Mutation Generation & Detection

Overview

To Set Up a User Account:

The Mutation Generation and Detection Core (MGD) Facility specializes in providing custom TALEN and Crispr-Cas9 DNA nucleases to induce targeted mutations in a genomic region of your interest. TALEN and Crispr-Cas9 DNA nucleases are a cutting edge technology for performing reverse genetic studies in multiple model systems, including, but not limited to Zebrafish, Drosophila, C. elegans, Mouse, and mammalian tissue culture.

The MGD Core also offers services to identify induced mutations using High Resolution Melt Analysis (HRMA). Our support includes hardware, reagents, and expert advice for optimizing and performing HRMA for your gene of interest. The MGD Core also offers Services for preforming custom HRMA analysis for mutation detection and genotyping. Please contact the Director of the MGD Core for details on this service.

Services

Mutation Generation and Detection Core Services and Rates

(Bulk pricing does not apply to internal or external CIHD members)

CRISPR-Cas9 Services

CRISPR-Cas9 is a customizable system for assembling DNA nuclease that are RNA:Protein hybrids. CRISPR-Cas9 is analogous to the TALEN DNA nuclease system and can be used to induce mutations at targeted regions in a model organism’s genome. Targeting of a gene’s coding region using CRISPR-Cas9 leads to null mutations and loss of that genes function and allows investigation of that genes role in the normal biology of a model organism. In addition to this basic “knockout” function CRISPR-Cas9 can be used to make small and large deletions of genomic regions and to increase the efficiency of incorporation of exogenous donor DNA (homology driven recombination). Together these different uses of CRISPR-Cas9 can be utilized to knockout a gene, tag a gene, delete genomic regions of non-coding RNA, or delete regulatory regions of genes such as promoters and enhancers.

Our Core offers a full line of CRISPR-Cas9 services and reagents to researchers, as well as help designing and implementing projects. All projects start with the Core designing optimal CRISPR-Cas9 target sites for your model organism and delivery of sequence confirmed plasmids. See the list below for a range of CRISPR-Cas9 services and pricing. Also provided are links to order forms if you are interested in services. For any questions and to submit order forms please contact the MGD Core at mutrus@genetics.utah.edu.

Utah Client CRISPR Request Form

Outside Utah Client CRISPR Request Form

CRISPR Order Directions

Pricing for our CRISPR Services

Custom CRISPR Services	U of U Researchers	External U of U Non-Profit Researchers	CIHD Members	External CIHD Members
1x CRISPR design & construction (includes custom sgRNA and Cas9 plasmids)	$250	$375	$73.83	$110.75
2x CRISPR design & construction (includes 2x custom sgRNA and Cas9 plasmids)	$480	$720	$147.66	$211.50

*Bulk Services are not available to CIHD members

Bulk Custom CRISPR Services	U of U Researchers	External U of U Non-Profit Researchers
3-4 CRISPR design & construction	$231	$346
5-6 CRISPR design & construction	$212	$318
7-8 CRISPR design & construction	$194	$290
9-10 CRISPR design & construction	$175	$262
11+ CRISPR design & construction	$160	$240

TALEN Services

TALEN-300x100
TALENs are customized DNA nuclease proteins used to induce mutations at targeted regions in a model organism’s genome. Targeting of a gene’s coding region using TALEN proteins leads to null mutations and loss of that genes function and allows investigation of that genes role in the normal biology of a model organism. In addition to this basic “knockout” function TALENs can be used to make small and large deletions of genomic regions and to increase the efficiency of incorporation of exogenous donor DNA (homology driven recombination). Together these different uses of TALENs can be utilized to knockout a gene, tag a gene, delete genomic regions of non-coding RNA, or delete regulatory regions of genes such as promoters and enhancers.

Our Core offers a full line of TALEN services to researchers, as well as help designing and implementing projects. All projects start with the Core designing optimal TALEN target sites for your model organism and delivery of sequence confirmed plasmids expressing TALENs. See the links below for the range of TALEN services provided, directions for ordering, order forms, and pricing. For any questions please contact the Core at mutrus@genetics.utah.edu.

Pricing for Our TALEN Services

Custom TALEN Protein Services	U of U researchers	Outside U of U Non-Profit Researchers	CIHD Members	External CIHD Members
TALEN plasmid pair design & construction	$750	$1,125	$218.23	$327.35
2x TALEN plasmid pair design & construction	$1,400	$2,100	$436.46	$654.70

*Bulk Services are not available to CIHD members

Bulk TALEN Ordering Rates	U of U Researchers	Outside U of U Non-Profit Researchers
5 or more TALEN pair design & construction	$675	$1,000
10 or more TALEN pair design & construction	$650	$950
15 or more TALEN pair design & construction	$625	$900

Utah Client TALEN Request Form

Outside Utah Client TALEN Request Form

TALEN Order Directions

High Resolution Melt Analysis (HMRA)

High Resolution Melt Analysis is powerful assay for the detection of sequence alterations that is a closed-tube, high-throughput, and highly sensitive system that can detect a wide range of sequence changes including: insertions, deletion, and SNPs. HRMA can be utilized to identify TALEN and Crispr-Cas induced mutations, for routine genotyping of model organism, or for SNP screening.

The Core provides all necessary knowledge, reagents, and equipment for designing, optimizing, running, and analyzing HRMA. The Core has a bank of Eppendorf MasterCycler Pro S PCR machine open for use and an Idaho Technology LightScanner High Resolution Melt Analysis Machine available for use. The Core also provides all the specialized reagents and materials necessary for preforming HRMA at a reduced cost to researchers (see link below). Contact the Core with any questions atmutrus@genetics.utah.edu
HRMA consists of two simple steps:

1) PCR of a heterogenous genomic sample that results in the formation of Heteroduplexes and Homoduplexes (~70 minutes)

2) Melt Curve Analysis of those duplex species over a temperature gradient (~4-7 minutes) to identify samples with induced mutations by deflected melt curves

HRMA has a lower detection level of 1.5% making identification of rare and mosaic induced mutations routine

Pricing for our HMRA Services

HRMA Services and Reagents	U of U Researchers & CIHD Members	External CIHD Members
Idaho Technology LightScanner Annual Access Fee	$100	$100
HRMA PCR plates (10 pack)	$42.60	$66.00
HRMCA PCR optical sealing films (10 pack)	$12.30	$19.50
Idaho Technology LightScanner MasterMix 100 rxns	$77	$115.50
Idaho Tecnology LightScanner MasterMix 500 rxns	$385	$577.50
Mineral Oil (500mL bottle)	$37.03	$55.55

Pricing and Priority

Referencing Us

The MGD Core exists to help researchers like you to further their science. If we could ask for a slight favor in return for that help we would greatly appreciate your help. The University of Utah determines the impact of individual Core’s on scientific research by how many papers mention the Core specifically in the Acknowledgements section.

If you have used any of the services or resources that the MGD Core provides (TALENs, Crispr, HMRA, or other) please add the following sentences to the Acknowledgements section of your paper:

Thank you to the University of Utah Mutation Generation and Detection Core.

Reagents Available

T7 sgRNA construct

Perfect for making CRISRP sgRNA RNA for injection into embryos, transfection into cells, or for use in vitro. Can be paired with commercially available Cas9 protein to produce a functional CRSIRP RNP (ribonucleoprotein) complex or with Cas9 mRNA. This construct can be linearized to produce dsDNA template for any RNA in vitro transcription kit.

t7crisprreagent

The following link is to a protocol on how to linearize the T7 Construct, how to make RNA in vitro, and how to purifiy that RNA. These protocols are only general guides.

Working With T7 CRISPR Construct

Transfection CRISPR Constructs

Perfect for transfection of the CRISPR-Cas9 system into cell lines using lipid reagents or electroporation. This Construct expresses both components of the CRISPR-Cas9 system, sgRNA and Cas9 protein, from one construct and comes in three different backbones:

Puromycin construct: contains a puromycin antibiotics selection gene attached to the Cas9 protein coding sequence via a 2A self-cleaving peptide sequence

Blasticidin Transfection construct: contains a blasticidin antibiotics selection gene attached to the Cas9 protein coding sequence via a 2A self-cleaving peptide sequence
GFP Transfection construct: contains a GFP fluorescent reporter gene attached to the Cas9 protein coding sequence via a 2A self-cleaving peptide sequence
mCherry Transfection construct: contains a mCherry fluorescent reporter gene attached to the Cas9 protein coding sequence via a 2A self-cleaving peptide sequence
Standard Transfection construct: does not contain either a selection or a reporter gene

transfectioncrisprreagent

Lentiviral CRISPR Constructs

Perfect for making virus particles for the transduction/infection of the CRISPR-Cas9 system into cell lines or primary cells. Transduction is used to achieve very high rates of expression per population, to infect hard to transfect cell lines, or to infect primary cells including primary T cells, hematopoietic stem cells, stem cells, iPSC, and others

These Constructs can be used in two ways

Can be transfected into cells using standard methods to validate that the CRISPR-Cas9 components are functional
After or before #1 these Constructs can be used to make lentiviral particles for transduction/infection of cells with the CRISPR-Cas9 components

These constructs come in four different backbones

Puromycin Lentiviral Construct: contains a puromycin antibiotics selection gene attached to the Cas9 protein coding sequence via a 2A self-cleaving peptide sequence

Blasticidin Lentiviral Construct: contains a blasticidin antibiotics selection gene attached to the Cas9 protein coding sequence via a 2A self-cleaving peptide sequence

GFP Lentiviral Construct: contains a GFP fluorescent reporter gene attached to the Cas9 protein coding sequence via a 2A self-cleaving peptide sequence

mCherry Lentiviral Construct: comtains a mCherry fluorescent reporter gene attached to the Cas9 protein coding sequence via a 2A self-cleaving peptide sequence

lentiviral-crispr-construct-cherry

Viral accessory and envelope constructs available upon request.

Model Organisms

Engineered DNA Nucleases systems, TALEN and CRISPR-Cas9 (ZFNs before them), have allowed certain specific experimental questions to be answered using Model Organisms or Vectors where this was not possible before. The basic thing to remember is if there is an embryo or cell that you can inject with mRNA/DNA or transfect plasmids into expressing an Engineered DNA nuclease then you can do any of the techniques mention on our website: targeted mutagenesis, deletion, tagging, etc. Engineered DNA nuclease have been successfully used to alter a genomic locus in 13 different invertebrate models organisms, 13 different vertebrate models organisms, and 12 different plant species Organisms List. Below is a listing of the Model Organisms for which our Core has produced successful DNA nucleases along links to papers for examples.

For our whole list of MGD Core publications, please click here.

Zebrafish

PLoS Genetics Zebrafish Paper

Drosophila

G3 Drosophila Paper

Aedes aegypti

PLoS One Aedes aegypti Paper

Mouse and Human Cells

PLoS One Human Cells

Contact

Hours of Operation

Monday-Friday 9am-5pm

Location

Room 7470 Eccles Institute of Human Genetics, Bldg. 533
15 North 2030 East
Salt Lake City, UT 84112

Staff

Crystal Davey Ph.D., Director
mutrus@genetics.utah.edu
801-585-0662

Oversight Committee

David J. Grunwald, Department of Human Genetics (Senior Faculty Advisor)
Dana Carroll, Department of Biochemistry
Ryan M. O’Connell, Department of Pathology
Charles L. Murtaugh, Department of Human Genetics

News

Friday September 9

Special Seminar Announcement

9-10 AM, 2nd Floor Conference Room, Dumke Bldg

“Using Crispr-Cas9 Engineered Nucleases in rodent model organisms.”

Dr. Timothy J. Dahlem

Director of the Mutation Generation and Detection Core

The MGD Core will be presenting a seminar at the weekly Renal Division Research Conference on applying the CRISPR-Cas9 gene editing system to rodent model organisms. This seminar will include a brief history and description of the CRISPR-Cas9 system, how it is used in rodent model organisms, and real life assessment of the efficiency and usefulness of the system to achieve varying experimental goals.

Disclaimer

The Mutation Generation and Detection Core guarantees delivery of sequence confirmed plasmids to express TALEN or CRISPR-Cas9 DNA nucleases or delivery of plasmids for the expression of TALE activator or repression proteins. However, we cannot and do not guarantee that these delivered plasmids will lead to the recovery of somatic or germ line mutations at your targeted genomic locus or will cause activation/repression of your gene of interest.

The MGD Core will do everything reasonably possible to assist you throughout the process. Our main goal has always been to support scientist and help further their research. If the plasmids provided by the MGD Core do not produce the expected affect please contact the MGD Core as we have protocols in place to deal with these specific situations.

As of May 2016 ~80% of the TALEN and Crispr constructs the MGD Core has made and delivered to researchers have confirm activity at the targeted locus.

4D Nucleofector CRISPR-RNP

Lonza’s 4D Nucleofector™ Technology is an improved electroporation technology that can help researchers achieve high transfection efficiencies in standard cell lines, primary cells, stem cells, and hard to transfect cell lines. With the 4D Nucleofector high efficiencies can be reached using much lower substrate amounts and with moderate impact on viability. The comprehensive way in which 4D Nucleofector™ Programs and cell type-specific solutions are developed enables nucleic acid and protein substrate delivery not only to the cytoplasm, but also through the nuclear membrane and into the nucleus. This allows for high efficiencies up to 99% and makes the transfection success independent from cell proliferation.

MGD Core Lonza 4D Nucleofector Units

The MGD Core has acquired a complete Lonza 4D Nucleofector System that any and all researchers can use. This complete System is made up of four unique functional parts:

Core Unit – The main control center for the 4D-Nucleofector System that controls the function of all other units. It has a 5.7’’ touch screen to operate all units and is loaded with intuitive operation software for designing and saving individual experimental setups.

X Unit – This base unit allows Nucleofection of cells in suspension in 20ul Nucleocuvette 16-well strips or in single-use 100ul Nucleocuvettes. Each well in a 16-well Nucleocevette strip is electroporated independently allowing for different conditions to be tested and re-use of the strips if wells are not used. This unit is perfect for testing individual conditions on cells and for small-scale experiments.

Y Unit – This unit allows Nucleofection of cells while still adherent to 24 well culture plates. This unit is perfect for working with adherent cells, such as neurons derived from stem cells, which are not transfectable in suspension. Transfection of adherent cells using the Y unit may lead to more physiological response in cells.

96-well Shuttle – Controlled by the Core unit the 96-well Shuttle is an add-on unit that allows for convenient optimization of conditions or large-scale screens to be preformed. Each individual well is processed independently allowing 96 different experimental conditions to be tested at one time.

Location and usage

The full Lonza 4D Nucleofector System is housed in Room 7470 of the Eccles Institute of Human Genetics, along with a cell culture hood for researchers to work with their cells in and a 37C incubator to store their cell.

To reserve time to use the Lonza 4D Nucleofector System please use the following link to login to the HSC Core Research Facilities resource page. Select the Mutation Generation page option and then select the 4D Nucleofector page option. Here you can reserve time to use the System. Researchers will be charged a $5.00 fee for every 30 minutes block reserved. Individual experiments do not require more than one 30-minute block.

University of Utah Core Labs

Lonza optimized cell line protocol Databases

Lonza maintains two databases with protocols (including Nucleofector Solution types and program numbers) of optimized protocols for a wide range of cell lines. Basically Lonza has already done the optimization experiment and determined the best conditions to achieve the highest transfection efficiency with the least amount of cell death. These databases are a good starting place to determine the most optimal protocol for working with your specific cell line.

Lonza’s public optimized cell line protocol database can be found at the following link: http://bio.lonza.com/6.html

Lonza also maintains a database of user-optimized protocols that is not publicly available. Please contact either Dr. Gregory Alberts or Haylee Erickson at the following information for access. Dr. Alberts is an expert on the use of the 4D Nucleofector System and a great resource for the best transfection protocols to use with your specific cell line. The following is a link to a seminar given by Dr. Alberts on using the 4D Nucleofection system: Dr. Alberts 4D Nucleofection System

Gregory Alberts, Ph.D. gregory.alberts@lonza.com

Global Subject Matter Expert

Lonza Pharma Bioscience Solutions

Haylee Erickson haylee.erickson@lonza.com

Sales Specialist, Rocky Mnt/Pacific NW

Lonza Pharma Bioscience Solutions

4D Nucleofection of CRISPR RNP

Original CRISPR-Cas9 experiments were performed using DNA vectors, viral vectors or RNA transfection to produce the components of the system: Cas9 protein and single guide RNAs (sgRNAs). New advances have demonstrated that these component can be produced and combined in vitro to form a ribonucleoprotein complex or RNP that is functional in vitro and in vivo without the need for transcription or translation. This CRISPR RNP complex a can be delivered directly to cells and results in immediate, efficient, and specific target cleavage by the CRISPR RNP.

Several labs have shown that combining the CRISPR RNP approach with the extremely high transfection efficiency of the Lonza 4D Nucleofection System can result in mutation frequencies reaching 90% of targeted gene copies in several different cell types. CRISPR RNP delivery is applicable to a wide range of cell types, including established cell lines, primary cells, adherent cells such as primary neurons, iPCS, and stem cells. With these cell types using the CRISPR RNP approach can dramatically shortened the time it takes to create targeted variants of your gene of.

Please contact the MGD Core if you have any questions concerning this approach or would like to discuss the possibility of using CRISPR RNP in your research. Also, the following link is a generalized protocol detailing how to combine the CRISPR RNP approach with the Lonza 4D Nucleofection System.

General Nucleofection Protocol