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General Questions About CRISPR

Carefully designed crRNA target oligos and avoiding homology with other regions in the genome are critical for minimizing off-target effects.

Unfortunately, PAM is a necessary requirement for CRISPR gene editing. However, in its absence, you could use our Invitrogen™ GeneArt™ Precision TAL effector-based nuclease.

There are several ways to increase efficiency, for instance, adding antibiotic selection and/or FAC sorting to enrich for the transfected cells will both help.

Invitrogen™ GeneArt™ CRISPR Nuclease Vector Kit

Please see the following recommendations: 

  • Use high-quality, purified plasmid DNA for sequencing. We recommend preparing DNA using the Invitrogen™ PureLink™ HQ Mini Plasmid Purification Kit. Add DMSO to the sequencing reaction to a final concentration of 5%.
  • Increase the amount of template used in the sequencing reaction (up to twice the normal concentration).
  • Use a 7:1 molar ratio of dITP:dGTP in your sequencing reaction.

Reason

Solution

Single-stranded (ss) oligonucleotides designed incorrectly

Make sure that each ss oligonucleotide contains the 5 nucleotides on the 3’ end required for cloning into the GeneArt CRISPR Nuclease Vector:

Top strand includes GTTTT on the 3’ end

Bottom strand includes CGGTG on the 3’ end

Double-stranded (ds) oligonucleotides were degraded

Store the 5 nM ds oligonucleotide stock in 1X Oligonucleotide Annealing Buffer

Avoid repeated freeze/thaw cycles; aliquot the 5 nM ds oligonucleotide stock and store at –20°C

Oligonucleotide annealing reaction inefficient

Ensure that the annealing reaction was performed as directed

If ambient temperature is >25°C, incubate the annealing reaction in a 25°C incubator.

Invitrogen™ GeneArt™ Genomic Cleavage Selection Kit

Please see the possible reasons and suggestions below:

Reason

Solution

Single-stranded (ss) oligonucleotide designed incorrectly

Make sure that each (ss) oligonucleotide contains the 4 nucleotides on the 5’ end required for cloning into the GeneArt Genomic Cleavage Selection vector:

Top strand includes AATT on the 5’ end

Bottom strand includes CTAG on the 5’ end

Double-stranded (ds) oligonucleotides were degraded

Store the 50 nM ds oligonucleotide stock in 1X ligation buffer at –20°C

Avoid repeat freeze/thaw cycles, aliquot 50 nM ds oligonucleotides stock and store at –20°C

Oligonucleotide annealing reaction was inefficient

Ensure that the annealing reaction was performed as directed

If ambient temperature is >25°C, incubate the annealing reaction in a 25°C incubator

This can occur if:

  • Oligos are designed incorrectly; make sure to include specific sequences for TALEN/CRISPR target
  • Transfection efficiency is too low; optimize conditions or use Lipofectamine 3000 or 2000 reagent for best results
  • Cell line–dependent; OFP does not express well in certain cell lines; we recommend using 293FT cells as a test for cleavage activity

Too much OFP background can occur if there is plasmid contamination, and/or can be cell line– and target-dependent. Make sure to pick single clones when culturing the cleavage selection plasmid, or try to reduce the amount of vector that is included in the transfection.

Results are locus-dependent. We recommend using the GeneArt Genomic Cleavage Detection Kit (Cat. No. A24372) to verify cleavage on the endogenous genomic locus.

Invitrogen™ GeneArt™ Genomic Cleavage Detection Kit

Please see the table below for common problems, possible causes, and recommendations:

DNA band appearance

Possible cause

Recommendation

Smear

Lysate is too concentrated

Dilute lysate 2- to 4-fold and repeat the PCR reaction.

Too faint

Lysate is too dilute

Double the amount of lysate in the PCR reaction. Do not use more than 4 µL of lysate in the PCR reaction. Lysate can inhibit the PCR reaction.

Disparity in band intensity between amplicons

Lysate concentrations vary between samples

Purify the PCR products with the Invitrogen™ PureLink™ PCR Purification Kit. For best results in comparing samples, purify PCR and use the same quantity of DNA in each cleavage assay. 50 ng to 100 ng of DNA is enough for each reaction.

No PCR product

Poor PCR primer design or GC-rich region

Redesign primers that are 18–22 bp, have 45–60% GC content, and a 52–58°C Tm range. For GC-rich regions, add 1–10 µL of 360 GC Enhancer in a 50 µL reaction and repeat the PCR amplification.

Please see the table below for common problems, possible causes, and recommendations:

Problem

Possible cause

Recommendation

No cleavage band visible

Nucleases unable to access target sequence or unable to cleave at target site

Transfection efficiency too low

Design new targeting strategy at nearby sequences.

Genomic modification too low

Optimize transfection protocol.

Omitted denaturing and reannealing step

Use Kit Control Template and Primers to verify the kit components and protocol.

Difficulty in analyzing gel data

Background interferes with the measurements of cleaved bands

Redesign the PCR primers to product a distinct cleaved banding pattern.

Nonspecific cleavage bands

Intricate mutations at the target site.

Digestion incubation too long.

Too much Detection Enzyme added.

Nonspecific cleavage by Detection Enzyme for certain target loci.

Redesign PCR primers to amplify target sequence. 

Use lysate from mock transfected cells or cells transfected with irrelevant plasmids as negative control to distinguish background from specific cleavage.

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