Recent Therapeutic Gene Editing Applications to Genetic Disorders

Round 1

Reviewer 1 Report

Comments:

In the manuscript (review), author reviewed the recent CRISPR based gene editing applications, including several gene editing methods, current development of CRIPSR methods, gene editing tool delivery methods, and many disease applications. This is a close-to-complete review paper to me, there are only a few suggestions I would like to give that may make this more general audience.

1.     Since authors started with gene editing methods, it worth to explain the editing mechanism, like ZFN, TALENs, then jump to CRIPSR.

2.     CRISPR is coming from bacterial phase defense system, this is important of its history, I did not find in the manuscript.

3.     Before introducing applications of HUMAN, author should consider introduce the process or milestone application/explorations on prokaryotic systems.

4.     Recombineering (MAGE) is another major gene editing tool now, author should consider adding that in.

5.     I did not find a statement of ‘why CRISPR’ is most applied into Human. It should be claiming the advantages (comparing with others) thus leading to human applications.

6.     In perspective, author should consider adding a discussion of different CRISPR systems (Cas12, 13 and even Cas14).

Author Response

Thanks to Reviewer 1 for the useful comments that have improved the manuscript. I have responded in bold below each comment:

1. Since authors started with gene editing methods, it worth to explain the editing mechanism, like ZFN, TALENs, then jump to CRIPSR.

In the section ‘‘1.1. Original gene editing tools’’, the second and the third paragraphs explain briefly how ZFNs and TALENs work in gene editing (already occupying >250 words), before jumping to CRISPR in the following section. Since none of the preclinical and clinical studies cited throughout the text have used ZFNs or TALENs, I don’t think that additional extensive explanation of ZFN/TALEN mechanisms would be of significant relevance.

2. CRISPR is coming from bacterial phase defense system, this is important of its history, I did not find in the manuscript.

In the section ‘‘2. Molecular mechanisms of CRISPR’’, the first two paragraphs explain in details how CRISPR was found in bacteria, as a defense mechanism against virus.

3. Before introducing applications of HUMAN, author should consider introduce the process or milestone application/explorations on prokaryotic systems.

Since the manuscript focus primarily on recent therapeutic gene editing applications to genetic disorders in human, I think it would be out of the scope to discuss applications in prokaryotic systems. An important part of its discovery in bacteria is explained in section ‘‘2. Molecular mechanisms of CRISPR’’.

4. Recombineering (MAGE) is another major gene editing tool now, author should consider adding that in.

I agree with this point and have included the following sentence at the end of the last paragraph of the section ‘‘3.2. Prime editing’’:

‘‘Furthermore, multiplex automated genome engineering (MAGE) in eukaryotes involves single-stranded DNA-mediated recombineering, and can incorporate fragments of several kb in length [53].’’         

5. I did not find a statement of ‘why CRISPR’ is most applied into Human. It should be claiming the advantages (comparing with others) thus leading to human applications.

The advantages of CRISPR, over other methods, that led to widespread applications in human are explained in section ‘‘1.2. The missing piece’’, as well as in section ‘‘2.1. Harnessing Cas9 for gene editing’’.

6. In perspective, author should consider adding a discussion of different CRISPR systems (Cas12, 13 and even Cas14).

The mechanisms of action of Cas12 and Cas13 are described in section ‘‘2.1. Harnessing Cas9 for gene editing’’, and are involved in several preclinical studies and clinical trials cited throughout the section ‘‘6. Applications of Therapeutic Gene Editing’’. Also, a new Cas13-based method is described in the fifth paragraph of the section ‘‘9. Future perspectives’’. Since a huge variety of Cas proteins have been identified, but are not relevant to this review, I referred the readers to a comprehensive classification system that was developed in a wiki format and is available online, as explained in the last paragraph of the section ‘‘2. Molecular mechanisms of CRISPR’’.

Reviewer 2 Report

The article entitled “Recent Therapeutic Gene Editing Applications to Genetic Disorders” is an excellent, exhaustive, timely and well written review of gene editing efforts. I have only minor remarks:

Perhaps the article Sequence-specific artificial photo-induced endonucleases based on triple helix-forming oligonucleotides by Perrouault L, Asseline U, Rivalle C, Thuong NT, Bisagni E, Giovannangeli C, Le Doan T, Hélène C. Nature. 1990 Mar 22;344(6264):358-60. doi: 10.1038/344358a0. PMID: 2156170 merits mention in the brief historical summary.

The author correctly avoids to write about gene editing as a strategy for correcting cancer driver mutations. Yet perhaps it could be useful to explicitly state that even CRISPR-Cas9 gene editing will not be able to correct a gene defect in all the cancer cells with the remaining cells rapidly reconstituting the tumor.

Undesired on-target effects should be cited (Unexpected CRISPR on-target effects. Lee H, Kim JS. Nat Biotechnol. 2018 Sep;36(8):703-704. doi: 10.1038/nbt.4207. Epub 2018 Jul 30. PMID: 30059492).

In the chapter on germline editing, the general hesitance towards germline gene manipulation, that is independent of the specific technical approach, should be mentioned.

Author Response

Thank you very much to Reviewer 2 for the constructive comments that have significantly enhanced the manuscript. I have responded in bold below each comment:

Perhaps the article Sequence-specific artificial photo-induced endonucleases based on triple helix-forming oligonucleotides by Perrouault L, Asseline U, Rivalle C, Thuong NT, Bisagni E, Giovannangeli C, Le Doan T, Hélène C. Nature. 1990 Mar 22;344(6264):358-60. doi: 10.1038/344358a0. PMID: 2156170 merits mention in the brief historical summary.

I agree with this comment and have included the following reference to Perrouault et al. about artificial photoendonucleases that can break DNA at a specific sequence, in section 1, on page 1 (see track changes in the text file):

‘‘…and that artificial photoendonucleases can break DNA at a specific sequence [2]’’

The author correctly avoids to write about gene editing as a strategy for correcting cancer driver mutations. Yet perhaps it could be useful to explicitly state that even CRISPR-Cas9 gene editing will not be able to correct a gene defect in all the cancer cells with the remaining cells rapidly reconstituting the tumor.

This is a good point, and I added the following sentence about the limitations of editing cancer driver mutations in section 5, on page 9:

‘‘Editing cancer driver mutations also represents an interesting approach for cancer therapies, however current CRISPR-Cas9 gene editing tools may not induce sufficient editing rates, leaving behind unedited cancer cells able to rapidly reconstitute the tumor.’’

Undesired on-target effects should be cited (Unexpected CRISPR on-target effects. Lee H, Kim JS. Nat Biotechnol. 2018 Sep;36(8):703-704. doi: 10.1038/nbt.4207. Epub 2018 Jul 30. PMID: 30059492).

I have included the following sentence and 2 references about undesired on-target effects in section 6, on page 9, although this issue was already discussed and referenced in section 8.1.:

‘‘Undesired damage may also include monoallelic and biallelic on-target indels, and even larger deletions and complex rearrangements [63,64].’’ 

In the chapter on germline editing, the general hesitance towards germline gene manipulation, that is independent of the specific technical approach, should be mentioned.

I agree with this interesting point, so I have included the following few sentences to discuss the general hesitance towards germline gene editing in section 8.4, on page 31:

‘‘This type of gene editing has been met with significant hesitance from the scientific community, ethicists, policymakers, and the public for a variety of reasons that are largely independent of the caution surrounding somatic gene editing, which targets non-reproductive cells and does not affect future generations. These reasons may include unforeseen genetic issues such as off-target effects persisting across generations, or the lack of consent from individuals who will inherit the edited genes. Many countries have legal and regulatory restrictions on germline gene editing, reflecting the reluctance and ethical concerns surrounding this practice.’’

Reviewer 3 Report

The article summarized CRISPR gene editing in therapeutic applications and also discussed their potential limitations. Minor revision is suggested. 

(1)     The article is well-written, but I think a review article should provide a take-home message to the authors, which seems to be missing in the article. 

(2)     There is no innovation in this review, considering that many CRISPR-related articles have been published. 

(3)     Please consider discussing potential work that can be done to overcome the limitations or safety concerns discussed in section 8. 

Author Response

Thank you to Reviewer 3 for the comments that contributed to improve the manuscript. I have responded in bold below each comment:

(1)     The article is well-written, but I think a review article should provide a take-home message to the authors, which seems to be missing in the article.

I agree with this suggestion and have included the following few sentences to reinforce the take-home message in the conclusion paragraph of the text:

‘‘In conclusion, the initial observations in the ‘80s of unusual clustered DNA repeats in bacteria led to the development of a variety of advanced gene editing technologies based on CRISPR. Cas9 nucleases are widely used and efficient to generate gene knockouts, however this advantage comes at the price of higher off-target effects. The invention of BE has enhanced the precision of gene editing while reducing off-target indels, but is limited to single nucleotide substitutions, and may induce bystander mutations as well as off-target DNA and RNA deamination. Perhaps PE is the most interesting approach for therapeutic gene editing so far since it is more versatile, addressing a wider range of mutation classes and sizes. Moreover, PE limits the likelihood of unintended changes elsewhere in the genome. High editing efficiencies, sometimes >75%, are achieved in blood and ophthalmic disorders due to greater accessibility of the target cells. On the opposite, in vivo editing efficiencies are lagging behind in most neurological disorders due to several challenges like the blood-brain barrier, the complex structure of brain cells, the inactive proliferation state of neurons, and potential immune responses.’’

(2)     There is no innovation in this review, considering that many CRISPR-related articles have been published.

Please see point 3 below.

(3)     Please consider discussing potential work that can be done to overcome the limitations or safety concerns discussed in section 8.

This is an interesting point, so I added the following paragraph at the end of section 8.1 to discuss more about what can be done to overcome the limitations of off-target effects, in addition to section 8.2 that already included suggestions to overcome limitations:

‘‘To overcome the limitations of off-target effects in therapeutic gene editing, more precise algorithms can be developed to design more specific sgRNAs and pegRNAs. New modifications in Cas proteins to alter their recognition properties can improve their fidelity. Developing targeted delivery systems that bring the gene editing components specifically to the cells or tissues of interest can reduce the risk of off-target effects in other parts of the body. Strategies for controlling the timing of gene editing can reduce off-target effects by limiting the duration of enzyme activity.’’