Gene Editing with the CRISPR-Cas9 System

Using one of the synthetic gRNA options has several advantages over expressing the gRNA, including improved editing efficiency and reduced labor intensity. Preformed guide RNA also does not continue to be expressed over a long period of time like plasmid-encoded guide RNA. By minimizing how long the gRNAs spend in the target cells, synthetic options help decrease the chance of toxicity. Despite that, chemical modifications also increase the amount of time the gRNA is in the cells. The goal is using the optimum time so that the gRNA is in the cells long enough to be effective, but not so long that the gRNA causes toxicity or increases off-target effects. IDT provides chemically synthesized guide RNAs with optimized formats.

All of the IDT Alt-R™ CRISPR RNA molecules contain chemical modifications, which protect from cellular endogenous nuclease activity. In addition, if you are still worried that high levels of endogenous, cellular nucleases might destroy the crRNA in your experiment, you can choose to use IDT's XT modification on crRNA. XT crRNA contains additional chemical modifications, making it longer-lived, nuclease resistant, safe for your cells, and still just as functional for CRISPR as regular crRNA. The standard Alt-R crRNA is end-blocked, whereas Alt-R crRNA-XT has both end-blocking and internal chemical modifications. Similarly, the Alt-R tracrRNA is heavily modified.

On- and off-target activity

The goal when designing a guide RNA is achieving the highest possible on-target activity of your CRISPR gene editing complex, while minimizing off-target activity. Off-target activity causes unwanted phenotypes, potentially including cell death.

PAM sequence

For Cas9 guide RNA designs, the target sequence must be next to a PAM sequence, NGG, where N is any base. It is important that you do not include the PAM sequence in the actual guide RNA design. The guide RNA recognizes and binds to 20 nucleotides on the DNA strand opposite from the NGG PAM site. The “N” of the NGG is immediately adjacent to the most 3’ base of the non-targeted strand side of the protospacer.

Length considerations

When you are using 2-part guide RNA, there are crRNA and tracrRNA components.

• crRNA: The crRNA is composed of a target-specific spacer region and another domain that hybridizes to the tracrRNA (green). The target-specific spacer sequence is 20 nucleotides long; if it is shorter than this, the on-target activity will be negatively impacted. IDT researchers have found that the optimal total length of the crRNA (the target-specific spacer region plus the domain that hybridizes to tracrRNA) is 36 nucleotides.
• tracrRNA: Part of the tracrRNA molecule hybridizes to the crRNA, and another part of it binds to Cas9. IDT scientists have found that the optimal, total length for tracrRNA is 67 nucleotides. This is shorter than the tracrRNA found in nature, but IDT scientists have found that when using 2-part guide RNA, shortening the tracrRNA to 67 nucleotides increases on-target activity.

When using sgRNA, the crRNA and tracrRNA are all part of one longer RNA molecule joined by a hairpin-like loop. Cas9 sgRNA for genome-editing purposes are typically 100 nucleotides in length.

The Alt-R S.p. HiFi Cas9 Nuclease V3 offers improved specificity over wild-type Cas9, greatly reducing the risk of off-target cutting events. This Cas9 variant also preserves the high level of editing efficiency expected from a Cas9 nuclease, maintaining 90–100% on-target editing activity at most sites. For applications that are sensitive to off-target events, combining the Alt-R S.p. HiFi Cas9 Nuclease V3 with optimized Alt-R CRISPR-Cas9 gRNA (crRNA:tracrRNA) is highly recommended.

Nickases are enzymes that produce single-strand breaks. Cas9 nickase variants have an alanine substitution within either of the 2 key catalytic domains, RuvC and HNH, of the WT Cas9 endonuclease. The RuvC mutant, D10A nickase, cuts on the targeted strand. The HNH mutant, H840A nickase, cuts the non-targeted strand. The trick to producing a DSB using nickases is to combine a Cas9 nickase – either D10A or H840A, together with two gRNA sequences-targeting sites that are close together but on opposite DNA strands. This creates a staggered DSB. For an experiment, you only need one type of nickase.

One benefit of using Cas9 nickases to introduce DSBs is that this can minimize unwanted off-target editing and support high on-target efficiency. Another reason to consider nickases in your experiments is that some Cas9 guide RNAs are less efficient than others. If you determine experimentally that there are no efficient Cas9 guides that target the site you want to mutate, then using a nickase with paired guides targeted at short distances from both sides of the desired mutation site may be a better approach.

For further information about Cas9 nickases and their use, please refer to the following articles or consult the corresponding chapter in IDT's CRISPR basics handbook:

When and how to use nickases               HDR and Cas9 nickase design               CRISPR basics handbook

The Alt-R S.p. Cas9-GFP V3 and S.p. Cas9-RFP V3 nucleases are high purity, recombinant S. pyogenes Cas9 enzymes that are expressed as fusion proteins with nuclear localization sequences and C-terminal 6-His tags. These enzymes have on-target functionality comparable to wild-type S.p. Cas9 and are designed for applications that require post-transfection visualization of the protein or enrichment of edited cells using fluorescence-activated cell sorting (FACS). These enzymes should be combined with Alt-R CRISPR gRNA in equimolar amounts.

Alt-R S.p. dCas9 Protein V3 has mutations that result in the loss of nuclease activity. This protein can form RNP complexes with Alt-R gRNAs and bind to the target region specified by the gRNA without cutting the DNA. The primary use of dCas9 protein is to block transcription at a specific site on the genome. This is known as CRISPRi and is an alternative to RNAi for knockdown instead of knockout of genes.