Why designing guides for CRISPR knockout experiments is more complex than just looking at every NGG PAM

  • 4 minute read
  • Gene editing

Thoughts around guide RNA design for specificity and functionality.

CRISPR was the first RNA targeted method of modifying DNA. Unlike zinc finger nucleases or TALENs you don’t have to redesign the whole protein to target a specific sequence. S. pyogenes Cas9 allows us to look through the whole genome and target any 20-nucleotide sequence adjacent to an NGG PAM motif. This is the borrowed elegance of the bacterial immune system, where the bacteria uses the CRISPR system to cut out and store, or “remember” sequences of viral pathogens that they later use to target and cut up viruses in a subsequent infection. The bacterial system is straightforward where the Cas9 searches for a protospacer adjacent motif (PAM), which is a NGG sequence (any nucleotide, guanine, guanine) then the 20 nucleotides adjacent are targeted using the targeting region of the CRISPR RNA sequence.

Now Available On-demand

Webinar: CRISPR knockout guide RNA design for optimal function and specificity

When implementing this in mammalian cells, we are using Cas9 to target and cut genomic DNA, which disrupts translation of the coding genes, when the cell imperfectly repairs the cut and introduces errors in the sequence. Instead of going through an entire gene and looking at all the NGG PAMs and creating guides that are going to target all possible sites, we need to predict which sequences will target, bind, and cleave most efficiently. Success of a gene editing experiment is measured through functional disruption or knockout of a gene. In addition to ensuring knockout, we need to make sure the cutting is specific and not disrupting other regions in the genome (no off-targeting).

Designing CRISPR-Cas9 RNA guides for potent knockout and high specificity, you need guidance beyond just looking at every NGG PAM.

A subset of design considerations include:  

  1. Melting temperature of the guide sequence
  2. The first position in the NGG PAM, that variable nucleotide can be important
  3. Dinucleotide content
  4. Location of the cut in the coding sequence (CDS)

Balancing all of the interdependent factors that contribute to what makes a highly functional and specific guide in an unbiased and high-throughput manner is complex. Picking CRISPR sequences can be an extremely time consuming endeavor and takes away from the biology you are studying. We have done the work upfront to help scientists focus on the biology and not the tools.

Using decades-long experience building algorithms for our RNAi product lines (ON-TARGETplus, ACCELL, SMARTvector), we employed our knowhow and built a comprehensive set of rules for CRISPR guide design otherwise known as the Dharmacon™ Edit-R™ algorithm. Initially we synthesized a "test set" of over 1,100 guides across 10 genes. We then analyzed the guides for functionality and specificity in an arrayed screening format with focus on the resulting phenotype, identifying the parameters that will predict guide RNA sequences highly functional and specific. The in silico predictions were then validated in multiple different assays and readouts to confirm the robustness of the algorithm.

These guide design rules are used for all of our predesigns. With our predesigns you can be assured that you don't have to spend time validating the reagents but can focus on your particular biology and genes of interest so you can achieve the learning goals from your gene editing experiments.

To learn more about how we developed the Dharmacon Edit-R CRISPRko algorithm watch Emily Anderson’s comprehensive webinar, hosted by the CRISPR journal. Register Now. 

Now Available On-demand

Webinar: CRISPR knockout guide RNA design for optimal function and specificity