Download (DNA, RNA, or DNA/RNA) Microinjection Service Form

CRISPR engineering (DNA, RNA, or DNA/RNA) Service Form
No guarantee is made for the number of transgenic mice produced since this depends on a number of factors
including:
1) The quality of DNAs/RNAs provided by the investigator (if provided by the investigator)
2) The ability of the guide to target the Cas9 to cut in vivo
3) The chromatin structure/accessibility of the DNA sequence to be targeted (highly variable and
unpredictable)
Please check the box next to the indicated responsibility as to whether the GTTR (Gene Targeting and
Transgenic Resource) is responsible, or the Investigator lab is responsible:
Task
Responsibility of GTTR Core
Responsibility of Investigator Lab
To provide DNA vectors and/or RNAs as
specified (see the form for specific quantities,
volumes and concentrations), to be
determined by mutual agreement with staff.
To provide a description of the DNA vectors
and/or RNAs involved, and a brief description
of the experiment.
To provide sequence of CRISPR RNAs used,
donor DNA molecules to be used for HDR, if
any (e.g. DNA oligos or fragments), and the
source of Cas for CRISPR injections. (Describe
origin of CRISPR backbone and Cas gene
being used.)
To assure that the DNA and/or RNA
fragment(s) will not produce any contagious
condition that may be harmful to animals or
to humans and that they do not exceed
containment requirements greater than
those specified by NIH BL-2 standards.
To inform the number of transgenic mice
obtained after tail DNA analysis by Southern
or PCR within 1 month of being notified that
tail samples are ready for screening.
Recommended number of injection days to request: Because the number of embryos available for injection each
day will vary, we guarantee at least 150-300 embryos will be injected per scheduled project. The actual number
injected may be more than this, but will not be less or the strain of the mouse chosen.
CRISPR Project: Request Information
Date:
Investigator:
Institute:
Address:
E-Mail:
Phone Number:
IACUC/animal welfare protocol number:
_
Descriptive Name for this CRISPR Project
Please provide a brief but clearly identifiable name. (Will appear on cage card as written here):
Brief description of desired outcome of project i.e. what is the mouse you want to make?
CRISPR-mediated cleavage can result in mutagenesis of both copies of the target gene. Could inactivation of the
target gene(s) cause lethality or abnormal phenotypes, either in the heterozygous or homozygous state? If so
please explain.
For CRISPR project: Please complete this section.
Targeted Gene(s) MGI ID:
Targeted Gene(s) Name:
Targeted Gene(s) Symbol:
Please list the names and protospacer sequences of any CRISPR guide RNAs to be injected (alternatively, if the GTTR
does the design and production, we will fill out and return back to you):
* For the embryonic expression of the CRISPR and Cas9 components, which types of nucleic acids will be injected:
either DNA plasmids, or in vitro transcribed RNAs? (Check one)
DNA
RNA
_ Both
_
If you answered DNA to the previous question marked by *, complete the following section. If you marked RNA,
skip this section.
Describe the DNA vector(s) used:
Vector size:
Method of vector DNA purification:
If you answered RNA to question marked by *, complete the following section. If you marked DNA, skip this
section.
Cas9 RNA: Provided by outside vendor? Yes
_
No
_
If Yes, list the vendor:
If No, please list the source template plasmid, synthesis and purification methods:
Cas9 Template:
Cas9 Synthesis and Purification methods:
Final concentrations of oligo/guide/nuclease will be as follows:
Each Injected oligo will be 100ng/ul;
Each Guide RNA will be 50ng/ul and
Cas9 will be 20ng/ul.
Please plan accordingly as they need to be resuspended together at a final injection volume of 50uL/day (at least).
Deviations from these concentrations, if requested, should be explained below:
For all CRISPR projects: Does this injection also require injection of additional DNA components, such as donor
molecules (oligos or DNA fragments) that are intended to edit, recombine with, or insert into the CRISPR genomic
target site(s), as via the homology-dependent repair pathway?
Yes
_ No
_
If the answer to the last question is yes, list and describe any DNA oligos or fragments used in combination with
CRISPR RNAs for gene editing (provide these DNA sequences in a separate document)
(alternatively, if the GTTR does the design and production, we will fill out and return
back to you):
Do you have any prior evidence that the exact same CRISPR target(s) can generate mutations with efficiency? (E.g.
prior injection or transfection data using the same CRISPR). If so please describe the experiment and recorded
efficiency:
Please describe the methods that will be used to screen and verify any CRISPR-generated mutations/genome
editing in the founder animals (e.g. Surveyor assays, direct sequencing, or other) (alternatively, if the GTTR
does the design and production, we will fill out and return back to you):
Strain of mice desired for injections:
_ (B10xC3H hybrid; C57BL/6J; other?)
Provide a brief description of the intended use of the engineered mice to be generated:
FOR CRISPR PROJECT REQUESTS, Please attach to this form (alternatively, if the GTTR does the design and
production, we will fill out and return back to you):
1. Sequences of guide RNAs and any DNA oligos/donor fragments being injected
2. If available, photos of gel analysis of RNAs.
3. Example of PCR demonstrating successful amplification of the wild-type mutation target from
mouse genomic DNA.
CRISPR-Cas Disclaimers:
• CRISPR-Cas-mediated “genome editing” is a rapidly emerging technology. Although the core has
demonstrated successful in vivo target cleavage in mouse embryos in a limited number of experiments,
not all targets may induce genome editing equally well, and so the GTTR cannot guarantee successful
cleavage/editing. It is the responsibility of the investigator to confirm whether mutagenesis and/or
genome editing has occurred successfully in the resulting mice.
• SgRNA-mediated cleavage has been reported to be prone to off-target mutagenesis. These events
have been observed in some CRISPR-modified mice, although not all CRISPR guide RNAs will be highly
prone to this problem. The GTTR does not hold responsibility for off-target mutations.
• DNA cleavage often occurs at the 1-cell stage, but may occur at the 2-cell stage or later. CRISPRmediated mutagenesis can therefore generate mosaic animals with regards to the proportion of cells
that carry specific mutations. On the other hand, more than 2 types of mutations at a single target site
can sometimes be observed in tissues from the injected mice. Thus, PCR detection of mutations in
liveborn mice does not guarantee germline transmission of particular mutations is efficient or possible
(although it typically should be).
• CRISPR-Cas-mediated mutagenesis can affect both alleles in a 1-cell embryo. Thus, increased lethality
may result from targeting essential genes, reducing yield of liveborn pups and/or correctly targeted
pups
• Mice will be released/shipped to the investigator only when payment for the project has been made,
and when proper animal transport paperwork is completed.
Investigator’s signature:
◎ Please submit the completed form to [email protected].
Beijing SBS Genetech Co. Ltd.
Fax: +86-10-82784290
Email: [email protected], [email protected]
Website: www.sbsbio.com