3) CRISPR Cas9 – gRNA Design

The RNA-guided CRISPR Cas9 system has revolutionized genetic engineering by allowing targeted genomic modification through the simple design of
a 20 base pair guiding sequence. This groundbreaking technology utilizes a short guide RNA (sgRNA) to direct the Cas9 nuclease to a specific genomic locus through complementary base pairing. The guide RNA or sgRNA is responsible for the specificity of the CRISPR Cas9 system,
and many considerations need to be taken during its design process. In our previous videos
we introduced the CRISPR Cas9 system briefly and presented the different tools and methods available for their expression in living systems; here we will go over the design criteria of
the gRNA and introduce you to the online programs that simplify this procedure. We invite you
to watch our previous videos first before continuing with this one. In bacteria and
archaea, the CRISPR RNA (crRNA) and transactivating CRISPR RNA (tracrRNA) form a complex which acts as the homing device for directing the Cas9 nuclease to the invading foreign genetic materials. The tracrRNA’s scaffolding ability along with the specificity of the crRNA can be combined into a single synthetic gRNA, simplifying guiding of targeted gene alterations
to only a one-component system all while maintaining equal or higher efficiency. The targeting
specificity of the CRISPR Cas9 system is determined by the 20 nucleotide sequence at the 5’
end of the gRNA. For the S. pyogenes CRISPR Cas9 system, the desired target sequence must
immediately precede a 5’-NGG protospacer adjacent motif (PAM). The gRNA base pairs
to the complementary strand of the target sequence where the Cas9 nuclease mediates
a double strand break around 3 nucleotides upstream of the PAM sequence. Note that the PAM sequence
is not a part of the 20 base pair gRNA sequence, however, its presence in the genomic DNA is
essential for CRISPR Cas9 genome editing. There are three points to consider when designing
your gRNA which relate to the sequence of the 20 base pairs guiding sequence: 1) GC
content: the typical range is between 40% – 80% GC content. A higher GC content stabilizes
the RNA: DNA duplex while destabilizing off target hybridization; 2) Length: the length
could be adjusted and range from 17-24 base pairs. A shorter sequence leads to minimized
off target effects (17 base pairs is the lowest limit on the length of the guiding sequence,
any sequence shorter than 17 has a statistical chance of targeting multiple genomic loci);
and 3) Potential Off Target Effects: Mismatch
tolerance between the gRNA and target site is what leads to off target effects of the
CRISPR Cas9 system and in general they depend on 1) position of the mismatch; 2) number
of mismatches in a given gRNA; 3) the sequence of the guide RNA; and 4) concentration of
the gRNA and Cas9 transfected. For the purpose of creating a small InDel mutations through
non-homologous end joining, there are many possible target sites across any protein.
Targeting closer to the N’ terminus of a protein coding region is more desirable, because
a frameshift is more likely to be deleterious if most of the protein has not yet been translated.
Also, both the coding and non-coding strand of the genomic DNA can be targeted as they
are both equally efficient at creating InDel mutations. If Homology Directed Repair is
needed for genome editing, however, the choice of target site is far more constrained by
the desired location of insertion. For more information about non-homologous end joining
(NHEJ) and Homology Directed Repair, please see our introductory video on the CRISPR/Cas9
system. With the tips in designing sgRNA taken into consideration, from PAM sequences to
target sites to GC content, we can now choose a sgRNA. Websites like Chop Chop Harvard provide
an easy-to-use system in designing the sgRNA against any gene of interest. To utilize this
technology, click on the link of chop chop Harvard in the description of this video.
Select the species of interest from the drop down menu. Species-specific gene IDs, genomic
coordinates or entire nucleotide sequences can be cut and pasted in for analysis. Click
on the Find Target Sites! Button and wait for the analysis to be completed. Once the
analysis is done, a graphic representation of the sequence will be provided, displaying
the various potential sgRNAs shown in their respective positions by base pairs. Scrolling
the mouse over these various sgRNA representations will show the website’s ranking with respect
to the best sgRNA to use for that gene. Below will be a list of all possible sgRNAs and
the breakdown of their rankings, based on genomic location, GC content, and Off-targets.
To ensure maximum sgRNA efficiency, knowing the number of off-target effects is very important.
For more information on each sgRNA candidate, one can click on each choice and view gRNA
and the PAM sequence in their genomic context. One can also view information on the off-targets,
if any, as well as information on the various primers that will encompass the sgRNA target
sequence and could be used for monitoring the genetic modification process. While Chop
Chop Harvard is one method of designing sgRNA, there are many other websites such as the
Broad Institute and BioTools that could also be utilized. To locate abm’s vast repertoire
of sgRNA libraries, start by finding us at our homepage at www.abmgood.com. From there
start by clicking on “CRIPSR/Cas9”. You will be taken to our CRISPR/Cas9 product page
which features everything required for carrying out successful genome modification through
the CRISPR Cas9 system. By clicking on Genome-wide sgRNA libraries, or alternatively scrolling
to the bottom of the page you will find our sgRNA products sorted by gene names. Our sgRNAs
are provided in different formats, including Lentiviral vectors, packaged lentiviruses,
adenoviruses, and even AAVs – which will be added soon. After choosing the vector you’d
like to use for sgRNA delivery, the search bar allows you to search by gene symbol or
accession number, or you can use the alphabetical sorting to find your gene of interest. Once
you’ve selected a gene, you can find all available sgRNA products relating to your
selection will be displayed. From here you can order your sgRNA products. Please leave
your questions and comments below and we will answer them as soon as possible. For more
information please visit our website. Thank you for watching!

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