CRISPR/Cas9 gRNA Vector

Design Guide
GenScript CRISPR gRNA/Cas9 Plasmids
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Vector Selection Plasmid (Non-viral) Lentiviral AAV

Enhanced Specificity Sp Cas9 Plasmids NEW!；Sp Cas9 Plasmids；

Deliverables/Cas9 Selection
Sp Cas9 Nickase Plasmids；Sa Cas9 Plasmids；Transcription Activation Plasmids

Model Organism Mammalian Cells

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Ready for Transfection

Multiplex Editing Versatility

Gene Editing/Expression Options Knock-out/Knock-in Transcription Activation

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Putting CRISPR into Practice:

With CRISPR/Cas9 genome editing, modified clonal cell lines can be derived within 2-3 weeks starting from
the gRNA design stage, while transgenic animal strains can be created in a single generation. The following
workflows and case studies describe best practices on how to use CRISPR in your laboratory.

CRISPR/Cas9 Vector Design Workflow

CRISPR/Cas9-mediated gene editing can be performed in the following steps:
1. Determine Genetic Modification
Select the application for your experiment (Table 3).
Table 1:

Genetic Nuclease
Application gRNA
Modification Activity

Permanently remove gene gRNA targeting 5′ exon or

Knock-out Cas or Cas9n
function essential protein domains

Generate a specific sequence

Knock-in Cas or Cas9n gRNA targeting region of interest
change

gRNA targeting gene promoter

Interference Reduce gene expression dCas-repressor
elements

gRNA targeting gene promoter

Activation Increase gene expression dCas-activator
elements

2. Select Expression System

CRISPR/Cas9 system components can be delivered in vivo using modified non-viral plasmid or
viral vector or delivery systems (Table 2).
Table 2:

Expression System Components ApplicaƟon

Constitutive or inducible Cas9

Plasmid Vector Constitutive or inducible gRNA Expression of Cas9 and gRNA
Reporter/selection marker

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Constitutive or inducible Cas9 Expression of Cas9 and gRNA

Lentiviral Vector Constitutive or inducible gRNA For infection of
Reporter/selection marker difficult-to-transfect cell types

Transient or stable expression

Constitutive or inducible Cas9
of SaCas9 and/or gRNA
AAV Vector Constitutive or inducible gRNA
For non-toxic infection of
Reporter/selection marker
dividing and non-dividing cells

Transient expression of CRISPR

Cas9 mRNA and gRNA In vitro transcribed Cas9 mRNA and gRNA
gene editing components

sgRNA/Cas9 Ribonucleoprotein Purified Cas9 protein and chemically synthesized Transient expression of CRISPR
(RNP) Complexes single guide RNA gene editing components

Modified recombinant AAV particles are attractive for transduction because of their low immunogenicity
and capability to infect both quiescent and dividing cells. While AAV vectors are a preferred vehicle for in
vivo gene delivery, the size of the SpCas9 gene (>4 kb) exceeds the typical cargo limit of AAV vectors.
Solutions that have been developed to date include:
• Creating transgenic animal lines that express Cas9, either constitutively or in an inducible manner, and
then to deliver only the guide RNAs and any necessary inducer at the time of the experiment.
• Developing a split-Cas9 system using split-inteins.
• Use smaller Cas9 orthologues from other species, such as Staphylococcus aureus (SaCas9), which
are small enough to be packaged along with a single guide RNA expression cassette into a single AAV
vector.

Some of the most widely-used model systems for biomedical research are primary mammalian cell
cultures or hard-to-transfect cell lines in which transfection efficiency can be quite low. For these cell
types lentiviral vectors are preferred.

Guide RNAs may be also introduced via U6-gRNA cassette expression, but the cleavage efficiency is
typically lower than when gRNA is expressed from a plasmid. However, U6-gRNA cassettes may be used
for rapid comparison of gRNA cleavage efficiencies so that the most optimal gRNA sequences can be
identified before subsequent cloning into your vector.

3. Gene Sequence Analysis

It is highly advisable to sequence the region of interest for the cell line or animal model you are using,
rather than assuming it matches with the NCBI reference sequence for your species/strain. Allele number
may also vary depending on species/strain. Variations can result in reduced cleavage efficiency.

4. Select Sequence for Modification

Select specific genetic sequences for modification depending on your application (Table 1).

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For loss-of-function mutations, design gRNAs against early exons to optimize expression disruption and
prevent the expression of truncated protein isoforms. Alternatively, targeting a functional site can
generate a loss-of-function mutant. For genes with multiple splice variants, care should be taken to
ensure that a constitutive exon is targeted if the goal is to knock out all splice variants.

For applications using paired Cas9n, opposite strands of the genomic DNA should be targeted, with a
40-60 bp offset between PAM sequences.

For interference and activation applications, promoter elements within 200 bp of the transcription start site
should be targeted.

5. Determine On/Off-Target Activity

Identify all PAM sequences within the region of interest: the PAM sequence will vary depending on the
Cas variant being used in the experiment (Table 3). A PAM sequence is required for targeting, so if none
are present, considering targeting a different location. The next 20 bps upstream of the PAM will
correspond with your putative gRNA sequence. Be sure to check for off-target sites, locations within the
genome where partial homology is present, which can result in off-target cleavage.

Table 3:

Cas Variant PAM Sequence

eSpCas9/SpCas9 NGG

SpCas9 VRER Variant NGCG

SpCas9 EQR Variant NGAG

SpCas9 VQR Variant NGAN or NGNG

SaCas9 NNGRRT
Cpf1 TTN

GenScript hosts free online human and mouse genome-wide databases developed by researchers at the
Broad Institute. These databases can be searched and accessed here:
http://www.genscript.com/gRNA-database.html

GenScript also host a free online gRNA design tool developed by researchers at the Broad Institute. The
design tool can be accessed here:
http://www.genscript.com/gRNA-design-tool.html

6. Designing Knock-In Constructs

To introduce specific changes within the genome it is necessary to supply a donor template that can be
used for HDR after Cas9 creates a DSB. HDR templates may be delivered as plasmids or as

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single-stranded oligos (ssODN). To detect successful HDR and quantify knock-in efficiency, donor
templates are often designed to include several synonymous mutations that can be distinguished from the
wild-type through sequencing. To prevent the cleavage of donor DNA after successful HDR, the donor
template should be designed with mutations in the PAM sequence.

As a general rule, eSpCas9/SpCas9 more efficient at mediating homologous recombination than Cas9
nickase.

7. Synthesize gRNA/Cas9 Vectors

Once you have determined your expression system, Cas nuclease and gRNA sequence your customized
vectors can be cloned or commercially ordered.

CRISPR-mediated Gene Edi ng in Model Systems

CRISPR/Cas9-mediated genome editing has been successfully conducted in many different species and
models (Table 6). Although the basic CRISPR/Cas9 components are the same regardless of the model
organism, the delivery method varies widely, and choosing the most appropriate vector for your host is
critical for success.

Table 4:

Host PAM Sequence

Lipofection-based transfection of DNA plasmids

Mammalian Cells Electroporation of DNA plasmids or RNP
Lenti or AAV virus-based transfection of DNA plasmids

Bacteria Transformation of plasmids into competent cells

Yeast Electroporation of plasmids and galactose induction of Cas9

Mouse: Direct injection into embryos

Germline Mutations Electroporation into zygotes

Mouse:
Direct injection of AAV into tissue of interest
Somatic Mutations

Danio rerio Direction injection into one-cell embryos

Drosophila melanogaster Direct injection into embryo germline

Danio rerio Direction injection into one-cell embryos

Caenorhabditis elegans Direct injection into hermaphrodite germline

Plants Agrobacterium-mediated transformation of gRNA/Cas9 vector

In vitro genome editing:

For easy-to-transfect cell lines, plasmids encoding gRNAs and Cas9 can be delivered with high efficiency via lipofec on.
CRISPR plasmids typically contain selec on markers such as genes conferring an bio c resistance, or fluorescent proteins for
easy visualiza on via FACS.

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For difficult-to-transfect cell lines or primary cells, len viral vectors are preferred. Guide RNAs may be delivered either via an
all-in-one plasmid that also encodes the Cas9 nuclease, or a separate plasmid that can be delivered into cells already
expressing Cas9 (Figure 8).

In vivo genome editing:

CRISPR/Cas9 system components can be delivered to germ line cells to create heritable mutations.
Stable, homozygous mutations at multiple loci can be achieved in a single generation in mice.
CRISPR-mediated genome editing can similarly be used to generate precise mutations in somatic tissues
of adult animals, and to modify multiple genes at once in the same cells. These tools are especially
valuable for creating clinically relevant in vivo cancer models, because human tumors often contain a
combination of gain-of-function mutations in oncogenes and loss-of-function mutations in tumor
suppressor genes42.

Verifica on of KO/KI and Off-target Effects

To identify successful cases of CRISPR-mediated KO/KI and determine whether a loss-of-function or
gain-of-function mutation has occurred, mRNA and protein gene products should be analyzed.
Techniques such as quantitative PCR, Northern blotting, and Western blotting can be utilized to determine
whether mRNA and protein concentrations are depleted or molecular weights are changed.

For difficult-to-transfect cells, it can be sufficient just to show that high KO/KI efficiency has been
achieved, without isolating clones for confirmation. For these cases genome editing efficiency is typically
assayed by next generation sequencing. A range of unique insertions and deletions will likely be
observed.

Best practices for managing off-target Cas9 activity:

• Use at least two independent gRNA sequences in parallel to derive distinct clones. Models created
through genome editing with distinct gRNAs that share the on-target locus, but do not share off-target loci
are an excellent way to create independent replicates.
• Isolate multiple, independent clonal cell populations for each gRNA used. The likelihood off-target DSBs
occurring at the same loci in independent clones is very low.
• Although few labs have the resources to do statistically powerful whole genome sequencing verification
protocols such as gUIDEseq, it is relatively easy to select the few predicted off-target sequences for each
gRNA you use and then sequence around those loci to ensure that off-target indels have not been
introduced.

To determine off-target effects, it is recommended to sequence predicted off-target sites, particularly those
with matches in the “seed” region of the 20 mer recognition site, which lies adjacent to the PAM. More
rigorous reviews of off-target cleavage can be performed using whole-genome sequencing.

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