Home Resources Selection Charts CRISPR Validation Tools Selection Chart

CRISPR Validation Tools Selection Chart

NEB offers reagents and kits for progressive levels of analysis to optimize CRISPR-based genome editing experiment designs. Mismatch assays are useful for initial, rapid screening of gene editing for targeting efficiency. Sanger sequencing with TIDE (Tracking of Indels by Decompositions) and ICE (Inference of CRISPR Edits) analyses estimate editing efficiency and the proportion of specific indels. Next-generation sequencing of amplicons containing target regions permits precise on and off-target analyses, including rare mutations. Whole-genome next-generation sequencing is the gold standard to validate genome editing with accurate genotyping.

 

Authenticase®

T7 Endonuclease I

EnGen® Mutation Detection Kit

Q5® High-Fidelity 2x Master Mix

or

OneTaq® 2X Master Mix

NEBNext® Ultra™ II DNA Library Prep Kit for Illumina®

NEBNext Ultra™ II DNA PCR-free Library Prep Kit for Illumina

NEBNext Ultra™ II FS DNA PCR-free Library Prep Kit for Illumina

APPLICATIONS TO VALIDATE CRISPR-BASED GENOME EDITING

Mismatch detection assay to determine the targeting efficiency with single-base pair mismatches

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Mismatch detection assay to estimate targeting efficiency with >2nt mismatches

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Sanger sequencing TIDE, or ICE analysis using clean, high-quality PCR amplicons to estimate editing efficiency and proportion of specific indels

      

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Amplicon seq to detect editing frequency, low-frequency edits, and characterize specific indels

        

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Whole genome seq to detect on and off-targeting with indel profiling from sheared DNA

          

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Whole genome seq to detect on and off-targeting with indel profiling from unsheared DNA

            

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Key

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Optimal, recommended for this application

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Works well for the selected application