CRISPR-mediated knock-in of a reporter gene

knock-in (KI)

The scarless targeted integration of functional DNA elements such as mutations, protein tags, fluorescent reporters and humanized genes into the genome for disease modeling as well as developmental and mechanistic studies is routinely achieved by RNA-guided nuclease technologies such as CRISPR-Cas. In addition to the ability to edit or delete DNA sequences within the genome, a repair process known as homology-directed repair (HDR) offers the ability to seamlessly insert DNA sequences.  In the presence of a double-stranded DNA break (DSB) created by the Cas endonuclease and an accompanying repair template (ssODN, lssDNA, donor vector) with sufficient homologous sequence, the cellular machinery may repair the DSB during the mitotic S-phase when homologous recombination proteins are active.  As a consequence, the repair template may become seamlessly integrated at the target site within the genome: this technology is known as a knockin (KI). 

Allow NovoHelix to leverage its expertise to build custom rodent models containing small knockins such as insertion of point mutations, altering amino acid codons, insertion of tags and barcodes, reporter genes (mKusabira-Orange, mCitrine, mVenus, mNeonGreen, GFP, mEmerald, mCherry, tdTomato, mScarlet, mCerulean, mTFP1, etc.) and whole gene replacement such as humanization. For large knockins, NovoHelix has routinely introduced large DNA constructs some upwards of 90-kb into traditional safe harbor sites Rosa26 and Aavs1 and has built a suite of flexible donor vectors such as Rosa26-lox-stop-lox (Rosa26-LSL). From the initial project concept, we can assist with a flexible design that includes building advanced genetic circuits for knockin to any genomic locus to advance your field.
model generation
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model generation

Service

Catalog Nr

Service Description

Timeline

Deliverables

Pricing

  • Rosa26 safe harbor knockin 
  • C57BL/6 mouse KI via CRISPR microinjection 
  • guaranteed founders 
KIMM001
 Services included are insertion of point mutations, altering amino acid codons, insertion of tags and barcodes, reporter gene knockins (GFP, mVenus, mCherry) and whole gene replacement such as humanization.  The service includes guide RNA design using the latest design guidelines, i.e. full-length guides, chemically modified gRNAs, truncated sgRNAs, hp-sgRNAs (hairpin-sgRNAs), that produces high on-target activity in our surrogate cell-based gene editing assay while limiting off-target activity. 
 3 - 4 months founders; 5 - 8 months GLT F1s

 At least 2 KI founders (or germline transmitted F1s) with the confirmed mutation

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  • Rosa26 safe harbor knockin
  • C57BL/6 mouse KI via CRISPR microinjection 
  • non-guaranteed service (per injection session)
KIMM002

 Services included are insertion of point mutations, altering amino acid codons, insertion of tags and barcodes, reporter gene knockins (GFP, mVenus, mCherry) and whole gene replacement such as humanization. Microinjection is performed as a per session service with a total of 500 zygotes injected.

 3 - 4 months founders 

 At least 500 embryos will be injected and implanted (2-3 microinjection sessions), which usually results in approximately 100 pups. Ear biopsies from all pups will be given to the investigator for genotyping. There is no guarantee that resulting pups will be gene edited.

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  • Rosa26 safe harbor knockin 
  • custom mouse strain - CRISPR-mediated knockin
  • guaranteed founders
 KIMM003
 Services included are insertion of point mutations, altering amino acid codons, insertion of tags and barcodes, reporter gene knockins (GFP, mVenus, mCherry) and whole gene replacement such as humanization.
 3 - 4 months founders; 5 - 8 months GLT F1s

 At least 2 KI founders (or germline transmitted F1s) with the confirmed mutation

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Custom knockin any locus
C57BL/6 mouse KI via CRISPR microinjection 
guaranteed founders 
KIMM004
Services included are insertion of point mutations, altering amino acid codons, insertion of tags and barcodes, reporter gene knockins (GFP, mVenus, mCherry) and whole gene replacement such as humanization.
 3 - 4 months founders; 5 - 8 months GLT F1s

 At least 2 KI founders (or germline transmitted F1s) with the confirmed mutation

Request Quote
  • Custom knockin any locus
  • C57BL/6 mouse KI via CRISPR microinjection 
  • non-guaranteed service (per injection session) 
KIMM005
 Services included are insertion of point mutations, altering amino acid codons, insertion of tags and barcodes, reporter gene knockins (GFP, mVenus, mCherry) and whole gene replacement such as humanization.
 3 - 4 months founders 

 At least 500 embryos will be injected and implanted (2-3 microinjection sessions), which usually results in approximately 100 pups. Ear biopsies from all pups will be given to the investigator for genotyping. There is no guarantee that resulting pups will be gene edited.

Request Quote
  •  Custom knockin any locus
  • custom mouse strain - CRISPR-mediated knockin
  • guaranteed founders 
KIMM006
 Services included are insertion of point mutations, altering amino acid codons, insertion of tags and barcodes, reporter gene knockins (GFP, mVenus, mCherry) and whole gene replacement such as humanization.
 3 - 4 months founders; 5 - 8 months GLT F1s

 At least 2 KI founders (or germline transmitted F1s) with the confirmed mutation

Request Quote
support services

Service

Catalog Nr

Service Description

Timeline

Deliverables

Pricing

  • Gene editing activity testing
  • Format - cell-based transfection
  • Assay - T7 endonuclease I/Cel-II/Surveyor
KIMM007
 NovoHelix offers a gene editing service to help clients test their CRISPR tools including guide RNAs, high-performance mutant Cas proteins and base editors in plasmid DNA or RNP formats.  A representative cell line will be transfected in triplicate and results will be generated by the mismatch-nucleases T7 Endo I or Cel-II as adopted from a protocol originally developed by Keith Joung's lab. Guide RNAs can also be tested in an vitro cutting assay if preferred, but we have found in vitro activity to supercede in vivo activity levels both in cellular and microinjection contexts; and hence in vitro mismatch nuclease assays often do not correlate to gene editing in vivo outcomes. 
 7-10 days

 Activity of up to 6 guide RNAs

  • Gene editing activity testing
  • Format - cell-based transfection
  • Assay - genetic reporter & flow cytometry
KIMM008
NovoHelix offers a gene editing service to help clients test their CRISPR tools including guide RNAs, high-performance mutant Cas proteins and base editors in plasmid DNA or RNP formats.  A representative cell line will be transfected in triplicate and results will be generated by a fluorescent reporter assay and flow cytometry.
 7-10 days

 Activity of up to 6 guide RNAs

 Genotyping Assay Development - knockin
KIMM009
 NovoHelix offers a knockout genotyping service to help clients develop robust genotyping protocols for screening animals after breeding and expansion of indidvidual mutant/KO founder lines.
 2-4 days

 genotyping protocol

 Genotyping Assay Development - complex knockin
KIMM010
 NovoHelix offers a knockout genotyping service to help clients develop robust genotyping protocols for screening animals after breeding and expansion of indidvidual mutant/KO founder lines.
 5-7 days

 genotyping protocol

 Development of guide RNAs - guide RNA cloning and design in a mammalian (U6, H1) expression vector or T7 in vitro transcription (T7- IVT) vector (DNA format)
KIMM011
 Service is for design and cloning of up to 6 guide RNAs for 1 target locust in a DNA plasmid format.
7-10 days
6 guide RNAs in plasmid vectors
 Development of guide RNAs - guide RNA cloning and design with T7 in vitro transcription (T7- IVT) for use in RNP format (RNA format)
 KIMM012
 Service is for design and in vitro transcription (IVT) of up to 6 guide RNAs for 1 target locust.
 7-10 days
6 guide RNAs ~ 500 ng/ul --- RNA format

 Client-provided guide RNAs - preparation for microinjection (DNA/RNA format)
 KIMM013
Service is for purification and for preparation of gRNAs for zygotic microinjection or slide-electroporation. Client should provide validated gRNA synthesis data such as small RNA electrophoresis results from Agilent's Bioanalyzer prior to microinjection and a minimum 200 ng/ul gRNA concentration. Purification is required to prevent the microinjection glass needle from clogging, to prevent embryo lysis and extensive delays in refitting and resetting microinjection setup.
 Client-provided dsDNA donor vector - preparation for microinjection 
KIMM014
 Purification of supercoiled plasmid by a proprietary anion exchange (AEX) chromatography, validation by restriction digestion and analysis by gel electrophoresis. Endotoxin levels are generally very low (0.05  – 0.5 ng LPS/µg) via our proprietary extraction method and are adequate for sensitive applications such as zygotic microinjection of mammalian embryos. The plasmid is predominantly in its supercoiled topology and free of RNA and protein contamination such as RNases and proteases.  Drop dialysis in nucleofection/electroporation/microinjection buffer is included in the purification service. Validation by additional restriction digestions (greater than 3) is available with commensurate fee schedule.  Purification is compulsory to prevent the microinjection glass needle from clogging, to prevent embryo lysis and extensive delays in refitting and resetting the microinjection setup.
 Knockin dsDNA donor vector construction
KIMM015
 Isogenic knockin dsDNA donor vector construction and Sanger-sequence verified with up to 5-kb insertion.  NovoHelix will provide an in silico map and provide restriction fingerprinting for plasmid structure via 3 digests. DNA is purified by a propriety AEX (anion exchange) chromotography and suitable for microinjection into mouse zygotes or nucleofection/electroporation.  The DNA is dialyzed on a membrane support in microinjection or electroporation buffer and then spun to remove particulate matter to obviate microinjection needle clogging.
 Knockin dsDNA donor vector construction - complex
KIMM016
 Isogenic knockin dsDNA donor vector construction and Sanger-sequence verified with up to 50-kb insertion.  NovoHelix will provide an in silico map and provide restriction fingerprinting for plasmid structure via 3 digests. DNA is purified by a propriety AEX (anion exchange) chromotography and suitable for microinjection into mouse zygotes or nucleofection/electroporation.  The DNA is dialyzed on a membrane support in microinjection or electroporation buffer and then spun to remove particulate matter to obviate microinjection needle clogging.
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 Mouse colony husbandry & management
KIMM017
 mouse colony management including breeding, weaning, sampling, monitoring, per cage per day for the production of founders and F-generation pups
technology
CRISPR-mediated knock-in of a reporter gene
CRISPR-mediated knock-in of a humanized allele—gene replacement knock-in
CRISPR-mediated knock-in of a point mutation
seamless knock-in of small protein tags or multiple point mutations by programmable nucleases
references
Liu Z, Schiel JA, Maksimova E, Strezoska Ž, Zhao G, Anderson EM, Wu Y, Warren  J, Bartels A, van Brabant Smith A, Lowe CE, Forbes KP. ErCas12a CRISPR-MAD7 for Model Generation in Human Cells, Mice, and Rats. CRISPR J. 2020 Apr;3(2):97-108.  doi: 10.1089/crispr.2019.0068. PubMed PMID: 32315227.
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