CRSIPRC/ as9 Gene Edtiing · CRSIPRC/ as9 Gene Edtiing Fragment Analyzer ™ Automated CE System Mutation analysis by capillary electrophoresis provides significant benefits over

Identify single-cell mutationsand determine mutation frequency.

CRISPR/Cas9 Gene EditingFragment Analyzer™ Automated CE System

Mutationanalysisbycapillaryelectrophoresisprovidessignificantbenefitsoverslabgelmethods.TheFragment Analyzer detects mutations generated from Non Homologous End Joining (NHEJ) and HomologyDirectedRepair(HDR)modificationstotargetedgenesequences.Asamultichannelcapillary electrophoresis instrument that uses performance tested non-denaturing Gel Kits coupled with a sensitive intercalating dye, the Fragment Analyzerautomatesworkflow,reduceshandsontime and decreases time to results while providing high detection sensitivity and separation resolution. PROSize® integrated analytical software package accelerates detection of mutation eventsbyefficientlydisplayingresults.

Forgeneeditingexperiments,theCRISPR-specificGelKitwillshowthemutationfrequencyofapooledcell population, determine the presence/absence of a mutation from a single cell compared to its wild type complement, and under certain circumstances, assist in calling the zygosity of alleles. Additionally, researcherscandeterminethequalityandquantityofisolatedgenomicDNApriortoamplification.

They can measure amplicon post-PCR to assist in combining equimolar amounts of pre-heteroduplexed wild type and experimental amplicon.

The Fragment Analyzer can be used in multiple ways related to CRISPR/Cas9 mutagenesis including single guide RNA (sgRNA) forcleavageefficiency,analyzingmutationeventsincelllinesorpools of individual cells, plant cells and microbes. Furthermore, time course studies or changes to cell lines during cell selective passage can be monitored.

ADVANCED ANALYTICAL > WWW.AATI-US.COM FRAGMENT ANALYZER™ Automated CE System

Instrument Capabilities


FRAGMENT ANALYZER WORKFLOW BENEFITS

Fragment Analyzer™ Automated CE System

Eliminate Slab Gel Usage

With its ease of use, ability to hold multiple 96 well sample plates, fast electrophoresis times, sensitivity and versatility, the Fragment Analyzer is the ideal instrument to analyze CRISPR/Cas9 mutated cells and cell lines.

Ease of Use

Simply “load and go”. With a long reagent shelf-life and room temperature stable reagents, prepared gel can be held worry-free on the instrument and pumped into the capillaries as needed. The long lasting capillary array does NOT require daily handling or maintenance and can be used for thousands of injections. Variable capillary array length (22cm, 33cm and 55cm) and number of capillaries (12, 48 or 96) provide the ultimateflexibilityinexperimentdesign.

Sample LoadingHolding up to 3 x 96 well plates, sample plates can be queued to run while new plates are loaded into the instrument as soon as samples have been injected. And with a single addition of a diluent post enzyme cleavage of the heteroduplexed amplicon, sample processing requires minimal effort and can be automated if desired. Duetotheminimumuptakeofsample,thesamesamplecanbeinjectedmultipletimesifwarrantedforconfirmationor reproducibility studies.

Time to Results

Withanautomatedworkflow,loadedsampleswillrununattendeduntilcomplete.Availableelectrophoresismethodsare designed for optimum resolution of fragments with some runs as short as 20 minutes. Electrophoresis voltage can be adjusted by the user to decrease total electrophoresis time as conditions and resolution requirements demand.

Sensitivity

Cleaved fragments can be detected down to 5pg/µL under the right experimental conditions. High sensitivity allows forthepotentialtoreducethenumberofamplificationcyclesand/orreducetheamountofcleavageenzymeused.

Versatility

WithmanyGelKitstochoosefrom,userscanqualifyorquantifyalltypesofDNAfragments.Specifically,GelKitsareavailabletoquantifyandqualifygenomicDNApriortoamplification.Postamplification,ampliconcanbesizedandthe concentration determined for correct pooling prior to heteroduplex formation and enzyme cleavage.


DeterminingwhichcellshavebeenmodifiedbytheCRISPR/Cas9machineryisacriticalfirststeppostmutagenesis.Slabgelbased methods, which can lack the sensitivity and resolution needed to see all mutation types as well as automation for large scale screening and analysis, also typically lack an integrated software analysis package.

PROSize®, the analytical software package within the Fragment Analyzer system seamlessly calculates fragment sizes, fragment concentration and fragment molarity. An additional software plugin specific for CRISPR/Cas9 mutation analysis shows the mutation frequency of pooled samples and zygosity of individual samples (diploid analysis).

DATA ANALYSIS SOFTWARE

Mutation Frequency Calculator

Users can input the expected uncut amplicon size and endonuclease cleaved fragment sizes either manually for each capillary or quickly through a CSV upload. The expected fragments are automatically compared to the observed fragment sizes and thencolorcodedforeasyidentification.Thepercentcleavageiscalculatedfromthemolarityofidentifiedfragmentsbasedonthe following formulation:

% cleavage =

CSV Upload of expected uncut and cut fragment sizes. The % error can be adjusted as needed to aid in fragment identification.

Average Molarity (fragment #1 and fragment #2)

Average Molarity (fragment #1 and fragment #2) + Molarity fragment uncut

DATA OUTPUT



Result data can be quickly exported into either PDF or CSV format. The CSV output, as shown below, provides immediate access to the pertinent information regarding the samples and can be used for further analysis when necessary. The PDF output provides a page by page report for each sample. All or some of the sample data can be exported in either report.

Data output showing the input expected sizes and the observed sizes. Full length and cut fragments are color coded for easy identification. The % cleavage (highlighted in yellow) is shown for each sample.

WellA1A2A3A4A5A6A7A8A9

A10A11A12

Name420054200642007420084200942010420114201242013420144201542016

(bp)125012501250125012501250125012501250125012501250

(bp)230230230

000

349350350437440441

(bp)100010001000

000

842837844715708712

(bp)127812881278125912781278127812781269124012591249

(bp)230230231

000

349350351437438437

(bp)994994994

000

842845845715719719

nmole/L27.6127.6231.9

64.7852.4650.5842.8450.4653.1417.0714.5617.89

nmole/L27.8525.2132.45

000

7.9510.5111.1350.3

41.1244.24

nmole/L24.7323.4729.21

000

8.5710.6210.8539.1533.3435.96

% Cleavage48.7846.8549.15NaNNaNNaN

16.1717.3117.1472.3871.8969.15

Estimated FullLength

EstimatedFragment 1

A B C D E F G H I J K L

123456789

10111213

Sample

EstimatedFragment 2

Measured Full Length

Measured Fragment 1

Measured Fragment 2

Full Length

Fragment 1

Fragment 2

CSV output of data. Expected and measured fragment size is shown as well as the molarity of the fragments. The % cleavage (highlighted in yellow) is calculated as described above.

CRISPR / MUTATION DISCOVERY KITS


The Gel Kit listed below has been optimized for detection of point mutations and other small mutations post heteroduplexed amplicon cleavage. The Gel Kit is available in a 1,000 sample kit size.

CRISPR Discovery Gel Kit, 1 – 6,000 bp, DNF-910-1000CP

a. Markers at 1bp and 6,000 bp; the fast running lower marker eliminates problems with primer dimers b. Input concentration range 5 ng/µL – 100 ng/µL c. Gel formulation suited for a wide range of fragments

OTHER AVAILABLE GEL KITS

Genomic DNA Analysis

Standard Sensitivity Genomic DNA Analysis Kit, DNF-487

a. Sizing range: 50 bp – 20,000 bp b. Input concentration range 5 ng/µL – 200 ng/µL

High Sensitivity Genomic DNA Analysis Kit, DNF-488

a. Sizing range: 50 bp – 40,000 bp b. Input concentration range 50 pg/µL – 5000 pg/µL

DNA / NGS Fragment Analysis

Standard Sensitivity Genomic NGS Fragment Analysis Kit, DNF-473

a. Sizing range: 25 bp – 5,000 bp b. Input concentration range 5 ng/µL – 100 ng/µL

High Sensitivity Genomic NGS Fragment Analysis Kit, DNF-474

a. Sizing range: 25 bp – 5,000 bp b. Input concentration range 50 pg/µL – 5000 pg/µL


Maximum Sample Throughput: Array dependent, either 12, 48 or 96 samples

Maximum Unattended Sample Capacity: Up to 288 samples

Minimum Sample Volume: 20 µL of liquid for injection; as little as 1 µL of actual sample required

Qualitative Kits: 35-500 bp; 35-1,500 bp; 35-5,000 bp; 75-15,000 bp; 75-20,000 bp

Quantitative Kits: NGS Fragment Analysis, Genomic DNA, Total RNA, mRNA, microRNA and TILLiNG

Resolution: Gel dependent; to as low as 2 bp

Sizing Accuracy: Typically 5% or better

Detection Sensitivity: As low as 5 pg/µL for fragments and 50 pg/µL for a smear

Light Source: 700 mA, 10 W LED, 470 nm excitation wavelength

Detector: High sensitivity CCD; 500-600 nm emission wavelength

Software: Fragment Analyzer instrument control, PROSize® 2.0 data analysis

Data Export Format: CSV,PDF,flexiblenumericalorbinaryoutputoptions

Environmental Conditions: Indoor use, normal laboratory environment 20-23°C

Relative Humidity Range: < 80% (non-condensing)

Electrical: 100-200VAC;50-60Hz;15A(alternateconfigurationsavailable)

Instrument Dimensions: Fullyconfigured61cmHx38cmWx51cmD(24x14x20in)

Instrument Weight: 38.5 Kg (85 lbs)

Automating genomic discovery

Advanced Analytical Technologies, Inc.

2450 SE Oak Tree Ct.Ankeny, IA 50021 USAPhone: +1-515-964-8500Fax: +1-515-964-7377E-mail: [email protected]

Advanced Analytical Technologies, GmbH.

Im Neuenheimer Feld 583D-69120 HeidelbergPhone: ++49 6221 868058-20Fax: ++49 6221 868058-99E-mail: [email protected]

Copyright © 2015 Advanced Analytical Technologies, Inc. All Rights Reserved. Reproduction, adaptation or translation without prior written permission is prohibited, except as allowed under copyright laws. Fragment Analyzer™ Automated CE System is a trademark of Advanced Analytical Technologies, Inc. PROSize® is a registered trademark of Advanced Analytical Technologies, Inc.



NUCLEIC ACIDS DETECTED BY THE FRAGMENT ANALYZER

PCR amplicons, restriction enzyme digests, SSR/microsatellite fragments, NGS library preparations from all platforms, total RNA, messenger RNA (mRNA), microRNA, small RNAs, high molecular weight DNA, genomic DNA, plasmids (supercoiled & linear), point mutations and indels (TILLiNG)

SPECIFICATIONS

Identify single-cell mutationsand determine mutation frequency.

Documents

CRSIPRC/ as9 Gene Edtiing · CRSIPRC/ as9 Gene Edtiing Fragment Analyzer ™ Automated CE System Mutation analysis by capillary electrophoresis provides significant benefits over