The bacterial CRISPR/Cas9 system (Clustered Regularly Interspaced Palindromic Repeats associated protein 9 system) is among the youngest players in the gene editing playground.
The target sequence-determining CRISPR RNA (crRNA) and the auxiliary trans-activating crRNA (tracrRNA), often fused to a chimeric single guide RNA (sgRNA), work together with Cas9 to become a promising and potent tool for genome engineering.
When a new tool is developed, it often means challenging and troublesome situations with no common solutions. Below we offer some useful tips to avoid these issues as well as solutions for when they arise.
Tip: Carefully Select Target Sites
- Avoid homology
Design the target sequence so that there is minimum similarity between your target and other regions in the genome.
- Ensure the 12-nt ‘seed’ region is adjacent to the PAM (Protospacer adjacent motif)
Targets are considered highly specific when the rest of the genome has no perfect matches to the 12-nt seed sequence directly adjacent to an NGG PAM sequence.Note: Strategic target selection can limit off-target cleavage.
Suggested Delivery Systems
- DNA plasmids
Injections of DNA plasmids encoding Cas9 and gRNA(s) work well for insertion/deletion (indel) generation during in-vivo transcription/translation.
- CRISPR components as RNA
Generating DNA as plasmids or PCR products yields templates for in-vitro transcription of gRNAs that can be used for observed efficient germline transmission.
- Cells expressing CRISPR components
It has been shown that there is a low toxicity of CRISPR/Cas9 components in the absence of crRNA. Thus, targeted modifications can be created via injecting only the targeting component of the system into a cell line stably expressing the common non-targeting system components Cas9 and/or tracrRNA.
Suggested Screening Methods for Targeted Events
- Molecular screening
PCR-based methods are a universal option for screening followed by either restriction enzyme usage, like protocols utilizing CEL-1 enzyme (Surveyor nuclease)/T7 endonuclease I (T7EI), or direct sequencing to quantify indel presence in CRISPR-treated cell populations.
- Negative screening
Generate deletions in cell lines that contain visibly marked elements in the target locus, so that successful treatment is easily detectable. With this screening method, indels might not be a sufficient means, so bigger deletions utilizing two double strand breaks by two flanking gRNAs may be needed.
- Positive screening
Insert a suitable marker that is either visible or that can easily be detected. This method mainly relies on homology directed repair (HDR) and requires ds DNA donor templates for the desired incorporation. Application of fluorescent tags flanked by FRT or loxP recombination sites allows for subsequent removal of the inserted marker sites.
Problem: No PAM Sequence Close to Your Target Sequence
The PAM is a necessary requirement for CRISPR gene editing. In case your target sequence of choice has no adjacent NGG, consider:
- ‘NAG’ as PAM in mammalian cells
NAG works as an alternative PAM when using Cas9 from Streptococcus pyogenes, mediating cleavage with approximately 1/5 the efficiency of NGG
- Using alternative Gene Editing tools
The use of other established nuclease based methods for gene editing, like the zinc finger nucleases (ZFN) or the transcription activator like effector nucleases (TALENs), help to circumvent the necessity for a PAM sequence with no or little drawbacks to editing efficiency.
Problem: Off-Target Activity
- Solution: Titration of sgRNA and Cas9
The amount of Cas9 and sgRNA can be titrated to optimize the on- to off-target cleavage ratio. However, increasing specificity by reducing the amount of transfected DNA can also lead to a reduction in on-target cleavage.
- Solution: Mutated Cas9
To reduce the potential for generating mutations at off-target cleavage sites, Cas9 can be mutated and supplied as a nickase. Nickases induce a single strand instead of a double strand break. Thus targeting is necessary, using two adjacent guide sequences, thereby raising specificity and lowering probability of off-target binding for both sequences. Off-target ss breaks are also less error prone during repair.
- Solution: PAM-proximal mismatches
At least two mismatches should lie within the PAM-proximal region of the off-target site, since SpCas9 tolerates single-base mismatches in the PAM-distal region to a greater extent than in the PAM-proximal region.
- Solution: Maxing mismatches
The maximal number of mismatches in identified off-target genomic sequences should be consecutive or spaced less than four bases apart.
Problem: Low Efficiency of Modification
Solution: Increase the tracrRNA length–there is a consistent increase in modification efficiency with increasing length of tracrRNA.
Solution: Try designing and testing 3 to 4 different DNA target sequences to increase modification efficiency for CRISPR nucleases (or nickases).
Solution: Efficiency can be increased by enriching the transfected cells via antibiotic selection and/or FAC sorting as follow-up processes to modification.