designed. This can be done through various online sgRNA
designing platforms. Once the sgRNA is designed or the
sequence is known, it can be either synthesized in the lab
using synthesis kits or ordered directly from companies that
can synthesize the required sgRNA.
2. There are a variety of Cas9 proteins commercially available. We
have only tested EnGen Cas9 NLS protein from New England
Biolabs, which works well in this approach with U2OS.EGFP
cells.
3. There is a range of commercially available delivery vehicles that
are primarily lipid and cationic polymer-based. We have tested a
few and found that lipofectamine CRISPRMAX works with
high efficiency in our approach with USOS.EGFP cells. Note
that this also depends on the types of cells being transfected as
this may work differently for primary cells or difficult-to-transfect cells.
4. The topic of genomic DNA purification is beyond the scope of
this chapter. Therefore, we only cover the methods until
extracting genomic DNA from the scaffold efficiently. Established protocol or commercial DNA purification kits can be
used to purify genomic DNA. We have tried Zymo DNA Clean
& Concentrator and it worked well.
5. The topic of genomic cleavage assay is beyond the scope of this
chapter. Established protocol or commercial genomic cleavage
detection kits can be used to detect genomic cleavage. We have
tried GeneArt Genomic Cleavage Detection Kit (Thermofisher) and it worked well. The primers here were designed
for EGFP and sgRNA presented in this protocol. For other
genes of interest, the primers shall be designed to flank the
cleavage site that the Cas9:sgRNA complexes bind to.
6. The topic of DNA gel electrophoresis is beyond the scope of
this chapter. We have used 2% agarose gel with TAE buffer.
Alternatively, TBE buffer can be used. Other established protocols or commercial systems such as E-Gel can also be used to
separate and detect the DNA fragments.
7. Ethidium bromide was used to visualize the DNA bands under
UV light. Ethidium bromide was added to the 2% agarose gel
to a final concentration of 0.5 μg/mL. Caution should be
exercised as ethidium bromide is a known mutagen. Alternatively, other dyes such as SYBR Green can be used.
8. Here, we describe a method to fabricate electrospun fibers
scaffold on the coverslips to simplify observation and imaging.
A similar protocol may be applied to the other scaffold designs
but optimization will be needed.
Scaffold-Based Delivery of CRISPR/Cas9
189
designing platforms. Once the sgRNA is designed or the
sequence is known, it can be either synthesized in the lab
using synthesis kits or ordered directly from companies that
can synthesize the required sgRNA.
2. There are a variety of Cas9 proteins commercially available. We
have only tested EnGen Cas9 NLS protein from New England
Biolabs, which works well in this approach with U2OS.EGFP
cells.
3. There is a range of commercially available delivery vehicles that
are primarily lipid and cationic polymer-based. We have tested a
few and found that lipofectamine CRISPRMAX works with
high efficiency in our approach with USOS.EGFP cells. Note
that this also depends on the types of cells being transfected as
this may work differently for primary cells or difficult-to-transfect cells.
4. The topic of genomic DNA purification is beyond the scope of
this chapter. Therefore, we only cover the methods until
extracting genomic DNA from the scaffold efficiently. Established protocol or commercial DNA purification kits can be
used to purify genomic DNA. We have tried Zymo DNA Clean
& Concentrator and it worked well.
5. The topic of genomic cleavage assay is beyond the scope of this
chapter. Established protocol or commercial genomic cleavage
detection kits can be used to detect genomic cleavage. We have
tried GeneArt Genomic Cleavage Detection Kit (Thermofisher) and it worked well. The primers here were designed
for EGFP and sgRNA presented in this protocol. For other
genes of interest, the primers shall be designed to flank the
cleavage site that the Cas9:sgRNA complexes bind to.
6. The topic of DNA gel electrophoresis is beyond the scope of
this chapter. We have used 2% agarose gel with TAE buffer.
Alternatively, TBE buffer can be used. Other established protocols or commercial systems such as E-Gel can also be used to
separate and detect the DNA fragments.
7. Ethidium bromide was used to visualize the DNA bands under
UV light. Ethidium bromide was added to the 2% agarose gel
to a final concentration of 0.5 μg/mL. Caution should be
exercised as ethidium bromide is a known mutagen. Alternatively, other dyes such as SYBR Green can be used.
8. Here, we describe a method to fabricate electrospun fibers
scaffold on the coverslips to simplify observation and imaging.
A similar protocol may be applied to the other scaffold designs
but optimization will be needed.
Scaffold-Based Delivery of CRISPR/Cas9
189