When it comes to gene editing, everyone will think of CRISPR technology. This famous technology in the past is still hot, especially our CRISPR god Zhang Feng. Recently published articles frequently appeared in well-known magazines, which caused a heated discussion. Over time, the CRISPR technology has not disappeared and has been online. But anything, including technology, has its advantages and disadvantages . Of course, CRISPR technology is no exception.

The CRISPR technology is so easy to use!

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After four years of technology, the familiar advantages are obvious: the reliance on ES cells is abandoned, and CRISPR technology enables editing of various species such as rats, pigs, monkeys, and zebrafish ; CRISPR technology relies on Cas9 and sgRNA. Achieve "precise" cutting, simplifying the experimental steps and greatly shortening the experimental cycle. Cycle is short, small amount of work, the attendant is low down the cost. In summary, the cost-effective technology of course will always be the fire.

CRISPR technology also has shortcomings?

Since its inception, CRISPR technology has been applied to scientific research in many laboratories around the world. While enjoying its advantages, its drawbacks are also well known: sgRNA and Cas9 protein are not always working diligently. Who is not tired yet? When the sgRNA that should be precisely positioned is also eye-catching, the Cas9 protein of the standard PAM sequence should be recognized, and occasionally it is not taken care of. The result is off-target , which affects the editing efficiency.

It is not terrible to have "disease". The key is "the right medicine"?

Doctors pay attention to the right medicine, and scientific research ideas are no exception. Find out the cause and solve the problem. Since there are problems with sgRNA and Cas9 protein, we will take different approaches to different diseases:

Optimize sgRNA

(1) Change the self: change the length of sgRNA , cut off 2-3 bases at the 5' end or add 2 guanines before the recognition sequence, the cutting activity is unchanged, and the off-target rate is reduced by 5000 times.

(2) Enhance self: increase the stability of sgRNA. For example , adding a pair of AU base pairings to gRNA . In addition, studies have shown that Cas9 orthologues are more specific for certain sites than SpCas9, which undoubtedly provides a new idea for improving specificity.

Improve the specificity of Cas9 protein itself

Specificity is important for the family and is important for CRISPR technology. Specificity is related to itself on the one hand and to the temptation of the environment on the other. Therefore, for the Cas9 protein, we must consider both internal and external factors.

(1) Brighten your eyes and identify who is your Mr/Mrs Right: Enhance the ability of Cas9 protein to recognize standard PAM sequences . Mutant D1135E is more specific for the recognition of standard PAM, thereby reducing the recognition of non-standard PAM sequences. Off target.

(2) It is good to be in love with the right person. Don't always smother the Cas9 protein : Site-directed mutagenesis to form eSpCas9 and SpCas9-HF1 . The mutant weakens the ability of Cas9 protein to bind to DNA non-specifically. Competitiveness to the sequence to bind to the Cas9 protein.

(3) Reduce the temptation of the outside world: regulate the expression level of Cas9 protein in cells. Inducible Cas9 protein is constructed by using an inducible promoter, inserting a 4-HT-dependent intein, etc. , reducing the time the genome is exposed to Cas9 and sgRNA complexes, ie reducing the probability of binding of Cas9 to non-targeting sequences, thereby reducing Off-target rate.

(4) Disengagement: Only retain its nucleic acid recognition ability and break its endonuclease activity. Of course, it is also necessary for itself: mutants H840A and D10A which reduce the function of HNH or RuvC domain ; construct CRISPRi and FokI-dCas9 which cause the Cas9 protein to lose endonuclease activity and perform gene editing by means of other endonucleases .

百奥赛图杀手锏——Southern blot detection gold standard

SgRNA design is rigorous

Discard sgRNAs that may be off target to functional genomic sequences

Pure RNA injection

Shorten the action time of Cas9 protein and sgRNA

Southern blot detection

Analysis of a large number of data statistics found that using ES cell method preparation, the random insertion probability is about 20%, using CRISPR/Cas9 technology, about 32% probability of random insertion. And 14% of these 32% random insertions could not be removed by mating passage, and the gold standard for random insertion was Southern blot . The Bao Sai map strictly controls the quality and insists on Southern blot detection to ensure that the animals obtained by genetic editing are not randomly inserted.

The purpose of Southern hybridization is to detect the insertion position and the number of copies of the gene of interest. Southern blot hybridization is a method for locating specific sequences of genomic DNA. The procedure is as follows: the enzymatically digested DNA fragments are separated by electrophoresis, and then the DNA on the gel is denatured and the single-stranded DNA fragments are transferred to the nylon membrane in situ or On other solid supports, they are fixed by dry baking or ultraviolet irradiation, and then hybridized with a corresponding structure of the DIG-labeled probe, and developed by autoradiography or enzymatic reaction to detect the content of a specific DNA molecule.

reference:

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[2] Kleinstiver BP, Prew MS, Tsai SQ, et al. Engineered CRISPR-Cas9 nucleases with altered PAM specificities[J]. Nature, 2015, 523(7561): 481.

[3] Terao M, Tamano M, Hara S, et al. Utilization of the CRISPR/Cas9 system for the efficient production of mutant mice using crRNA/tracrRNA with Cas9 nickase and FokI-dCas9[J]. Experimental animals, 2016, 65 (3): 275-283.

[4] Müller M, Lee CM, Gasiunas G, et al. Streptococcus thermophilus CRISPR-Cas9 Systems Enable Specific Editing of the Human Genome [J]. Molecular Therapy, 2016, 24(3): 636–644.

[5] Qi L, Larson M, Gilbert L, et al. Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression [J]. Cell, 2013, 152(5):1173.

[6] Luke A. Gilbert, Matthew H. Larson, Morsut L, et al. CRISPR-Mediated Modular RNA-Guided Regulation of Transcription in Eukaryotes [J]. Cell, 2013, 154(2):442.

[7] Hsu PD, Scott DA, Weinstein JA, et al. DNA targeting specificity of RNA-guided Cas9 nucleases [J]. Nature Biotechnology, 2013, 31(9): 827-32.

[8] Fu Y, Sander JD, Reyon D, et al. Improving CRISPR-Cas nuclease specificity using truncated guide RNAs [J]. Nature Biotechnology, 2014, 32(3): 279.

[9] Kleinstiver BP, Vikram P, Prew MS, et al. High-fidelity CRISPR-Cas9 variants with undetectable genome-wide off-targets: [J]. Nature, 2016, 529(7587): 490-495.

[10] Slaymaker I M, Gao L, Zetsche B, et al. Rationally engineered Cas9 nucleases with improved specificity[J]. Science, 2016, 351(6268):84.

[11] Chiang T W W, Sage C L, Larrieu D, et al. CRISPR-Cas9 D10A nickase-based genotypic and phenotypic

Screening to enhance genome editing[J]. Scientific Reports, 2016, 6:24356.

[12] Pan Y, Shen N, Jungklawitter S, et al. CRISPR RNA-guided FokI nucleases repair a PAH variant in a phenylketonuria model[J]. Scientific Reports, 2016, 6:35794.

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