Of the 23-base standard CRISPR genomic target sequence, the bases actually required for target recognition are the first 20 bases and the last 2 bases (...GG). Combined, this target is sufficiently long enough that most targets of interest will turn out to be unique in mammalian genomes. However, Cas9 can tolerate mismatches, leading to concerns about off-target cleavage.
Off-target cleavage: Off-target cleavage events can occur and are well documented for CRISPR/Cas9. The “seed region” of approximately 12 bases proximal to the PAM motif are most crucial for pairing and DNA cleavage, while mispairing in the distal bases can sometimes be tolerated . The frequency of off-target CRISPR cleavage events is currently controversial, and is probably highly target- and system-dependent. The most current data relevant to mouse embryos is from Yang et al . For 5 different guide RNAs designed to unique targets, they identified all potential off-target (OT) regions (N=47) in the mouse genome that had up to 3 or 4 mismatches within the 20 bp coding sequences of the guide RNAs. 6-10 mice or ES cell lines were screened per guide RNA. Of all the OT locations, mutations were induced at 3 of 47 OT sites screened. They noted that the only OT sites with detectable mutations had only 1 or 2 mismatches compared to the target. This correlates with the observation that multiple mismatches reduce CRISPR cleavage efficiency. A very useful online CRISPR target design tool is available that provides data on all off-target sites for predicted targets is found at: http://crispr.mit.edu.
There are a few reasons to believe that off-target cleavage issue seems is less of an overall concern for injected mouse embryos as compared to tissue culture-based CRISPR experiments, where off-target cleavage events have been studied in more detail . Some of this reduced rate of off-target effects in mouse embryos may be due to the more transient expression of the CRISPR/Cas RNAs following embryo injections, as opposed to the longer duration of expression from transfected plasmids in cell culture. However it should be kept in mind that off-target cleavage may occur. Also note that, for mice, the potential effects of off- target mutations could potentially be removed by backcrossing the resulting mice to the parent strain.
Mutations can be created using a “nickase” variant of Cas9 in which one of the two strand-specific DNA cleavage domains is inactivated by a single amino acid change [11, 14]. Single targets are not mutagenized at high efficiency since the single strand “nicks” are usually repaired in vivo by ligase. However, by using two targets on opposite strands in fairly close proximity, NHEJ or HDR can be induced at moderate efficiency. This scheme should reduce off-target mutations since off-target nicks will be isolated in the genome, and thus will usually be quickly repaired by ligase. However, the complexity and constraints on target selection are increased in “paired-nickase” experiments.
• CRISPR/Cas9 expression in mouse embryos: In the Vanderbilt Transgenic Mouse / ES Cell Shared Resource (TMESCSR), we have successfully performed CRISPR/Cas9 mutagenesis in mouse embryos by injecting either (1) cytoplasmic injection of CRISPR/Cas9 RNAs or, (2) pronuclear injection of plasmid DNAs for transient expression of CRISPR/Cas. We have recently adopted injection of the PX330 plasmid (Addgene #42230), which can be easily modified to express customized guide RNAs for targets of interest. This allows easy customization of the reagent and a simple miniprep protocol to prepare the DNA for injection. PX330 is a bifunctional plasmid that also expresses Cas9 mRNA.
For those who are interested in making the RNAs themselves, we have used a Cas9 in vitro mRNA expression vector created by Dr. Wenbiao Chen here at Vanderbilt . This Cas9 vector has been codon-optimized and incorporates nuclear localization signals, and is known to be highly functional in zebrafish and mouse embryos. Several other versions of Cas9 expression constructs are also available from Addgene.
• TALENs as alternatives to CRISPR/Cas: TALENs also can direct DNA cleavage at desired targets, and so share many conceptual and outcome similarities to CRISPR/Cas . The primary advantage TALENS afford compared to CRISPR/Cas is more flexibility in target site choice. The primary disadvantages are twofold: first, TALENs appear usually to have lower targeting efficiency than CRISPR/Cas reagents; second, a new TALEN vector has to be designed and created for each target, which increases the time and cost. Customized TALEN vectors can be purchased from commercial vendors, who also usually provide design assistance. Although the TMESCSR does not provide TALEN design assistance, we can perform pronuclear injections with TALEN reagents provided by the investigator; please contact the TMESCSR if you wish to pursue this.
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