How to overcome the CRISPR off target effect

For the past two years, the CRISPR-Cassystem has been increasingly used for gene knockout, exact changes in specificbases, and transgenes.

The CRISPR-Cas system has many advantages:simple design, high efficiency and relatively low cost. Compared with thetraditional method, it is more important that it can correct the mutationcaused by genetic disease, to avoid errors caused by random integration.However, CRSIPR-Cas9 itself has a tendency to target mutations.

Although the current technology has beengreatly improved, but some researchers still believe that the specificity ofCRISPR-Cas is relatively low. In this paper, we will discuss the mechanism ofCRISPR-Cas in genome editing applications and the factors that affect itsspecificity, the CRISPR potential target mutations reported in the literature,the method of enhancing CRISPR-Cas target specificity, and the methods used toenhance CRISPR -Cas specific solution.

CRISPR-Cas Target recognitioncapability

CRISPR-Cas,which is widely used in genomic editing, consists of two parts:

1.Streptavirus-derived Cas9 nuclease.

2.AsingleguideRNA(sgRNA) .sgRNA is a chimera of two RNA molecules in the CRISPR targetingrecognition system: CRISPRRNA (crRNA), transactivatingCRISPRRNA.

AsgRNA contains 20 variable nucleotide regions with target site specificity. Inaddition, the specific binding of Cas9 to the target sequence requires theidentification of the 3 nt N-G-G sequence, PAM (ProtospacerAdjacentMotif).

When thesgRNA forms a complex with Cas9, the complex is localized to the targetsequence and the PAM site, the target DNA is desorbed, and the length of the 20nt guide RNA is annealed to the antisense strand. Finally, under the action ofCas9 nuclease, the target DNA double-strand breaks. If DSBs are not repairedimmediately, the cells will die. The preferred DSB repair pathway is arelatively error-free repair mechanism under homologous recombination, but thismechanism is hardly present in mitotic cells because it must be present in thelate post-divisional chromosome replication and sister chromatid pairing.

Also,homologous recombination is usually not present in non-dividing cells. Inaddition to homologous recombination, the DSB was repaired by non-homologousend-of-the-line (NHEJ). NHEJ is prone to errors, leading to sequence insertionor deletion of mutations, as if the broken chromosome ends were recitedtogether. CRISPR-Cas, as a simple and efficient gene knockout tool, was used byresearchers to explore the tendency of gene insertion or deletion.

SgRNA Bindingspecificity

Consideringthat the target recognition sequence length of CRISPR-Cas9 is 20 nt, if thebinding of RNA to the chromosomal target site is strictly based on thesequence, these target sites should be unique in the eukaryotic genome, plusthe need Identification of PAM loci, CRISPR-Cas error is unlikely to be cut.

However,factors that affect CRISPR specificity are not just sequences. YanfangFu andhis colleagues have concluded that even if one or more mismatches are formed,sgRNAs are able to direct the Cas9 nuclease to act on three different targetsof the GFP reporter gene integrated into the chromosome. It is acceptable thata plurality of mismatches existing or adjacent to each other between the targetRNAs and the target sequence are dispersed. The mismatch in the 10 nucleotidesnear the PAM site in the Guide RNA is more likely to be accepted than themismatches in the 10 nucleotides away from the PAM site, and the acceptable rangeof the mismatch distribution still depends on the target sequence The

Inaddition, Fu et al. Prepared three human endogenous genes of the Guide RNAs,and predicted the structure of each gene in the genome on the off target site.There are multiple mismatches between almost all target sites and target RNAs.Detection of insertion or deletion using the T7 endonuclease I test, found thatthese predicted off target sites usually contain insertions or deletions thatare as many or more as the target site. There are still detectable insertionsor deletions that can be detected by up to 5 mismatches between the Guide RNAand the chromosome. The degree of mismatch acceptable depends on the Guide RNAsequence, the chromosome target sequence and the cell line used.

Earlytrials of CRISPR-Cas specificity (eg, Fu et al., 2013) raise questions fromresearchers about the potential application of this technology. New studieshave reported that CRISPR-Cas systems are more specific.

Forexample, EricLander Laboratories uses large-scale mutant screening of humansgRNAs based on lentiviral vectors. Screening of the anti-DNA damage mechanismshowed that there was a higher incidence of gene mutation in the mismatchrepair pathway when correctly targeted to sgRNAs compared to the mispectedsgRNAs (Wang et al., 2014). In addition, a sgRNA that was used to successfullyrestore the wild-type function of the Crygc gene and to cure cataracts in micehad no effect on 10 of 12 transgenic animals (10 containing target sites) Eachsite has a mutation (Wu.etal., 2013). The results of the study (Li et al.,2013; Yang et al., 2013), in combination with optimized software tools, predictthat sgRNAs have high potential specificity.

(Ran, etal., 2013a; Xiao, et al., 2014).

Thisresult shows that our long-held view that "CRISPR-Cas9 has off-targetmutative preference" is exaggerated.

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