Increasingly the literature suggests that CRISPR's potential for off-target effects is not as “bad” as originally thought. Here are a few things to set your mind at ease if you’re working with the CRISPR-Cas9 system:
Early on in our CRISPR use at Horizon, we learned that that when it comes to guide RNA design not all sgRNAs are created equal. In fact what makes a good guide is still the topic of a great deal of research in the field – and aside of the consensus that a guide with a guanine at position -1 is more likely to have a higher activity, we are still a way from being 100% confident in predicting in silico guide activity in vitro/vivo.
The CRISPR/Cas9 system has been rapidly adapted to practically every model system for its ease to generate and high efficiencies to cleave target DNA. But unlike our experience with Zinc Finger Nucleases, in the human, rat and mouse cell lines we tried successful co-transfection of Cas9 mRNA and sgRNA was cell-line dependent, and often resulted in either very low or no cleavage activities.
However, sequential transfection of cells with Cas9 DNA first, and sgRNA followed 24 hrs later, reliably produced good level of activity, indicating the requirement of Cas9 presence at the time of introduction of sgRNA. Not surprisingly, creation of a cell line stably expressing Cas9 led to consistently high cleavage activities upon transfection of sgRNAs. Transfection of recombinant Cas9 protein pre-complexed with sgRNA (ribonucleoprotein particles, or RNPs) led to efficient cleavage as well.
On the other hand, when Cas9 mRNA and sgRNAs are co-microinjected into single cell embryos, it produces target cleavage as efficiently as RNPs to produce straight KOs and large deletions between two target sites, again raising a question of local concentrations of Cas9 protein and sgRNA.
Below we summarize some of the work we've done optimizing delivery of CRISPR-Cas9, which which can be read in full publication form in Human Gene Therapy here.
Transfection or electroporation is used to efficiently introduce the nucleic acids required for CRISPR cell line engineering into a cell line. The type and number of nucleic acids (usually plasmids) being introduced into a cell line will depend on the engineering event being undertaken. At its most simple, gene knockouts can be achieved by transfection of a plasmid expressing wild-type Cas9 along with a guide RNA (gRNA) to the gene that is being knocked out.
If you've followed our guide to planning a successful genome editing experiment, then you'll hopefully be working in optimal conditions, and have a good idea of your guide's editing efficiency. This number should give you a rough idea of how many clones you're going to have to screen to find a targeted clones. Keep in mind the following:
Once a clone has been identified positive for targeting in the initial screen, the cells should be expanded to a 48-well plate. Expansion of the cells can usually occur at 1-4 days post screen.
While CRISPR-Cas9 has made gene editing cheaper, easier and more accessible than ever before, using the system can in some cases still be challenging, and no scientist can yet be 100% certain of success. With careful preparation and planning however, chances of success can be significantly boosted.
There exist now a range of techniques to perform genome editing, such as ZFN, CRISPR, TALENS and AAV, each with their own strengths and weaknesses. However, one consistent element that has a significant impact on the success of that editing event when generating an isogenic cell line is the choice of parental cell line to be engineered.
Recombinant adeno associated virus (rAAV) is a precise and effective method to introduce defined changes into endogenous genes and rAAV vectors can stimulate homologous recombination (HR) up to 1000-fold over that seen using plasmids.
Nuclease based approaches like CRISPR-Cas9, ZFNs and TALENs facilitate targeted modification of genomes by inducing double-strand breaks (DSBs) within chromosomes at specified locations. This stimulates the natural DNA-repair mechanisms of homologous recombination and non-homologous end joining.
Plasmid DNA, PCR products, and single stranded oligonucleotides are routinely used as donors to introduce specific changes at the DSB site. The efficiency of introducing a desired change is dependent on many factors including
- the type of donor
- the length of homology
- the complexity of the desired change
- characteristics specific to the cell line
We have looked at whether rAAV vectors can be used as donors for DNA modification to obtain higher efficiencies than seen with other donor approaches.