How to reduce off-target effects and increase CRISPR editing efficiency?

As an emerging editing tool, CRISPR has attracted extensive attention in recent decades. However, its use has been largely limited due to its natural off-target effect. Therefore, in recent years, a large number of scientists have been trying to optimize the system in order to reduce off-target effects and increase editing efficiency. Mainly from the following aspects:

1. Improving sgRNA specificity

sgRNA near 10 ~ 12 bp base pairs is usually called PAM seed area, which determine the specificity of sgRNA and target DNA recognition. And the remaining sequence in different extent affects the off-target effects. Ren et al. found that sgRNA gene editing efficiency is proportional to the area of GC content with sgRNA seeds. When there were 3 or more base mismatches between sgRNA seed region sequence and off-target site DNA sequence, the off-target effect decreased or even disappeared. Therefore, when designing sgRNA sequence, the GC content can be selected at 40%~60%. The sgRNA sequences with low homology other than the target gene sequences can improve the specificity of sgRNA. The sgRNA sequences at the distal-PAM region can cause changes in Cas9 configuration and promote target DNA fragmentation. When the 15^th base of the distal end of PAM is cytosine, it can increase the specificity of sgRNA and reduce the off-target. However, when designing the proximal sgRNA sequence of PAM, guanine is preferred in the first base of the seed region, cytosine is avoided, because guanine-enriched sgRNA is easier to fold, forming a more stable structure and reducing off-target effect. 

The length of sgRNA is also closely related to its specificity. The use of shortened sgRNA sequences, i.e., less than 20 nucleotide sequences, can effectively reduce the off-target effect without affecting the gene editing effect. However, Kleaver et al. found that shortening sgRNA sequences does not increase specificity, and may reduce gene editing efficiency. Therefore, the strategy to reduce the off-target effect by shortening the length of sgRNA remains to be further verified. Another simple modification to increase the specificity of sgRNA is called "GG20" strategy, that is, two guanines (called ggX20 sgRNAs) are added at the 5 'end to replace the corresponding GX19 sgRNAs when sgRNA is designed, which can greatly reduce the off-target effect and increase the specificity.

In addition, using the online provision of sgRNA design and off-Target evaluation sites,Such as the CRISPR design tool (http://crispr.mit.edu), ZiFiT, Cas9 (http://zifit.partners.org/ZiFiT/)The design (http://cas9.cbi.pku.edu.cn/), E - CRISPR (http://www.e-crisp.org/), the Cas – offinder (http://www.rgenome.net/cas-offinder/), CHOPCHOP (https://chopchop.rc.fas.harvard.edu/index.php). At the same time, sgRNA can be designed or potential off-target sites can be found in any given genome or sequence by using locally downloaded and installed software tools (such as CasOT) to assess potential off-target sites and setting parameters as required.

2. Controlling the dosage of Cas9-sgRNA

Cas9 - sgRNA the concentration of the compound is a kind of strategy, had the potential to reduce the effect of miss distance is by controlling Cas9 protein or sgRNA expression quantity to reduce the effect of miss distance. Pattanayak et al. through the genomes of HEK293T cells cut, miss loci of five potential high throughput sequencing test found that short sequence, low activity of sgRNA sgRNA than sequence length, high activity has more specificity, and high levels of sgRNA - Cas9 compounds can cut PAM sequence or internal sites nearby, Shalme et al. transfected Cas9 protein and sgRNA into melanoma cells using lentivirus vector, and when Cas9 protein was stably expressed for 7~14 days, the mutation rate of an off-target site containing 3 base mismatch increased from 40% to 50%. This study suggests that continuous cell expression of Cas9 will increase the risk of off-target, and thus the activity of Cas9 can be reduced by blocking antibodies or inhibitors of Cas9 protein, thus reducing the off-target effect. Although the off-target risk can be reduced by regulating the concentration of sgRNA and Cas9 nucleases, the corresponding genomic editing ability will also be weakened after the concentration is reduced. Therefore, the balance between gene editing efficiency and off-target effect must be considered. Existing studies suggest that when the gRNA:Cas9 complex ratio is 2:1 or 3:1, the target gene knockout efficiency is higher and the off-target effect can be effectively reduced.

3. Transforming Cas9

The specificity of THE CRISPR/Cas9 system was improved by modifying the wild-type Cas9.One strategy is to inactivate one of the restriction sites in Cas9 and obtain mutant D10A Cas9 and H840A Cas9 enzymes. Since mutant Cas9 can only cut single strand, two sgRNA are required to guide simultaneously, and two adjacent incisions are generated in different strands of DNA to cause double-strand DNA fracture. Therefore, off-target occurs only when both sgRNA are mismatched, which greatly reduces the off-target effect. Genome editing using Cas9 single-notch enzyme and "paired gRNAs" can effectively improve the gene knockout efficiency and significantly reduce the off-target effect in cell lines (about 50-1,500 times), and this method is applicable to genome editing of human cells, animals, plants, bacteria and other biological models. Another kind of similar to the above strategy, is the nuclease Cas9 protein structure domain mutations, dCas9 inactivated, and then with Fok I nuclease form fusion protein (Cas9 – Fok I), when two sgRNA guide Cas9 - if Fok I combined into located 15 ~ 20 bp target DNA regions, Fok I polymerization and activate the second protein, DNA of intermediate cutting, forming double chain end fracture.The application of this nuclease can induce efficient gene editing in human cells and greatly reduce the level of off-target mutations. Highly specific mutations of Cas9 protein have been developed to avoid most off-target mutations induced by wild-type Cas9.For example, the enhanced mutant eSpCas9, the high-fidelity mutant SPCAS9-HF1, and the high-precision mutant HypaCas9 et al. Therefore, the researchers can reduce the off-target effect by selecting optimized Cas9 nucleases.

4. Selecting the appropriate delivery carrier

Currently Cas9 by plasmid DNA, mRNA and protein is passed to the target cells, the three ways of direct delivery Cas9 mRNA and protein effect of these two approaches can significantly reduce the miss distance CRISPR/Cas9 delivery way mainly includes the physical methods (microinjection, liposome transfection, DianZhuan, high-pressure tail intravenous injection, etc.) and viral vector (slow virus, adenovirus, adeno-associated virus, etc.).Existing studies have shown that the use of a delivery system of Cas9/sgRNA ribonucleoproteins (RNPs) could not insert exogenous DNA sequences into edited cell genomes, effectively reducing off-target effects. In the evaluation of 11 off-target sites, it was found that cationic liposome-coated RNPs delivery system increased SpCas9 protein specificity by nearly 10 times compared with plasmid DNA transfection. In a similar study, the RNPs delivery system increased the ratio of on-target and off-target efficiency of the two sgRNas by about 3 times. When Cas9 mRNA or Cas9 protein and 2 '-o-methyl 3' -thioprecipitates (MS) modified sgRNA was co-transported by the RNPs system, the ratio of on-target and off-target efficiency of the three sgRNA targeting different genes was significantly improved. In addition, CRISPR-Gold, composed of nano-gold particles and cationic polymers, can deliver Cas9 ribonucleoprotein and donor DNA to different cells to correct mutated DNA of Duchenne muscular dystrophy mice and reduce off-target effect. Using other delivery systems, such as active P22 - Cas9 nanometer carrier of self-assembled systems, stylized Cas9 imminent and sgRNA packaging in phage P22 virus-like particle (VLP), and can also realize Cas9 nuclease target sequence specificity of dsDNA cutting, and reduce the miss efficiency, and the work shows the development of P22 as Cas9 cell specific targeting carrier potential. Therefore, when CRISPR/Cas9 system is used for gene editing, delivery vectors such as RNPs, cationic liposomes and gold nanoparticles can be selected to increase the specificity of Cas9 nuclease and reduce off-target effect. 

5. Using the Cas9 enzyme "off switch"

Pawluk et al. for the first time discovered the CRISPR/Cas9 "off switch", the team identified three naturally occurring, can inhibit Cas9 enzyme protein family, the CRISPR resistant proteins to specific block of neisseria meningitidis Cas9 cleaves DNA. Rauch, et al. in listeria was found in a variety of called CRISPRs inhibitors, including AcrllA2 and AcrllA4 2 inhibitors to block from streptococcus pyogenes enzyme or SpyCas9 Cas9 enzyme activity. Subsequently, Huang Zhiwei's research group studied how the "off switch" inhibits the CRISPR/Cas9 system. By comparing the crystal structure of SpyCas9:sgRNA complex binding to AcrllA4, it was found that AcrllA4 competitively binds to the PAM interaction site, blocking SpyCas9's recognition of the double-stranded DNA substrate, and THAT AcrllA4 further inhibited the endonuclide activity of SpyCas9 by blocking the RuvC active site. In recent years, Doudna team found broad spectrum CRISPR/AcrllC1 Cas9 inhibitors and AcrllC3 respectively by different strategies inhibit Cas9 nucleases. AcrllC1 by combining directly to the conservative HNH Cas9 enzyme catalytic domain, to prevent a variety of homologous Cas9 cutting of double-stranded DNA can block the activity of individual Cas9 AcrllC3, through two polymerization induced Cas9 stop its combination with double-stranded DNA. Therefore, in order to avoid continuous expression of Cas9 protein in cells, the above inhibitors can be used to reduce off-target effect.

In addition to the above methods, chemical modification of sgRNA can also effectively reduce off-target effect.For example, crRNA can be chemically modified by 2-fluorine ribose, 2 '-O-methyl 3' -thiophosphate (MS), 2 ', 4 'BNANC [N-ME] bridging nucleic acid and LANlock-nucleic acid, which can improve the stability of sgRNA and increase the specificity of Cas9 endonuclease.DNA nucleotides can also be used to replace some RNA nucleotides at the 5 'and 3' ends of crRNA to guide Cas9 nuclease and reduce the off-target effect of Cas9 nuclease in human genome editing.In addition, the method to optimize the detection of off-target effect plays an important role in ensuring the accuracy of gene manipulation. For example, the lentiviral vector (IDLV) with integrase defect is used to detect the off-target effect of CRISPR/Cas9, and the method can detect the off-target frequency of 1% at least.