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Thank you for approaching NUCLEICA.

If you are interested in a CRISPR screen or a heavily customizable project, please do not use this form and contact us directly. If you are looking for a knockout, knockin or a deletion, please proceed as follows:

1) COMPLETE THE FIELDS BELOW;

2) SEND THE FILE TO mail@nucleica.tech and we will be in touch within 24 hours.

Please fill in this table the best way you can. If you don’t know the details we ask, don’t waste any time searching for information we can find easily. We are mostly interested in avoiding communication mistakes and getting any additional information that you may have that is impossible to find elsewhere (for instance, any tricks to keep your cell line happy, a splice form you found but did not publish that could escape a knockout strategy, a published knockout strategy in some obscure or old publication that you would like to mimic, etc.).

CONTACT INFORMATION

First Name

Last Name

Company/Institution Name

Address

Phone

TIN - Taxpayer Identification Number of your institution (essential for the invoice):

How did you find us? Please check the box here and throughout the document

How did you find us?

Google search

Group email sent by us

Word-of-mouth

From a former client* Please type the name of the client:

*This information is important for our referral rewards.

YOUR PROJECT

PROJECT DESCRITION

Please describe your project in this box. You may be succinct about the science, but be as precise as possible about the genomic edition. Add links to the gene, a type of edition you may want to mimic, etc. If you have specific requests about the editing (HR-based versus base editor-based, etc.), time constraints or specific concerns, mention them here.

Please note that the project can be CRISPR knockouts, deletions, knockins, CRISPR-based gene activation/repression, CRISPR-based screens, stably transfected/transduced cell lines and others.

PREFERRED CELL LINE

NAME OF THE CELL LINE YOU WILL SHIP

Insert name + ATCC link or ref if possible:

Number of allelic copies (related to ploidy)

If you know the karyotype of the cell line and the chromosome of your target gene:

Relevant papers and any useful information not found in the literature. Please include complete information about the culture medium and any relevant details to handle the cells:

Is there a selectable marker in the cell line of choice. Which one? The cell may have been engineered to have a puromycin resistant gene, express GFP, etc., which could be relevant for the CRISPR approach.

Here we refer to modifications (knockins, knockouts, stable transfectants, etc.) not described in the link to the cell line:

Detailed culture conditions, including how to culture, split and freeze the cells. Please mention how you handle the cells. Send us protocols as detailed as possible:

Additional non-trivial tips on how to handle the cells:

CRISPR PROJECT: SINGLE GENE EDIT

STARTING PRICE:

Isolated edited clones (4200 €)

Clones in bulk cultures (2450 €)

“Do it yourself” package (1250 €, the client has to purchase the reagents)

Strategy design only (250 €, within 3 days)

ABOUT THE EDIT:

Isolated edited clones (4200 €)

Deletion - add 1900 € for isolated clones ☐

Knockin SNP or small tag

add 2300 € for isolated

or add 550€ for bulk cultures

Knockin large insert (1 allele, e.g. GFP ) -

add 3100 € for isolated clones

or add 1350€ for bulk cultures

EXTRA FEES:

iPSC or any cell line requiring an expensive medium (add 400 € for bulk cultures and 2000 € for clones per guide used)

Extra fees for the number of targeted alleles in the isolated clone package (there is no extra fee for the bulk cultures):

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For knockouts, deletions, SNPs, and short inserts (e.g. a small tag), the starting price is for the targeting of two alleles (sufficient for a KO in a diploid cell). Add 1500 € per extra allele (e.g., triploid cell) for any of these modifications.

For large insert knockin the starting price is for a single allele (as in a diploid cell heterozygous for the insert). Add 1500 € per extra allele para (e.g., homozygous diploid cell).

TARGETED GENE OR REGION

GENE OR GENOME COORDINATES

For genes, please use a link describing your gene in at least one of the following databases:

NCBI

UCSC genome browser gateway

UNIPROT

KNOCKOUT OR DELETION (Ignore this section if you are looking for a knockin)

PROJECT DESCRITION

Please mention the most critical domain of the protein, the isoforms and any report describing the knockout you plan to make (even if from a different species and using different technology).

PLEASE BE CLEAR ABOUT THE ISOFORM(S) YOU WANT TO KNOCKOUT. By default, we target all isoforms:

Is the knockout expected to affect cell growth/viability?

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Yes

Unknown

If yes, please give details (e.g., a reference) here:

Do you also want +/- (het) clones besides the knockout (-/-) clones?

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Yes (add 500 €)

KNOCKIN (Ignore this section if you are looking for a knockout or deletion)

Type of knockin

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Point mutation or small insertion (e.g. a FLAG tag of loxP)

Large insertion (e.g. GFP)

Genotype of knockin

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Homozygous

Heterozygous

For protein fusions, specify the fusion partner from this menu: Choose an item.

If you want to insert a different insert, please write the protein sequence here:

STARTING ATG. Please notice that if you do not tick one of these boxes we will by default keep the starting ATG (most people agree that there is no strong reason to remove it). In the case of the fusion partner gene added to the C-terminus of the gene, the starting ATG:

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will be kept

will be removed

Position of added protein

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Fused to the N-terminus of the target protein

Fused to the C-terminus of the target protein

Protein linker: for an overview, check this review. Please select your linker using the dropdown menu (Choose an item.) or write the protein sequence here:

FOR knockin projects: can the PAM site or the spacer sequence in the template be altered? To avoid retargeting the allele after the introduction of the right edit, and also to facilitate the genotyping of the clones, a common strategy is to mutate the PAM sequence or the spacer of the template, so that Cas9 will not cut the edited allele. This is done by introducing silent mutations that will not change the protein sequence and respecting codon usage as much as possible. If for some reason you prefer not to introduce these additional changes, please be aware that the efficiency of targeting may be affected.

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Yes

FINAL IMPORTANT DECISIONS ABOUT YOUR SINGLE-GENE CRISPR PROJECT
Please check the section of our page describing the EXTRAS. If you don’t check any box, we will apply the default option

Type of Cas9

By default, we use a commercial conventional Cas9. If you want an engineered Cas9 that reduces the number of off-targets, please select the option “Hifi” Cas9.

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The default option (conventional Cas9)

“Hifi” Cas9*(add 500 €)

*Cas9 - click here to know more about this protein.

Wild-type controls

By default, we will provide 2 control clones that were exposed to Cas9+sRNA but did not acquire the mutation. Essentially, these clones are identified when we genotype the collection of clones for the presence of the mutation. Since the method is not 100% efficient, there will always be clones that were not edited in the target region but may have been edited elsewhere in the genome (off-targets). If you prefer another wild-type control, please select it.

Extra guide

By default, we test more than one guide RNA but the edited clones come from cells transfected with the same guide RNA. Including an extra guide may be helpful because the best control for off-target effects is the confirmation that the phenotype is the same for clones edited with different guide RNAs.

Confirmation of the knockout

By default, we confirm that the two alleles acquired frameshift mutations. Further confirmation at the RNA and/or protein level can be provided

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The default option (no extra control)

Controls from a parallel mock transfection (add 500 €)

Controls from a parallel transfection with only Cas9 (no guide) (add 500 €)

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The default option (no extra guide)

Use extra guide (add 750 €)

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The default option (no extra guide)

The default option (no extra confirmation)

RT-qPCR (add 400 €)

Western blot* (add 400 €)

*You have to provide the antibody

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CRISPR PROJECT: GENE ACTIVATION OR REPRESSION:*

CRISPR PROJECT: CRISPR-BASED SCREEN*

STABLE TRANSFECTION (with plasmids)/ TRANSDUCTION (with viruses)*

PLASMID BUILDING*

*Details will be discussed over a zoom meeting.

Thank you for approaching NUCLEICA.

CONTACT INFORMATION

How did you find us?

YOUR PROJECT

PROJECT DESCRITION

PREFERRED CELL LINE

CRISPR PROJECT: SINGLE GENE EDIT

STARTING PRICE:

ABOUT THE EDIT:

EXTRA FEES:

TARGETED GENE OR REGION

KNOCKOUT OR DELETION (Ignore this section if you are looking for a knockin)

PROJECT DESCRITION

KNOCKIN (Ignore this section if you are looking for a knockout or deletion)

FINAL IMPORTANT DECISIONS ABOUT YOUR SINGLE-GENE CRISPR PROJECTPlease check the section of our page describing the EXTRAS. If you don’t check any box, we will apply the default option

FINAL IMPORTANT DECISIONS ABOUT YOUR SINGLE-GENE CRISPR PROJECT
Please check the section of our page describing the EXTRAS. If you don’t check any box, we will apply the default option