Validation of CRISPR-based Gene Editing

Research using CRISPR/Cas nuclease-based genome editing, such as modeling diseases or identifying potential therapeutics, relies on methods to validate targeting efficiency in vitro and in vivo. NEB offers reagents for in vitro enzymatic indel detection assays and next-generation sequencing of edited nucleic acids.

Cas9 digest and enzymatic mismatch assays

Cas9 digests and enzymatic mismatch cleavage assays are simple and sensitive methods for validating editing efficiency. DNA isolated from editing experiments typically contains a mixture of edited and unedited sequences. PCR amplification followed by re-annealing can produce heteroduplex DNA containing mixtures of edited and unedited strands. Enzymes such as T7 Endonuclease I and Authenticase^® can cleave these mismatched indel sequences and provide an estimate of editing efficiency. The Cas9 nuclease itself can be used to estimate editing efficiency as it can digest fully matched unedited sequences, but not most edited sequences.

• Authenticase (NEB #M0689) is a mixture of structure-specific nucleases that outperforms T7 Endo I in detecting CRISPR-induced on-target mutations across a broad range of mutations/wild-types.
• EnGen^® Mutation Detection Kit (NEB #E3321) provides optimized reagents for conventional T7 Endonuclease-based mutation detection of genome editing events.
• Cas9 Nuclease, S.pyogenes (NEB #M0386) detects indels with the advantage of assessing locus modification at genome editing targeting efficiency above 50%.
  
  
  

Sequencing to detect CRISPR on- and off-targeting

NEB carries a broad range of next-generation sequencing kits to analyze gene editing events with automation compatibility. Targeted or whole genome CRISPR validation approaches permit accurate genotyping, and large-scale CRISPR screening of genomes, pools and clone analyses. NEB recommends PCR-free NGS library preparation to produce the highest quality, high-yield libraries of whole genomes without PCR bias.

• NEBNext^® Ultra™ II DNA Library Prep Kit for Illumina^® (NEB #E7645) or NEBNext Ultra™ II DNA Library Prep with Sample Purification Beads (NEB #E7103) are recommended for amplicon sequencing.
• NEBNext Ultra II DNA PCR-free Library Prep Kit for Illumina (NEB #E7410) enables whole genome libraries with sheared DNA.
• NEBNext Ultra II FS DNA PCR-free Library Prep Kit for Illumina (NEB #E7430) enables whole genome libraries with unsheared DNA which fragments, end repairs, and dA-tails DNA, all in a single tube with no cleanups or transfers.