Crispr Cas9
CRISPR Cas9 is a gene editing system that uses a single guide RNA (sgRNA) to direct the Cas9 nuclease to a specific DNA sequence, where it creates a double strand break (DSB). The cell then repairs the break by either error prone non homologous end joining (NHEJ) or high fidelity homology directed repair (HDR). This guide is for molecular biologists, lab technicians, and graduate students who want a practical, source bounded framework for designing and executing CRISPR Cas9 experiments. It covers core concepts, decision points, step by step workflows, quality checks, common mistakes, and the limits of interpretation.
The core machinery consists of two components: the Cas9 protein and a synthetic sgRNA that combines a CRISPR RNA (crRNA) with a trans activating crRNA (tracrRNA). The sgRNA contains a 20 nucleotide spacer that binds complementary DNA adjacent to a protospacer adjacent motif (PAM), which for SpCas9 is NGG. The Cas9 then cuts both DNA strands three base pairs upstream of the PAM. These fundamentals are described in detail in the free biomedical resources of the NCBI Bookshelf.
At a Glance
| Component | Role | Key detail |
|---|---|---|
| Cas9 nuclease | Cuts DNA | Requires PAM sequence NGG (for SpCas9) |
| sgRNA | Guides Cas9 to target | 20 nt spacer + scaffold, no PAM on sgRNA |
| PAM | Required for recognition | Located on non target strand, 3` of target |
| NHEJ | Error prone repair | Induces indels, disrupts gene function |
| HDR | Precise repair | Uses donor template, inserts or corrects sequence |
| Donor template | Required for HDR | Single or double stranded DNA with homology arms |
Decision Criteria
Choosing the right approach depends on your experimental goal. If you want to knock out a gene, NHEJ mediated indel generation is the simplest and most efficient path. If you want to knock in a specific mutation or a reporter tag, you must use HDR. Key decisions include:
Target location: Exons near the 5` end or functional domains give the best knockout results. For HDR, the cut site should be within 10 to 20 base pairs of the desired edit. Use the EMBL EBI Training resources for guide design tools and target validation exercises.
Cell type and delivery: Dividing cells are required for efficient HDR. Hard to transfect cells may need viral delivery (lentivirus, AAV). Non dividing cells rely almost exclusively on NHEJ.
Off target tolerance: Use high specificity Cas9 variants (e.g., eSpCas9, SpCas9 HF1) and select guides with few predicted off targets. Always validate with targeted sequencing.
Donor template choice: For HDR, single stranded oligonucleotide donors (ssODNs) work for small edits (<50 bp). Larger inserts require double stranded DNA donors with long homology arms (typically 500 to 1000 bp each).
Practical Workflow or Implementation Steps
The following sequence guides you from design to validation. It is adapted from the Galaxy Training Network open workflows.
Identify target site: Obtain your gene sequence. Choose an early constitutive exon or a domain critical for function. Check for unique target sites using genome browsers.
Design guide RNAs: Use a guide design tool (e.g., CRISPick, CHOPCHOP). Input the 20 bp target sequence immediately upstream of an NGG PAM. Filters: avoid poly T runs >4, matches to off target sites, and low on target efficacy scores. Select three to five guides per target.
Synthesize and clone sgRNAs: Order sgRNA as annealed oligonucleotides and ligate into a plasmid with a U6 promoter and Cas9 expression cassette. Alternatively, use synthetic sgRNAs or in vitro transcribed RNAs.
Deliver reagents into cells: Choose transfection, electroporation, or viral transduction based on cell type. Include a fluorescent marker or antibiotic resistance for enrichment. For HDR, co deliver the donor template at a 1:1 molar ratio with the CRISPR plasmid.
Harvest and test: 48 to 72 hours post delivery, extract genomic DNA. Screen for editing by Sanger sequencing or mismatch cleavage assay (T7E1 or Surveyor). For HDR, use PCR spanning the edit with primers outside the homology arms.
Isolate clones: Dilute cells into single clones or use FACS sorting. Expand clones and confirm editing by sequencing. Ensure at least two independent clones per condition.
The Bioconductor project offers R packages for analyzing sequencing data from CRISPR screens and validating off target hits.
Quality Checks
To ensure reliable editing results, include these checks at each stage.
Guide efficacy: Before performing large scale experiments, validate each guide in a pilot study using a mismatch cleavage assay or next generation sequencing. Aim for at least 20% editing efficiency for knockout, higher for HDR.
Off target analysis: Use computational prediction tools to identify the top 10 candidate off target sites. Re sequence these regions in edited cells. For published work, off target profiling by whole genome sequencing is recommended.
HDR verification: A common error is to assume HDR succeeded based on a PCR product. Always sequence across the edited region. Use a restriction site that is introduced or removed by the edit as a rapid screen. The NCBI Sequence Read Archive provides raw data from many validated editing projects that can serve as positive controls.
Cell line identity: Mycoplasma contamination can affect editing outcomes. Test cells before and after editing. Passage cells for a short time to avoid genetic drift.
Common Mistakes
Ignoring PAM proximity: The cut site is 3 bp upstream of PAM. Designing a guide far from the intended edit location reduces HDR efficiency. Always check the distance between cut and target mutation.
Using mismatched guides: Guide spacer sequences must be exactly complementary to the genome. A single mismatch near the PAM distal end (seed region) can abolish activity. Double check your design against the reference genome.
Low HDR efficiency: Many researchers assume HDR will occur at a high rate. In practice, HDR rarely exceeds 10 to 20% even in optimized cells. Use HDR enhancers (e.g., SCR7, L755507) or enrichment strategies like co selection with a resistance marker.
Forgetting PAM requirement for donor: The donor template must not contain the PAM sequence if it will be reintroduced. Mutate the PAM in the donor to prevent recutting after HDR (silent mutation).
Overlooking repair pathway choice: Some cell lines strongly favor NHEJ. Knockout experiments can proceed with NHEJ, but for HDR you may need to transiently suppress NHEJ. Recent work in Knockdown of XRCC5 and XRCC6 activity using CRISPR/Cas9 technology enhances homology directed DNA repair at the CHST6 locus in HEK293 cells shows that silencing key NHEJ factors can boost HDR rates significantly.
Limits of Interpretation
CRISPR Cas9 is a powerful tool but its results must be interpreted with caution. Some key limitations:
Off target editing: Even with high specificity variants, off target cuts can occur at sequences with 3 to 5 mismatches. Always validate with unbiased methods like GUIDE seq or whole genome sequencing for critical applications.
Mosaicism: When editing embryos or multicellular organisms, not all cells will carry the same edit. This complicates phenotypic analysis. The Functional characterization of Acyl CoA Synthase 1 in tomato branch angle development demonstrates how mosaicism requires screening multiple independent transformants.
Incomplete knockout: A single allele may be edited while the other remains wild type. Protein function may persist at low levels. Use western blot or functional assays to confirm knockout.
Epigenetic effects: Cas9 binding alone can block transcription without cutting, a phenomenon called CRISPR interference. For gene knockout studies, confirm mRNA reduction by qPCR. The review Epigenetic editing makes its mark describes how catalytically dead Cas9 fused to epigenetic modifiers can alter gene expression without DNA cleavage, which could confound results if not controlled.
Cell type variability: Editing efficiency and repair pathway choice differ drastically between cell lines and primary cells. Ferroptosis induction via genetic approaches CRISPR/Cas9 based disruption on key anti ferroptotic genes highlights that even in the same cell type, different guides can yield widely different editing rates.
Not a therapeutic guarantee: While CRISPR holds therapeutic promise, in vivo delivery, immune responses, and long term safety are major hurdles. Studies like RBM20 variants disrupt Ca(2+) handling and metabolism in dilated and non compaction cardiomyopathy stem cell models show that disease phenotypes can be rescued in vitro, but translation to patients requires extensive validation.
Frequently Asked Questions
What is the PAM sequence and why does it matter?
The PAM (protospacer adjacent motif) is a short DNA sequence immediately downstream of the target site on the non target strand. For the most common Cas9 from Streptococcus pyogenes (SpCas9), the PAM is NGG. The Cas9 must recognize this PAM to bind and cut. If the PAM is absent or mutated, editing fails. When designing a donor template for HDR, you must alter the PAM to prevent the Cas9 from cutting the repaired locus.
Can I use CRISPR Cas9 for gene activation or repression?
Yes. A catalytically dead Cas9 (dCas9) fused to transcriptional activators (e.g., VP64) or repressors (e.g., KRAB) can modulate gene expression without cutting DNA. This approach is called CRISPRa or CRISPRi. It does not permanently change the genome, so effects are reversible. See the Epigenetic editing makes its mark article for current advances.
How do I choose between Cas9 and Cas12a (Cpf1)?
Cas9 cuts both DNA strands bluntly and requires a G rich PAM. Cas12a cuts staggered ends and uses a T rich PAM. Cas12a also processes its own crRNA array, making it useful for multiplex editing. Choose Cas9 for standard knockout and HDR, and Cas12a for multiplexing or when you need overhangs for directional cloning.
Why is my HDR efficiency so low?
HDR is naturally suppressed in most cells. Efficiency depends on cell cycle phase (S/G2 is permissive), delivery method, and donor template design. To improve HDR, synchronize cells, add nocodazole or other cell cycle regulators, use a longer homology arm, or knock down NHEJ factors as shown in Knockdown of XRCC5 and XRCC6 activity using CRISPR/Cas9 technology. Also, co delivery of a fluorescent HDR reporter can help sort edited cells.
References and Further Reading
- NCBI Bookshelf A comprehensive collection of open access biomedical textbooks covering CRISPR mechanisms and applications.
- EMBL EBI Training Interactive tutorials on guide design tools and genome editing data analysis.
- Galaxy Training Network Reproducible workflows for CRISPR sequencing analysis and quality control.
- Bioconductor R packages for analyzing CRISPR screen data, off target detection, and variant calling.
- NCBI Sequence Read Archive Repository for raw sequencing data from CRISPR editing experiments, useful for benchmarking.
- Functional characterization of Acyl CoA Synthase 1 in tomato branch angle development Example of CRISPR editing in plants, including mosaicism handling.
- Epigenetic editing makes its mark A Nature review on dCas9 based approaches and their interpretation.
- PLK1 inhibition enhances Brentuximab vedotin efficacy in CD30 positive T cell lymphoma via spindle assembly checkpoint activation Demonstration of CRISPR knockout used to validate a drug target.
- RBM20 variants disrupt Ca(2+) handling and metabolism in dilated and non compaction cardiomyopathy stem cell models Use of CRISPR in induced pluripotent stem cells for disease modeling.
- Ferroptosis induction via genetic approaches CRISPR/Cas9 based disruption on key anti ferroptotic genes Practical protocol for generating knockout lines in a specific pathway.
- Knockdown of XRCC5 and XRCC6 activity using CRISPR/Cas9 technology enhances homology directed DNA repair at the CHST6 locus in HEK293 cells Method to boost HDR by targeting NHEJ factors.