CRISPR Case Studies and FAQs
CRISPR Case Studies
- Case study: High Gene knock-in Efficiency and Reduced off Target Integration with single-stranded DNA HDR Template
- Case Study: Using CRISPR to Generate a KRAS Knock-Out Human Colon Cancer Cell Line
- Case Study: Using CRISPR to Generate a Knock-Out Cell Pool
- Case Study: Using CRISPR for E.coli Gene Editing
CRISPR stands for Clustered Regularly Interspaced Short Palindromic Repeats. CRISPR sequences were originally identified in the Escherichia coli (E. coli) genome, and were found to function as part of an RNA-based adaptive immune system to target and destroy genetic parasites at the DNA level. CRISPR-associated protein (Cas) is an endonuclease that cuts foreign DNA, allowing integration into the host genome. Cleavage only occurs when there is a protospacer adjacent motif (PAM) around the targeted sequence of the invading DNA, ensuring highly accurate targeting. Researchers studying CRISPR have adapted it for use as a tool for genetic modification of the target host genome. CRISPR/Cas9 has recently become a popular genome editing tool, due to its simplicity and versatility.
Of the other gene editing technologies available, CRISPR/Cas9 has stood out for its simplicity and efficacy. The CRISPR system requires only a few simple DNA constructs to encode the gRNA and Cas9, and if knock-in is being performed, the donor template for HR. As a result, CRISPR gene editing is an approachable technique for use in any lab regardless of molecular biology expertise. The table below outlines a few of the key differences between CRISPR gene editing and other popular techniques.
Once Cas9 nucleases are guided to the target DNA and create a double strand break 3-4 bases upstream from the PAM sequences, there are two ways the double strand break (DSB) can be repaired. If there is no donor DNA present, resolution will occur by error-prone non-homologous end joining (NHEJ), resulting in an indel that effectively knocks out protein function. Alternatively, if donor DNA sequences are available, the DSB is repaired by homology directed repair (HDR) for precise knock-in of the target gene.
Using CRISPR Plasmids for Genome Editing:
- gRNA design: GenScript's proprietary gRNA design algorithm uses the most current genome assembly data available from NCBI and other publicly available sources, and selects the best target sequences to avoid off-target effects. We search for an ~20 bp locus in the endogenous genome of interest for which a highly-similar match does not appear elsewhere in the genome. Off-target Cas9-mediated cleavage can occur even with up to 3 mis-matches between the gRNA and the endogenous genome, though most papers have reported little to no off-target effects.
- Nuclease / targeting strategy: Most researches use the Cas9 nuclease isolated from Streptococcus pyogenes. Cas9 WT induces double-strand breaks that are typically repaired through non-homologous end joining (NHEJ), which introduces small insertions or deletions that lead to frame-shifts and total loss of protein expression. This has proven to be an easy and effective way to introduce phenotypic KO in every cell line and organism attempted to date. Another strategy employs a mutant version of this enzyme, Cas9-D10A (Nickase), which can be used to induce two single-strand breaks flanking a region you want to delete for a more specific or comprehensive knock-out. If you require gRNA sequences for use with a different enzyme, or have other special requests for your CRISPR targeting strategy, please email your request to us.
- Number of unique gRNA sequences used: Based on our in-house experience using our design tool to create knock-out cell lines, a single gRNA construct is typically sufficient to knock-out your gene of interest; however, we recommend ordering at least two gRNA constructs per gene that you want to target in order to increase your chance of successful genome editing without off-target effects.
- 4μg of research grade plasmid DNA
- Electronic vector map
- Sequence chromatograms encompassing your custom insert
- Quality assurance certificate
- Determining the target gene locus
- Finding suitable sequences for Cas9 targeting
- Checking the potential for off-target binding
- Selecting gRNAs sequences that lie within your preferred binding region.
- Cells only need to be transfected once.
- gRNA/Cas9 expression is driven in an ideal 1:1 ratio.
- In most easy-to-transfect cell lines non- viral vectors can work well.
- Lentiviral transfection is typically necessary in cells with low transient transfection efficiency, such as primary cell cultures or hard-to-transfect cell lines.
- AAV vectors have low immunogenicity and are preferred for in vivo gene delivery. Since the cargo limit of AAV vectors is generally smaller than other vectors (<5 kb), packaging the SpCas9 gene into these vectors can be challenging. The Staphylococcus aureus Cas9 orthologue (SaCas9) is smaller than SpCas9 and is the preferred Cas9 variant for AAV vectors.
Factors that can affect CRISPR targeting efficiency and specificity are:
When you order gRNA clones from GenScript, we deliver a sequence-verified plasmid containing all elements required for gRNA expression and genome binding: the U6 promoter, spacer (target) sequence, gRNA scaffold, and terminator. We guarantee sequence accuracy for gRNA clones we deliver; however, given the complexity of creating genomically edited cell lines including transfection and selection, we cannot guarantee the outcome of experiments performed using our gRNA constructs. If you prefer to receive sequence-validated KO or KI cell lines created using CRISPR technology, please refer to our GenCRISPR™ mammalian cell line service.
Choosing the vector(s) you'll use to express the two critical components needed for CRISPR/Cas9 genome editing, the guide RNA and the Cas9 nuclease, is an important step in your experimental design. Many researchers prefer to use an all-in-one vector that will drive expression of gRNA and Cas9 in a 1:1 ratio. All-in-one vectors may also contain selection markers, such as fluorescent proteins or genes conferring antibiotic resistance, which can make it easier to isolate desired genome-edited clones. You may prefer to express the gRNA and Cas9 from separate vectors, for example if you want to vary the gRNA:Cas9 ratio, or if you want to screen a pool of gRNAs or use a larger gRNA library.
A minimum of 3 gRNA sequences are recommended to ensure knock-out and experimental accuracy. Independently obtained knock-out mutants provide redundancy to safeguard against any hidden off-target effects.
Designing your gRNA sequences involves 4 steps:
GenScript's gRNA database and online design tool will take out much of the guesswork when you're choosing gRNA sequences, by providing off-target scores and chromosomal location.
All-in-one vector systems have two main advantages:
Dual vectors, where Cas9 and gRNA are expressed independently on separate constructs, are more suitable if you plan to express multiple gRNAs for multiplex targeting. For these applications, Cas9 should first be stably expressed in the cell line, after which the cells can be transfected with different gRNA vectors to generate a cell pool.
Vector selection for CRISPR gene editing should consider both application and cell type.
SpCas9 nickase vectors are advantageous to use in experiments which are more sensitive to off-target editing. However, it is important to remember, that two gRNAs will need to be designed to target both forward and reverse strands. These gRNAs must be oriented so that PAM sites are distal to each other. gRNA sequences should be offset with windows of up to 100bp between them.
For robust SAM transcription activation, gRNAs must target the first 200bp upstream of the transcription start site (TSS). To decrease the degree of transcription activation, design gRNAs that target the SAM complex to greater distances upstream of the TSS. To repress transcription, design gRNAs that target SAM to +50 relative to the TSS, which will effectively block the TSS.
GenScript maintains a genome-wide SAM gRNA database which contains 6 SAM gRNAs designed to activate each coding region of the human genome. For other species, our scientists offer complimentary SAM gRNA design help. Request custom SAM gRNA design here.
Using CRISPR RNP Format for Genome Editing:
- DNA free
- Detectable at high levels shortly after transfection
- Quickly cleared from the cell for less off-target effects
- Highly efficient even in hard-to-transfect cells
- Best for in vivo studies
A minimum of 3 crRNA sequences are recommended to ensure knock-out and experimental accuracy. Independently obtained knock-out mutants provide redundancy to safeguard against any hidden off-target effects.
Unlike the traditional plasmids or lentivirus delivery methods, CRISPR/Cas9 RNP are delivered as intact complexes, and do not require cellular expression, thus has many advantages.
When using sgRNA, there is no need for annealing crRNA and tracrRNA duplex prior to use. More importantly, several studies have showed that sgRNA has better stability than crRNA:tracrRNA when duplexed with Cas9, thus leading to higher editing efficiency1,2.
1. Hendel, et al., Chemically modified guide RNAs enhance CRISPR-Cas genome editing in human primary cells. Nat. Biotechnol., 33 (2015) 985-989.
2. Ryan et al., Improving CRISPR–Cas specificity with chemical modifications in single-guide RNAs. Nucleic Acids Research, 46 (2018) 2: 792–803.
Synthesis of 100 nt long sgRNAs was traditionally possible through in vitro-transcription (IVT) using phage RNA polymerase. These in vitro transcribed sgRNAs contain a 5’-triphosphate, which was thought to trigger immune response in many cell types. A recent study showed that sgRNAs with 5’-triphosphate modifications produced through in vitro-transcription can indeed induce innate immune responses and lead to cytotoxicity in human and murine cells. However, chemically synthesized sgRNAs without the 5’-triphosphate modifications demonstrated much better editing efficiency in cells, thus supporting that chemically synthesized sgRNAs are the most ideal reagent for CRISPR genome editing up till now3.
3. Kim et al. CRISPR RNAs trigger innate immune responses in human cells. Genome Res. 2018. 28: 367-373.
Our modified sgRNA has 2’-O-methyl and phosphorothioate modifications at the first three 5’ and 3’ terminal RNA residues.2′ O-Methyl oligo modification is best characterized as a RNA analog which offers stability against hydrolysis and nucleases. The phosphorothioate (PS) modification renders the internucleotide linkage more resistant to nuclease degradation.
Using ssDNA as CRISPR HDR Precise Gene Knock-In Template:
- Carefully design your sgRNA. The sgRNA cleavage site should be close to the mutation site and it is best to be within 15bp of each other
- Optimize the transfection efficiency before transfection of gRNA/Cas9 and donor template.
- Use FACS sorter to enrich the transfected cells.
- Use drug or marker to select KI clones if feasible (e.g tag a puromycin resistance gene at C-termini of insertion gene for selection).
- For more information, please view GenScript’s webinar on Strategies to Efficiently Generate CRISPR KO/KI Cell Lines.
- HDR efficiency is highly correlated with Cas9 cutting efficiency, thus can be improved by carefully designing and choosing sgRNAs.
- Use long ssDNA, rather than dsDNA, for significantly reduced off-target integration.
- While generating conditional alleles via the integration of LoxP sequence, use one long ssDNA is more efficient than using two short ssDNAs.
It depends on the experimental purpose and host cell line. When compared to double-stranded DNA donors, ssDNAs demonstrated significantly improved editing efficiency and specificity, as well as reduced off-target integration, especially in editing primary cells, stem cells, and developing transgenic animal model.
|HDR efficiency||Medium||High||Comparable to dsDNA|
For point mutation, it is suggested to use asymmetric ssDNA design. You can read this paper for more design tips. For large gene insertion, homology arms with 300-1000bp flanking the insertion gene have been reported. In most cases, 500bp long homology arms should work. It is important to design the ssDNA template containing a silent mutation to mutate the sgRNA PAM sequence in order to avoid secondary cleavage. KI donor design can be complicated. It is highly suggested to use a software (e.g. snapgene) to view and edit the sequence.
Homology arms with 300-1000bp flanking the insertion gene have been reported. In most cases, 500bp long homology arms should work.
CRISPR/Cas9-mediated null allele production in mice is highly efficient, however, the generation of conditional alleles has proven to be more difficult.
You can refer to the following workflow to identify issues and solutions:
At GenScript, sequences that are less than 3000nt long in length are routinely produced. We have also successfully synthesized ssDNA that are up to 5000nt long with motifs of extremely high GC content and repeat regions in the past. If you want to synthesize ssDNAs longer than 3000nt long, please contact our Technical Support Team to get a quote.
At GenScript, the maximum delivery quantity for sequences < 1 kb long is 40 µg; for sequences within 1-3 kb long is 20 µg; and for sequences >3 kb long, the maximum yield is 5 µg.
We carry out two major QC examinations on the final ssDNA product prior to lyophilization: 1) Sanger sequencing to ensure the accuracy of the ssDNA sequence; 2) Purity test of the final ssDNA product via gel electrophoresis.
During the production of ssDNA, we do two rounds of sequencing to guarantee the sequence accuracy. We first pick sequence verified plasmid DNA template via sequencing to ensure the purity and sequence accuracy of the final ssDNA product. In addition, we use direct sequencing on the final ssDNA product to confirm the ssDNA product homogeneity.
We combine the advantages of using magnetic beads and agarose gel purification to ensure high yield, absolute purity, and zero chemical contaminations.
No. The final ssDNA product only contains full length, sequence-verified, ssDNA molecules. By using our proprietary, patent-pending, enzymatic synthesis approach, GenScript guarantees that our ssDNA product has a non-detectable dsDNA level.
Some ssDNA products may have strong aggregation tendency due to the nature of their nucleotide sequences. These aggregates are most likely formed due to intramolecular and/or intermolecular forces.
To test whether the extra band in a gel image is ssDNA aggregates or dsDNA contamination, you can perform a digestion test using S1 Nuclease, which degrades ssDNA, but not dsDNA. If the ssDNA product is 100% pure, with no dsDNA, then all samples should be digested with S1 Nuclease and no band should be observed after running gel electrophoresis. However, if a product has dsDNA contamination, then it can’t be fully digested after the addition of S1 Nuclease and still have bands present on the gel image.
To further confirm whether the extra band is indeed ssDNA aggregates, you can cut out the ssDNA band on the gel and run a second around of gel electrophoresis. If the extra band is aggregated ssDNA molecules, it will still show up on the second gel after purification. In addition, the ratio of the extra band over the ssDNA band in the two gels would remain similar.