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How To Use CRISPR: Your Guide to Successful Genome Engineering

Chapter 03


THE COMPLETE GUIDE TO UNDERSTANDING CRISPR SGRNA

We are currently experiencing a biotechnology revolution. Advances in genomics,
spearheaded by CRISPR-Cas9 technology, have greatly accelerated genome
engineering research. As CRISPR is opening the door for an increasing number of
applications each day, more and more researchers are adopting this technique for
their studies.

As with every new technology, the path to CRISPR expertise can be blocked by
confusing, and sometimes intimidating, jargon. Don’t let that discourage you! We
are committed to making CRISPR accessible to all. This includes providing
educational resources - your personalized CRISPR dictionary, so to speak - to
simplify all the relevant concepts for you. In this article, we discuss a common
and important concept: single guide RNA (sgRNA).

--------------------------------------------------------------------------------


INTRODUCTION TO CRISPR-CAS9 TECHNOLOGY

Before we delve into the depths of our discussion on single guide RNAs, let us
first review the CRISPR-Cas9 gene-editing mechanism. The popularity of CRISPR is
largely due to its simplicity. As shown in Figure 1, the CRISPR-Cas system
relies on two main components: a guide RNA (gRNA) and CRISPR-associated (Cas)
nuclease.

 * The guide RNA is a specific RNA sequence that recognizes the target DNA
   region of interest and directs the Cas nuclease there for editing. The gRNA
   is made up of two parts: crispr RNA (crRNA), a 17-20 nucleotide sequence
   complementary to the target DNA, and a tracr RNA, which serves as a binding
   scaffold for the Cas nuclease.
 * The CRISPR-associated protein is a non-specific endonuclease. It is directed
   to the specific DNA locus by a gRNA, where it makes a double-strand break.
   There are several versions of Cas nucleases isolated from different bacteria.
   The most commonly used one is the Cas9 nuclease from Streptococcus pyogenes.


Figure 1. The CRISPR-Cas9 System. The CRISPR-Cas9 system comprises a guide RNA
(gRNA) and Cas9 nuclease, which together form a ribonucleoprotein (RNP) complex.
The presence of a specific protospacer adjacent motif (PAM) in the genomic DNA
is required for the gRNA to bind to the target sequence. The Cas9 nuclease then
makes a double-strand break in the DNA (denoted by the scissors). Endogenous
repair mechanisms triggered by the double-strand break may result in gene
knockout via a frameshift mutation or knock-in of a desired sequence if a DNA
template is present.



COMPETITIVE ANALYSIS OF GUIDE RNA FORMATS

--------------------------------------------------------------------------------

COMPETITIVE ANALYSIS OF GUIDE RNA FORMATS

Guide RNAs come in two formats: two-piece cr:tracrRNA and single guide RNA
(sgRNA), both with their advantages and disadvantages. At Synthego, we
understand how important the right guide RNA format is for a successful CRISPR
experiment. In this application note, we compare the editing efficiencies of
Synthego’s cr:tracrRNA, cr:tracrRNA from other vendors, and Synthego’s sgRNA.
Learn which is the best option for your experiment needs.

Download


WHAT IS THE DIFFERENCE BETWEEN GRNA AND SGRNA?

The crRNA part of the gRNA is the customizable component that enables
specificity in every CRISPR experiment. But you may have noticed another term,
sgRNA, commonly used in CRISPR-related resources. So what exactly is the
difference between gRNA and sgRNA?

sgRNA is an abbreviation for “single guide RNA.” As the name implies, sgRNA is a
single RNA molecule that contains both the custom-designed short crRNA sequence
fused to the scaffold tracrRNA sequence. sgRNA can be synthetically generated or
made in vitro or in vivo from a DNA template.

While crRNAs and tracrRNAs exist as two separate RNA molecules in nature, sgRNAs
have become the most popular format for CRISPR guide RNAs with researchers, so
the sgRNA and gRNA terms are often used with the same meaning in the CRISPR
community these days. However, some researchers are still using guide RNAs with
the crRNA and tracrRNA components separate, which are commonly referred to as
2-piece gRNAs or simply as cr:tracrRNAs (pronounced CRISPR tracer RNAs).

The term “sgRNA” has been previously used elsewhere to refer to different types
of CRISPR RNAs, including synthetic guide RNA and short guide RNA. In this
guide, we have used the conventional definitions to avoid confusion: gRNA is the
term that describes all CRISPR guide RNA formats, and sgRNA refers to the
simpler alternative that combines both the crRNA and tracrRNA elements into a
single RNA molecule.





DESIGNING SGRNA FOR CRISPR EXPERIMENTS

The CRISPR guide RNA sequence directly impacts the on-target DNA cleavage
efficiency and unintentional off-target binding and cleavage. Therefore,
designing the right guide RNA is a critical step for the success of your CRISPR
experiments and there are several important parameters to consider while
designing a guide RNA.


WHAT PAM SEQUENCE DOES YOUR CAS NUCLEASE USE?

Each Cas nuclease binds to its target sequence only in presence of a specific
sequence, called protospacer adjacent motif (PAM), on the non-targeted DNA
strand. Therefore, the locations in the genome that can be targeted by different
Cas proteins are limited by the locations of these PAM sequences. The nuclease
cuts 3-4 nucleotides upstream of the PAM sequence.

Cas nucleases isolated from different bacterial species recognize different PAM
sequences. For instance, the SpCas9 nuclease cuts upstream of the PAM sequence
5′-NGG-3′ (where “N” can be any nucleotide base), while the PAM sequence
5′-NNGRR(N)-3′ is required for SaCas9 (from Staphylococcus aureus) to target a
DNA region for editing.

Note that although the PAM sequence itself is essential for cleavage, it should
not be included in the single guide RNA sequence.


USING SOFTWARE TO DESIGN CRISPR SGRNAS







Once the target gene and Cas nuclease have been selected, the next essential
step is to design the specific guide RNA sequence. Several software tools exist
for designing an optimal guide with minimum off-target effects and maximum
on-target efficiency. The following tools are the most popular guide RNA design
tools available which have GUIs for ease of use.

 * Synthego Design Tool
 * Broad Institute GPP sgRNA Designer
 * CRISPOR
 * CHOPCHOP
 * Off-Spotter
 * Cas-OFFinder
 * CRISPR-Era
 * Benchling CRISPR Guide RNA Design tool
 * E-CRISP - also has a CRISPR library designer for the batch design of sgRNA
   libraries

Several of these tools, such as Off-Spotter and Cas-Offinder, are specifically
developed for detecting potential off-target editing. Others, like CHOPCHOP, are
not only for Cas9 but also provide options for alternative Cas nucleases and PAM
recognition. For more details on all the different platforms available, this
2018 paper reviews all the available tools for sgRNA design.

Synthego’s design tool offers fast and easy design of sgRNAs that generate up to
97% editing efficiency and the lowest off-target effects from a library of over
120,000 genomes and over 8,300 species. The tool can also be used to validate
guides designed using other methods.

> “The Synthego design tool is extremely fast and the user experience is unlike
> anything I've seen before - very sleek and visually appealing. It allows me to
> rapidly commence my CRISPR experiments by reducing significant time in the
> design process.” -Dane Hazelbaker, a researcher at the Broad Institute of MIT
> and Harvard.

Read more about these design tools on our blog post


IMPORTANT CONSIDERATIONS AND LIMITING FACTORS FOR SGRNA DESIGN

There are several things you should consider when designing sgRNA for CRISPR
experiments:

 * The GC content of the sgRNA is important, as higher GC content will make it
   more stable - it should be 40-80%.
 * The length of the sgRNA should be between 17-24 nucleotides, depending on the
   specific Cas nuclease you’re using. Shorter sequences can minimize off-target
   effects, however, if the sequence is too short, the reverse can also occur.
 * Mismatches between gRNA and target site can lead to off-target effects,
   depending on the number of mismatches and their position/s.
 * It may be necessary to design multiple sgRNAs for each gene of interest, due
   to the fact that activity and specificity can be unpredictable.


COMPARISON OF DIFFERENT SGRNA FORMATS

Once the guide sequence has been designed, the next step is to actually make it.
This can be achieved by synthetically generating the sgRNA or by making the
guide in vivo or in vitro, starting from a DNA template. The method used to make
the sgRNA influences the experimental editing efficiency.


Table 1. Comparison of Time, and Labor Costs Associated with Different Guide RNA
Formats.





PLASMID-EXPRESSED SGRNA

One of the original methods of making sgRNAs involves expressing the guide RNA
sequence in cells from a transfected plasmid. In this method, the sgRNA sequence
is cloned into a plasmid vector, which is then introduced into cells. The cells
use their normal RNA polymerase enzyme to transcribe the genetic information in
the newly introduced DNA to generate the sgRNA.

Cloning the guide RNA plasmid generally requires about 1–2 weeks of lab time
prior to the actual CRISPR experiment. The plasmid approach is also more prone
to off-target effects than other methods because the guide is expressed over
longer periods of time. The plasmid DNA can integrate into the cellular genome,
which can result in adverse effects and problematic for downstream applications,
and can even cause cell death.


5 REASONS WHY RIBONUCLEOPROTEINS ARE A BETTER ALTERNATIVE TO CRISPR PLASMIDS

--------------------------------------------------------------------------------

5 REASONS WHY RIBONUCLEOPROTEINS ARE A BETTER ALTERNATIVE TO CRISPR PLASMIDS

Several researchers use plasmid transfection for their CRISPR genome editing
experiments, but is that the best strategy? High off-target effects, variable
editing efficiencies, and integration in host genome are just a few of the
reasons why RNPs have quickly become the more efficient alternative to plasmids.





IN VITRO-TRANSCRIBED SGRNA

Another method for making sgRNA, termed in vitro transcription (IVT), involves
transcribing the sgRNA from the corresponding DNA sequence outside the cell.
First, a DNA template is designed that contains the guide sequence and an
additional RNA polymerase promoter site upstream of the sgRNA sequence. The
sgRNA is then transcribed using kits that contain reagents and recombinant RNA
polymerase.

Synthesizing sgRNA using the IVT approach requires about 1–3 days. However, the
method suffers from certain drawbacks. It is labor-intensive and prone to
errors, and the in vitro-transcribed sgRNA generally needs additional
purification before it can be used in CRISPR experiments. These limitations
result in highly variable editing efficiencies and increased risk of off-target
effects.

Another issue associated with IVT sgRNA is that it can have negative effects on
certain cell types. Several studies have found that IVT sgRNA can trigger innate
immune responses in human and murine cells, causing cytotoxicity and apoptosis.
This is thought to be attributed to the 5’ triphosphate group of IVT sgRNAs,
which is recognized in the cytoplasm by DDX58, an antiviral innate immune
response receptor. DDX58 then produces type I interferons and proinflammatory
cytokines, leading to cell death.


SYNTHETIC SGRNA

The plasmid and IVT methods for sgRNA generation suffer from limitations such as
variable editing efficiencies, high off-target effects, and cumbersome
implementation. The need for a better alternative-fueled commercial production
of synthetic sgRNA.

Synthego generates high-quality synthetic sgRNA using chemical synthesis.
Transfecting synthetic sgRNA pre-complexed to the Cas protein in the
ribonucleoprotein (RNP) format generated the highest CRISPR editing
efficiencies.

While the cost of generating synthetic sgRNAs was initially a barrier for
researchers who wanted to improve their CRISPR editing using synthetic sgRNAs,
recent technological developments have alleviated those concerns. Synthego’s
high-throughput, automated, and scalable RNA synthesis platform enables the
production of high fidelity sgRNAs that demonstrate improved CRISPR
accessibility across all labs.

If you’re considering a change from IVT-generated to synthetic sgRNA, you can
download our guide on how to switch from IVT to synthetic sgRNAs, including
optimization of experimental conditions.


4 ADVANTAGES OF SYNTHETIC SGRNA


Figure 2. Synthego’s sgRNA Cited in Scientific Publications. The graphs show the
distribution of Synthego’s synthetic sgRNA usage in different application areas
(top) and research areas (bottom).


Synthego’s high-quality synthetic sgRNA has been cited in over 217 peer-reviewed
publications from a variety of research areas including oncology, immunology,
genetic disease, and neuroscience (Figure 2). Synthetic sgRNA has several
advantages over other sgRNA formats, four of which are listed here:

 * Improved Editing Efficiency
 * Minimum Risk Delivery System
 * Increased Experimental Convenience
 * Increased Stability With Chemically Modified Guides

> “We always get significantly higher editing efficiencies with Synthego's
> sgRNAs compared to in vitro transcribed sgRNAs.” - Aamir Mir, Ph.D.
> Postdoctoral Associate, Umass Medical School.


IMPROVED EDITING EFFICIENCY WITH SYNTHETIC SGRNA

The editing efficiency in cells transfected with synthetic sgRNA has been
experimentally confirmed to be higher than that of non-synthetically derived
sgRNA. Synthego's high-quality sgRNA achieves up to 97% editing efficiency and
enables highly reproducible experiments. CRISPR editing with synthetic sgRNA is
more efficient than with IVT guides, as shown in Figure 3.





Figure 3: Editing Efficiencies of sgRNA vs IVT. The same sgRNA sequences were
generated by either in vitro transcription (IVT) or synthesized by Synthego as
chemically modified sgRNAs. Indel frequency from experiments using Synthego
sgRNA was always >90% while editing by the IVT guides was always less than 10%
and sometimes undetectable. Data courtesy of Shondra Pruett-Miller, Ph.D., St.
Jude Children’s Research Hospital.

The consistent editing efficiency likely results from the difference in purity
between the two sgRNA formats. As shown in Figure 4, synthetic sgRNA shows
higher purity compared to IVT sgRNA.



Figure 4. Purity of Synthetic sgRNA vs IVT-derived sgRNA. Comparison of mass
spectrometry traces shows Synthego sgRNA has higher purity than IVT-derived
sgRNA for the same gene target. Note: The IVT-derived guide is slightly longer
due to the required additional transcription and terminator nucleotides.


MINIMUM RISK DELIVERY SYSTEM

One caveat of DNA-based methods for generating sgRNA, especially the plasmid
format of CRISPR components, is the continual expression of guide RNAs inside
the cell. This could result in unwanted effects in random or unexpected places
in the genome. Introducing the CRISPR machinery in the ribonucleoprotein format
into cells alleviates these concerns as the RNP exists transiently inside the
cell and shows reduced toxicity and off-target effects (Figure 5).


Figure 5. Plasmid vs RNP Cell Viability Comparison. Cell viability was assessed
using the CellTiter-Glo® Luminescent Cell Viability Assay. For the cell
viability assay, cells were nucleofected without RNP or plasmid as a mock
control. RNPs were constituted with different ratios of sgRNA and Cas9, 50 pmols
sgRNA:10 pmols Cas9, or 90 pmols sgRNA:10 pmols Cas9.


INCREASED EXPERIMENTAL CONVENIENCE

Even if an experiment is complicated, its preparation does not need to be.
Plasmid and IVT-derived RNA, in the best-case scenario, require a few days to a
week for preparation time before cell transfection. An important advantage of
synthetic sgRNA is that it arrives ready to use, thus saving the valuable time
and effort of researchers for the actual experiments - there is no cloning or
sequencing required.


DOWNLOAD APPLICATION NOTE

--------------------------------------------------------------------------------

EXCEPTIONAL SGRNA PURITY WITHOUT HPLC

This application note demonstrates the high editing performance of Synthego’s
chemically modified sgRNAs without HPLC purification. The results presented here
indicate that our high-quality synthesis removes the need for an HPLC
purification step required by other vendors, decreasing both turnaround time and
cost of Synthego’s sgRNAs. 

Download





INCREASED STABILITY WITH CHEMICALLY MODIFIED GUIDES

Stem cells are popular in therapeutics, having been widely applied in studying
disease models of different cell types. After the introduction of CRISPR,
modifying stem cell genomes was an obvious next step for researchers to test
further gene therapy options. However, stem cells, just like primary cells,
proved difficult to transfect with regular RNA guides.

> “Synthego’s chemically modified sgRNA provides a critical tool for our CRISPR
> research when it comes to difficult stem cell gene targets. Our research into
> stem cell-based human therapeutics presents editing challenges that require
> the highest efficiency guides,” - Andy Scharenberg, MD.

The advantage of synthetic single guide RNA is that it allows chemical
modifications that prevent degradation of the CRISPR machinery by the
intracellular immune response. Researchers have achieved up to 90% editing
efficiency with these challenging cell types using Synthego's chemically
modified sgRNAs. Moreover, these modified guides achieve exceptional editing
efficiencies even without HPLC treatment, required by other vendors, thus
decreasing turnaround time and cost of our product. Find data and further
details in our free Application Note.




MULTI-GUIDE SGRNA: IMPROVED KNOCKOUT STRATEGY


Figure 6. Fragment Deletion with Multi-guide sgRNA. Synthego’s multi-guide sgRNA
includes up to 3 modified sgRNAs (grey bars) that target a single gene of
interest. When co-transfected, the sgRNAs create concurrent double-stranded
breaks (vertical dotted lines) at the targeted genomic locus and consequently
induce one or more 21+ bp fragment deletions.


Achieving high-efficiency CRISPR knockouts can be challenging. Since individual
sgRNAs aim to generate random indels, they may not always result in complete
gene disruption. To overcome this problem, Synthego now offers multi-guide
sgRNAs for CRISPR knockouts - three separate sgRNAs that are spatially designed
for large fragment deletion in your gene/s of interest, targeting early exons.
This creates several double-stranded breaks in the gene and results in one or
more deletions of up to 20 base pairs, significantly increasing the chances of a
successful knockout. This technique is able to consistently generate deletions,
with knockout scores of up to 98.9%.

Multi-guide sgRNA can be particularly useful in generating clean knockouts in
cells that are difficult to genetically manipulate. A recent pre-print described
using Synthego multi-guide sgRNA to perform knockouts on human monocyte-derived
dendritic cells, which are typically difficult to edit. For more information,
you can watch this brief video about how our multi-guide sgRNA method works.

You can also download our application note explaining the method in detail and
discussing the advantages of this technique.




SYNTHEGO SYNTHETIC SGRNA KITS

An increasing number of researchers are using synthetic sgRNA in their CRISPR
experiments, due to their higher efficiency and the reproducibility of results.
Therefore, there is a great need among genome engineering researchers for
high-quality products supporting these assays.

Synthego’s CRISPRevolution CRISPR kits address this issue by offering economical
access to a full range of synthetic RNA products for high fidelity editing and
increased precision in genome engineering. 

To learn more about synthetic sgRNAs, you can check out our guide to choosing
the right CRISPR guide format.


SYNTHETIC SGRNA KIT

Synthego’s sgRNA kits result in indel frequencies of up to 99%, consistently
outperforming cr:tracrRNA, plasmids, and IVT. With high purity and necessary
chemical modifications for increased stability and decreased cellular innate
immune responses, synthetic sgRNA kits are the best approach for generating
knockouts or knock-ins in almost any type of cell, from immortalized cell lines
to primary cells.

“Synthetic sgRNAs, in addition to their standard benefits (speed of preparation,
consistency, stability) are the simplest way to go from a sequence to a hands-on
experiment, and are an extraordinary pedagogical tool for teaching the basics of
genome editing.” - Arnaud Martin, Ph.D. Assistant Professor, George Washington
University.


GENE KNOCKOUT KIT V2

Our Gene knockout (GKO) kit v2 increases the likelihood of generating CRISPR
knockouts in any cell type. The kit utilizes our multi-guide sgRNA strategy to
generate high-efficiency knockouts for any gene (see the multi-guide sgRNA
section above for more details).


Figure 7. Editing Efficiency of Individual vs. Multi-guide sgRNA. Two gene
targets (TNF, TLR4) in dendritic cells (transfected via nucleofection) were
edited using individual sgRNA and multi-guide sgRNA. Editing efficiency was
analyzed by sequencing the targeted loci on a MiSeq and sequencing outcomes were
categorized based on editing type (no indel, large deletion ≥50bp, small
deletion <50bp, insertion). Stacked bars represent the percentage of read
sequences assigned to each outcome. The multi-guide sgRNA for each target
resulted in >75% large deletion outcomes. Dr. Marco Jost and Dr. Jonathan
Weissman, University of California, San Francisco, and Dr. Amy Jacobson and Dr.
Michael Fischbach, Stanford University.


Check out our tips and tricks guide with all the information you need to get
started with multi-guide. Curious about how researchers have used GKO Kit v2 in
their research? Find out more in our researcher video presentation series.

“A constant challenge for our research is trying to reduce the number of clones
we need to screen to find a desired targeted modification. In our tests,
Synthego’s multiple modified synthetic guide RNAs, including the Gene Knockout
Kit, gave us greater than 80% knockout rates for seven guides,” - Shondra
Pruett-Miller, Director of Center for Advanced Genome Engineering, St. Jude
Children’s research hospital.




SGRNA ARRAYED SCREENING LIBRARIES

The CRISPR-Cas technology has great potential in loss-of-function screening
experiments. In an arrayed CRISPR screening, individual genes are targeted in
each well across a multiwell plate. Such assays help correlate genes to function
and identify novel drug targets for developing therapeutics.

Synthego’s chemically modified sgRNA screening libraries include up to three
sgRNAs specifically designed to cooperatively knock out each target gene.
Multi-guide sgRNAs greatly increase the probability of creating a functional
knockout. Moreover, each arrayed library requires minimal set-up time and
deconvolution time and can be also used for multiple cell types including
primary human cells. Both custom arrayed CRISPR screening libraries and over 30
standard libraries are offered by Synthego, and all libraries arrive ready for
transfection.

You can read about how Synthego’s sgRNA library was used to screen for kinases
that regulate cancer phenotypes in this case study, where the authors were able
to identify potential false-negatives missed in previous screens using cr:tracr
RNA library screens.

“As CRISPR screening in cell lines is becoming routine, the next frontier is to
implement these approaches in more complex model systems, such as primary cells.
Synthego's Arrayed CRISPR Screening Libraries and sgRNA kits have been
transformative for our high-throughput screening efforts to answer central
questions in innate immune recognition, antigen presentation, and T-cell
polarization, among others, in primary human dendritic cells. The quality of
Synthego's reagents and the resulting knockout efficiencies are truly
outstanding.” - Jonathan Weissman, Ph.D. Professor, Cellular Molecular
Pharmacology, School of Medicine, UCSF.

Prior to CRISPR, researchers largely used RNAi to perform loss of function
screens. The ease and minimization of false negatives with CRISPR are quickly
replacing RNAi as the preferred choice for screening assays. Learn more about
the differences between RNAi and CRISPR methods in our blog post.


ADVANCED RNA

At Synthego, we also offer advanced RNA, which is synthetic RNA tailored to be
compatible with any Cas nuclease, including novel and engineered variants such
as peg RNA, based on your unique research requirements. Advanced RNA is
available at any production scale, at high purities, with flexible length and
any necessary modifications.


GMP-COMPLIANT SGRNA 

In addition to providing sgRNAs and knockout kits for small-scale testing and
research, Synthego has developed a Good Manufacturing Practice (GMP) production
facility, complete with ISO 9001 certification, for manufacturing GMP-grade
sgRNA for use in clinical trials. We can provide sgRNA to any purification
specifications, at any scale, using validated processes, making us the ideal
partner on projects from discovery to any stage of clinical development. 

For more information, you can read about how Synthego sgRNA is being used in
cell and gene therapies. You can also download our large-scale synthetic sgRNA
flyer detailing our GMP manufacturing capabilities.


THE CRISPR REVOLUTION HAS JUST BEGUN

The introduction of the CRISPR-Cas9 system ushered in simplicity and efficiency
in the field of genome engineering. Similarly, the development of high-quality
synthetic sgRNA has been revolutionary in further simplifying CRISPR
experiments. Synthego strives to further refine the technology through
innovative products to enable CRISPR for all. This will allow researchers to
execute their experiments faster and with better precision, accelerating genome
editing research like never before.

THE BENCHMARK REPORT

--------------------------------------------------------------------------------

AN INSIDE LOOK AT WHAT'S HAPPENING AT THE BENCHTOP

We conducted a blind survey that contained 36 questions covering the challenges,
applications, success levels, and satisfaction levels around the CRISPR
workflow. 

Download Report

--------------------------------------------------------------------------------

To Chapter 4 To Chapter 2

--------------------------------------------------------------------------------

To Beginning
 * Chapter 01
   How To Design Guide RNA for CRISPR
 * Chapter 02
   How to Design CRISPR Guide RNAs with the Synthego Design Tool
 * Chapter 03
   The Complete Guide to Understanding CRISPR sgRNA
 * Chapter 04
   Importance of the PAM Sequence in CRISPR Experiments
 * Chapter 05
   How to Choose the Right Cas9 Variant for Every CRISPR Experiment
 * Chapter 06
   How to Select the Best CRISPR Transfection Protocol
 * Chapter 07
   How to Pick the Best CRISPR Data Analysis Method for Your Experiment
 * Chapter 08
   Step-by-Step Guide for Analyzing CRISPR Editing Results with ICE


HOW TO USE CRISPR


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