C for up to one week, or at -20°C for up to one month. Note that RNPs
stored at 4°C may become susceptible to contamination from microbial growth after long periods of time.
Dissolving RNPs in P3 Nucleofector® Solution is an option to avoid dilution of the Nucleofector® Solution.
Nucleofector® Solution is animal component-free and non-toxic.
D. HDR Template
Our Tips and Tricks guide: Design and Optimization of CRISPR Knock-in Experiments offers general guidelines
for planning your knock-in experiments.
The quality and the concentration of DNA HDR donor template used for Nucleofection® plays a central role forthe efficiency of gene transfer. We strongly recommend the use of high-quality plasmids from vendors or, if
isolated within the lab, use plasmid purification kits like EndoFree® Plasmid Kits (Qiagen, Cat. No. 12391 Giga Kit,
12362 Maxi Kit, 12381 Mega Kit). The purified DNA HDR donor template should be resuspended in deionizedwater or TE buffer with a concentration between 0.2-1 µg/µl. Please check the purity of each plasmid preparation by measuring the A260:A280 ratio, according to manufacturer’s protocol. Other DNA donor templates could
be used but will require optimization by the user.
If you wish to try alternative knock-in strategies, please be aware that we have no specific recommendations.
There are limitations on the amount of donor DNA template you can use and the editing efficiency of eachknock-in may vary.

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Contact Technical Support
If you require further technical support, please reach out to our Scientific Support Teams:• Synthego (support@synthego.com):° sgRNA and HDR template design, sgRNA resuspension° RNP formation° ICE analysis• Lonza (Scientific.Support@lonza.com (US), Scientific.Support.EU@lonza.com (EU and International)):° T-cell preparation and cell handling° Nucleofection® Process, protocols and format

Citations
1. Albanese M et al. Rapid, efficient and activation-neutral gene editing of polyclonal primary human resting CD4+
T cells allows complex functional analyses. Nat Methods. 2022 Jan;19(1):81-89. doi: 10.1038/s41592-021-013288. Epub 2021 Dec 23. PMID: 34949807; PMCID: PMC8748193.

Additional Information

About Synthego

For an up-to-date list of all Synthego
Protocols and other resources,
please visit Synthego.com/resources

Synthego is the leading genome engineering
innovation company. The company’s
automated, full stack genome engineering
platform enables broader access to CRISPR
to accelerate basic scientific discovery,
uncover cures for diseases, and develop novel
synthetic biology applications. Headquarteredin Silicon Valley, Synthego is used by scientists
from the largest global biotechnology
companies and global biology universities to
unlock the potential of gene editing.

For technical assistance,
contact our Scientific Support Team:
Ph: 888.611.6883

Email: support@synthego.com

Lonza Cologne GmbH – 50829 Cologne Germany
For research use only. Not for use in diagnostic procedures.
The Nucleofector® Technology is covered by patent and/or patent pending rights owned by the Lonza Group Ltd or
its affiliates. All trademarks belong to Lonza, registered in USA, EU or CH or to third party owners and used only for
informational purposes. All third party copyrights have been reproduced with permission or belong to Lonza. The
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