CRISPR Submission
CRISPR targeting
consists of a Cas protein (usually spCas9) which creates a double strand break
at a defined location and a guide RNA (gRNA) which complexes with the Cas
protein to target the double strand break. If the goal of the mutation is to
introduce a defined sequence such as a base change, tag or transgene, a DNA
vector containing the desired mutation along with arms of homology flanking the
mutation is also required. The DNA is often referred to as the homology directed
repair (HDR) template
Cas protein: The Core supplies spCas9 which is the
Cas used for the vast majority of work. There are some situations where an
alternate Cas is needed. In these cases, please contact Chip to discuss your experiment.
Guide RNA: The gRNA is a short RNA composed of a scaffold sequence
necessary for Cas-binding and a user-defined 20 nucleotide spacer that
targets the double strand break. The 20mer must be adjacent to a Cas defined
PAM site which for Cas9 is NGG. The double strand break is made between the 3rd
and 4th nucleotide of the spacer from the PAM. The goal for choosing
a gRNA is to have the break be within 10 nucleotides of the desired mutation. You
also need to identify gRNAs that have a low likelihood of creating breaks in
undesired locations. There are numerous web based
applications to assist with finding gRNA 20mers. Most give the same results
but present the options in different ways. One frequently used by the Core is CRISPOR. If you are not able to identify a
suitable gRNA that meets your goals, please contact Chip to explore other options.
In practice,
there are 2 available gRNA formats – a single fused gRNA or a 2 component RNA
consisting of a crRNA containing the user-defined sequence and a tracrRNA which
doesn’t change. At the Core, we mostly use the 2-component format. The Core
provides the tracrRNA. The crRNA is supplied by the client and can be purchased
from many vendors such as IDT. When purchasing,
please order the 2nmol quantity and do not resuspend the RNA prior to
submitting.
HDR Template: The format of the HDR Template is chosen
based on the desired mutation. It can be single or double stranded and consists
of the desired mutation which is flanked by arms of homology. Usually, smaller
is better when injecting DNA into embryos so a goal is to use the smallest DNA
possible to achieve your goal. The goal also drives the format of the DNA to
use…
Small Knockins
or nucleotide mutations: The
best HDR template format for insertions or nucleotide changes under ~130 bases is
a small single strand DNA oligo. The design includes the desired mutation
flanked by 35-50 nucleotide arms of homology. It’s important to disrupt CRISPR
targeting along with introducing your desired mutation by mutating one of the
Gs in the PAM or by making 2 or more base changes in the 20mer. If your
mutation doesn’t create these conditions, use codon swaps to achieve this. The
oligos are most often purchased from a vendor such as IDT.
Purchase the highest purity available from the vendor to avoid contaminants
that can kill the embryos. Purchase 2-4 nmol quantity and do not resuspend prior
to submitting.
Large Knockins: The HDR template for Large Knockins is usually
double strand DNA although single strand DNA works as well. The design includes
your transgene flanked by arms of homology. There is still some debate on the
best size of arms – short 36 base have been shown to work well for knockins in
the 1000 nucleotide range however for larger transgenes arms that are 100-500
nucleotide are probably a better choice. It’s important to disrupt CRISPR
targeting along with introducing your desired mutation by mutating one of the
Gs in the PAM or by making 2 or more base changes in the 20mer. If your
mutation doesn’t create these conditions or insert inside the 20mer+PAM, use
codon swaps to achieve this. There is flexibility on the source of the
template. When purchasing or preparing the template, always keep in mind that
the reagents will be injected into embryos. Impurities will prevent clog the
injection pipet or kill the embryos in utero. If preparing your own template,
follow the guidelines here.
If purchasing from a vendor, pay attention to how the DNA is purified. For
example, IDT gBlocks
are a poor choice for embryo injection whereas IDT Megamers
usually work well. When choosing a vendor, please discus the goal of embryo
microinjection with them. The desired quantity for submission is 3ug. If
purchased from a vendor, do not resuspend the prior to submitting. If preparing
yourself, be sure to use injection buffer provided by the Core and the
concentration should be ~100ng/ul.