Edit-R synthetic sgRNA negative controls

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ゲノム編集
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Edit-R synthetic sgRNA negative controls

Edit-R synthetic sgRNA negative controls

ヒトおよびマウスのゲノム中のどの遺伝子も標的としないように、バイオインフォマティクスに基づき設計されたnon-targetingコントロール

CRISPR-Cas9実験におけるベースライン細胞応答の決定に、non-targetingコントロール化学合成sgRNAの使用をお薦めします。

AAVS1 and Rosa26 cutting controls are designed to target regions of the genome that do not result in functional knockout of a protein or known phenotypic changes. These regions are sometimes known as safe harbor regions. These guides are validated for mismatch detection assays and are recommended for screening applications as an additional control that results in Cas9 cutting without a phenotypic readout.

Non-targeting controls are recommended as negative controls for experiments using synthetic sgRNA. All Edit-R non-targeting controls are designed to have a minimum of three mismatches or gaps to all potential PAM-adjacent targets in the human or mouse genomes. Changes in viability or gene expression levels in cells treated with these controls likely reflect a baseline cellular response that can be compared to the levels in cells treated with target-specific sgRNAs.

High percentage of gene editing using the T7E1 assay to determine indel formation of the Edit-R AAVS1 cutting controls

Jurkat cells (250,000/96w) were nucleofected with (SE buffer, program CL-120) 50 pmol of RNP (40 µM NLS-Cas9 + 80 µM sgRNA) in triplicate. U2OS cells hEF1α-Cas9 were transfected using 0.15 µL Dharmafect 4/96w + 50 µM sgRNA (incubated at room temperature for 20 minutes in MEM solution).

After 72 hours cells were harvested for direct lysis (+ Rnase + proteinase K). Primers per spacer region were used to amplify 450-800 bp genomic amplicon from the edited and unedited samples. A hot-start Phusion II touch-down PCR program was used to generate amplicons for each sample (Tm ~65 for each). PCRs were directly incubated with T7 endonuclease I and ran on a 2% agarose SyberSafe gel @ 80V for 90 minutes.

TIDE analysis confirms high efficiency of indel formation using Edit-R AAVS1 cutting control guides

Jurkat cells (250,000/96w) were nucleofected with 50 pmol of RNP (40 µM NLS-Cas9 + 80 µM sgRNA) in SE buffer and program CL-120, in triplicate. U2OS cells were transfected using 0.15 µL Dharmafect 4/96w + 50 µM sgRNA (incubated at room temperature for 20 minutes in MEM solution).

After 72 hours cells were harvested for direct lysis. Primers per spacer region were used to amplify 450-800 bp genomic amplicon from the edited and unedited samples. A hot-start Phusion II touch-down PCR program was used to generate amplicons for each sample. PCRs were sent for Sanger sequencing. Sequencing files were analyzed by tracking of indels by decomposition (TIDE).

High efficiency of indel generation using Edit-R Rosa26 cutting controls by T7EI

NIH/3T3 Cas9 stable cells were plated at 10,000 cells/well in a 96 well plate and transfected using 0.2 µL Dharmafect 1 with 25 nM sgRNA (incubated at room temperature for 20 minutes in MEM solution). Cells were harvested at 72 hours post-transfection for direct cell lysis. Primers per spacer region were used to amplify 450-800 bp genomic amplicon from the edited and unedited samples. A hot-start Phusion II touch-down PCR program was used to generate amplicons for each sample. PCR samples were directly incubated with T7 endonuclease I and ran on a 2% agarose SyberSafe gel @ 80V for 90 minutes.

High efficiency of indel generation using Edit-R Rosa26 cutting controls by TIDE

rosa26 cutting sgrna tide graph

NIH/3T3 Cas9 stable cells were plated at 10,000 cells/well in a 96 well plate and transfected using 0.2 µL Dharmafect 1 with 25 nM sgRNA (incubated at room temperature for 20 minutes in MEM solution). Cells were harvested at 72 hours post-transfection for direct cell lysis. Primers per spacer region were used to amplify 450-800 bp genomic amplicon from the edited and unedited samples. A hot-start Phusion II touch-down PCR program was used to generate amplicons for each sample. PCR samples were sent for Sanger sequencing. Sequencing files were analyzed by tracking of indels by decomposition.

Yes, these controls have the 2xMS modification pattern, as described in the Stabilizing modifications on Edit-R crRNA and tracrRNA blog article.

Our neutral cutting controls target an inactive region of the genome to provide a quantitative measure of cutting efficiency by mismatch detection assays, while maintaining a negative or negligible phenotype.

The neutral cutting controls target safe harbor genomic loci: human AAVS1 located on chromosome 19 and mouse Rosa26 on chromosome 6. Safe harbor loci are well-characterized, widely accepted, inactive genomic regions.

Our non-targeting control guide RNAs do not target any region in the genome. Non-targeting designs control for phenotypic changes that may be caused by the delivery of the CRISPR-Cas9 reagents in the absence of cutting. However, the non-targeting controls cannot indicate changes caused by Cas9 cutting because they do not result in cutting by the Cas9 enzyme.

Our positive controls target established housekeeping genes to offer insight into phenotypic changes that might be due to Cas9 cutting but are not specific to cutting within the gene of interest. However, there may be cell types or culture conditions under which cutting at the housekeeping gene locus could result in a phenotype, or experimental situations where disrupting a gene is not desired. To overcome this potentially confounding effect, the neutral cutting control can be included to provide more robust results and increased confidence in the phenotypes observed following editing of the target gene.

Neutral cutting controls can be helpful if there is concern that an observed phenotype is not gene-specific. In particular, neutral cutting controls are helpful as an additional control in screening applications and when a phenotype is observed with a positive control guide RNA. Additionally, this guide can be used in discovery settings where disruption of a gene could affect downstream phenotypes and results.