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!