There are over 75,000 pathogenic genetic variants that have been identified in humans and catalogued in the ClinVar database. Previously developed genome editing methods using nucleases and base editors have the potential to correct only a minority of those variants in most cell types. A new technique from David Liu’s lab at the Broad Institute could add more precision and flexibility to the CRISPR editing world.
This new approach, published in Nature earlier this week, is called prime editing. It’s a “search-and-replace” genome editing technique that mediates targeted insertions, deletions, and all possible base-to-base conversions. And, it can combine different types of edits with one another. All of this is possible without double strand breaks (DSBs) or donor DNA templates. How does this work? First, an engineered prime editing guide RNA (pegRNA) that both specifies the target site and contains the desired edit(s) engages the prime editor protein. This primer editor protein consists of a Cas9 nickase fused to a reverse transcriptase. The Cas9 nickase part of the protein is guided to the DNA target site by the pegRNA. After nicking by Cas9, the reverse transcriptase domain uses the pegRNA to template reverse transcription of the desired edit, directly polymerizing DNA onto the nicked target DNA strand. The edited DNA strand replaces the original DNA strand, creating a heteroduplex containing one edited strand and one unedited strand. Lastly, the editor guides resolution of the heteroduplex to favor copying the edit onto the unedited strand, completing the process.
Let’s examine the parts in more detail.
The prime editor: A fusion between Cas9 and reverse transcriptase
To decrease the components prime editing would introduce into the cell, the team fused the M-MLV reverse transcriptase (RT) with the Cas9 H840A nickase to create the prime editor (PE). They found that orientation matters: fusing the RT to the C-terminus of the Cas9 nickase resulted in higher editing efficiency. They called this complex PE1.
Building upon prior reverse transcriptase research, (Baranauskas et al., 2012; Arezi and Hogrefe, 2009), the Liu lab created and evaluated 19 PE1 variants with RT mutations known to increase activity, enhance binding between the template and primer binding site, increase processivity, or improve thermostability. What came out on top? The Cas9 nickase fused to a pentamutant of M-MLV RT. They called this system PE2, which had prime editing efficiencies on average 2.3- to 5.1-fold (though up to 45-fold) higher across different genomic sites compared to PE1.
The pegRNA: A template and guide all in one
The other important component of prime editing is the prime editing guide RNA (pegRNA). The pegRNA is a guide RNA that also encodes the RT template, which includes the desired edit and homology to the genomic DNA locus. Sequence complementary to the nicked genomic DNA strand serves as a primer binding site (PBS). This PBS sequence hybridizes to the target site and serves as the point of initiation for reverse transcription.
To optimize pegRNAs, the team found that extending the pegRNA primer binding site to at least eight nucleotides enabled more efficient prime editing in HEK293T cells.
Prime Editor 3 (PE3): Resolving mismatched DNA to favor the edit
Once the prime editor incorporates the edit into one strand, there’s a mismatch between the original sequence on one strand and the edited sequence on the other strand. To guide heteroduplex resolution to favor the edit, the Liu lab turned to a strategy they previously used when they developed base editing (Komor, et al, 2016). By nicking the non-edited strand, they can cause the cell to remake that strand using the edited strand as the template.
A third prime editing system called PE3 does just this by including an additional sgRNA. Using this sgRNA, the prime editor nicks the unedited strand away from the initial nick site (to avoid creating a double strand break), increasing editing efficiencies 2-3 fold with indel frequencies between 1-10%.
Advantages of prime editing
Less constrained by PAM sequence location
The prime editor extends the reach of CRISPR genome editing as it can edit near or far from PAM sites making it less constrained by PAM availability like other methods. The PAM-to-edit distance can be over 30 base pairs for prime editing. Since PAM sites occur every ~8 base pairs on either DNA strand, many previously developed base editors (Table 1 from Rees and Liu, 2019) with a <8 base pair editing window cannot edit within what Fyodor Urnov refers to as “PAM deserts” in the genome.
More versatile and precise than base editing (in certain circumstances)
Base editors developed thus far can only create a subset of changes (C->T, G->A, A->G, and T->C). Prime editing allows for all 12 possible base-to-base changes.
Prime editing is also more precise. Base editors, for example, will edit all the C’s or A’s within the base editing window, while prime editors make a specific edit defined by the pegRNA. In cases when bystander editing is unacceptable, prime editors can be used to avoid this possibility.
However, there are instances where traditional base editors are preferred. For instance, if target nucleotides are positioned within the canonical base editing window, base editing has higher efficiency and fewer indels than prime editing. But for positions that aren’t well positioned within the editing window, prime editing is more efficient due to its lower dependence on PAM placement.
Fewer byproducts and more efficient than homology directed repair
Homology directed repair (HDR) stimulated by double strand breaks has been widely used to generate precise changes. However, the efficiency of Cas9 cleavage is relatively high while the efficiency of HDR is relatively low, meaning that most Cas9-induced DSBs are repaired by non-homologous end joining. As a result, Cas9 treatment causes most products to be indels while the efficiency of HDR is typically less than 10%. In contrast, prime editing can offer ~20-50% efficiency in HEK293T cells with 1-10% indels. In other tested cell types, including post-mitotic primary mouse cortical neurons, the authors report lower prime editing efficiencies, but still see much higher ratios of desired edits to indel byproducts than Cas9-initiated HDR.
What’s next for prime editing?
While prime editing is an exciting step towards more versatile genome editing, it’s new at this point and warrants many additional studies. In their paper, the Liu lab points out the need to investigate off-target prime editing in a genome-wide manner, identify any inadvertent effects the prime editors may have on the cells, and assess in vitro and in vivo delivery strategies. It’s exciting to see the amount of discussion on Twitter about prime editing (here, here, and here) and we look forward to seeing what comes next for prime editing.
Application of Prime Editing
Editing from 1 to 44 bases
Prime editing allows point change to maximum 44-nt long knock-in and maximum 80-nt long knock-out.
Though tagging of fluorescence is not possible, the prime editing platform can add a flag tag and a 6-histine tag that would be useful to isolate endogenous protein in native complexes. It is also a solution when an antibody is not available to locate the protein into the cells.
We can also note that the CRE-Lox system requires only 34 nt with 2 recognition regions of 13bp and 1 spacer region of 8bp. Thus, larger insertion can be performed with a successive combination of the prime editing and the CRE-Lox system using CRE mRNA.
KO of up to 26 codon is also possible with prime editing platform.
Knock-in with no donor
CRISPR-CAS9 gene editing leads to KI using a donor template to repair the double stranded break cause by the endonuclease activity of the CAS9. Without a donor, the classic CRISPR-CAS9 system leads only to KO and so lost of function.
Prime editing provides means to generate changes from 1 up to 44 bases without a donor. Thus, a transient expression of the prime editing complex is enough. There is no risk of genotoxicity caused by random insertion of a plasmid donor and the delivery into the cells is simpler and so, it should more efficient. These 2 key points provide interesting therapeutics perspectives for the up to 75000 genetic diseases.
Further reading: https://www.genengnews.com/insights/genome-editing-heads-to-primetime/
References
Anzalone, Andrew V., et al. “Search-and-replace genome editing without double-strand breaks or donor DNA.” Nature (2019): 1-1. PubMed PMID: 31634902.
Arezi, Bahram, and Holly Hogrefe. “Novel mutations in Moloney Murine Leukemia Virus reverse transcriptase increase thermostability through tighter binding to template-primer.” Nucleic acids research 37.2 (2008): 473-481. PubMed PMID: 19056821. PubMed Central PMCID: PMC2632894.
Baranauskas, Aurimas, et al. “Generation and characterization of new highly thermostable and processive M-MuLV reverse transcriptase variants.” Protein Engineering, Design & Selection 25.10 (2012): 657-668. PubMed PMID: 22691702.
Komor, Alexis C., et al. “Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage.” Nature 533.7603 (2016): 420. PubMed PMID: 27096365. PubMed Central PMCID: PMC4873371.
Rees, Holly A., and David R. Liu. “Base editing: precision chemistry on the genome and transcriptome of living cells.” Nature reviews genetics 19.12 (2018): 770-788. PubMed PMID: 30323312. PubMed Central PMCID: PMC6535181.
By: https://blog.addgene.org/prime-editing-crisp-cas-reverse-transcriptase
内容介绍：
人类的致病遗传变异既有点突变又有碱基插入缺失突变【1】。2016年，哈佛大学David Liu实验室开发出新型单碱基编辑器CBE（Cytosine base editors），实现了C·G–T·A碱基对的自由转换【2】，2017年底他们又构建出ABE（Adenine Base Editor），可实现A·T–G·C碱基对的转换，这对众多点突变遗传病的治疗有重要意义，因而在基因治疗领域的应用前景相当广阔【3】（详见：突破丨Nature长文发表基因编辑最新成果——无需切割DNA也能自由替换ATGC）。不过，除了C·G–T·A和A·T–G·C碱基转换，对其它类型的碱基突变以及碱基的插入缺失突变，目前依然缺乏有效的研究工具：传统的同源重组修复（HDR）需要外源的双链/单链DNA模板，系统复杂且效率低下，这极大地限制了相关工作的开展，因此开发更加高效且广谱的精准基因编辑工具迫在眉睫。
2019年10月21日，哈佛大学David Liu实验室在Nature杂志上发表了题为Search-and-replace genome editing without double-strand breaks or donor DNA的论文。文章开发出了全新的精准基因编辑工具PE （Prime Editors），新工具PE无需额外的DNA模板便可有效实现所有12种单碱基的自由转换，而且还能有效实现多碱基的精准插入与删除（最多可插入44bp的碱基，可删除80bp的碱基），这一全能性的工具为基因编辑领域带来了重大变革。
新工具PE是以CRISPR-Cas9系统为基础，在两方面加以改造：首先是改造单链引导RNA （sgRNA），其3’末端增加了一段RNA序列，新获得的RNA被称作pegRNA；第二则是将Cas9切口酶（H840A突变型，只切断含PAM的靶点DNA链）与逆转录酶融合获得新的融合蛋白。pegRNA的3’端序列有双重角色，一段序列作为引物结合位点（PBS），与断裂的靶DNA链3’末端互补以起始逆转录过程，另一端序列则是逆转录的模板（RT模板），其上携带有目标点突变或插入缺失突变以实现精准的基因编辑（图1）。
图1 改造后的pegRNA结构
基因编辑工具PE的基本原理如图2所示，首先是在pegRNA的引导下，Cas9 H840切口酶切断含PAM的靶点DNA链，断裂的靶DNA链与pegRNA的3’末端PBS序列互补并结合，之后逆转录酶发挥功能，沿RT模板序列开始逆转录反应。反应结束后DNA链的切口处会形成处在动态平衡中的5’-和3’-flap结构，其中3’flap结构的DNA链携带有目标突变，而5’flap结构的DNA链则无任何突变。细胞内5’flap结构易被结构特异性内切酶识别并切除，之后经DNA连接和修复后靶位点处便实现了精准的基因编辑。
图2 基因编辑工具PE的基本原理
在经体外验证和酵母中的验证之后，研究者将野生型的鼠白血病病毒（M-MLV）逆转录酶融合在Cas9 H840切口酶的C末端，构建出了第一代精准基因编辑工具PE1，在293T细胞中PE1的点突变效率为0.7~5.5%, 碱基的增加/删除效率则为4~17%，依然有更大的提升空间。之后研究者通过优化M-MLV逆转录酶得到第二代编辑工具PE2，其点突变效率和碱基的增删效率较PE1有两倍以上的提高。
PE1/2系统只编辑双链DNA的一条链，另一条非编辑链需进一步的DNA修复以完成精准编辑。传统上，通过Cas9切口酶切断非编辑链可以有效提高该链的修复效率。为此研究者在PE2的基础上，增加可切断非编辑链的sgRNA，最终获得新的PE3和PE3b系统（图3）。新系统的编辑效率较PE2提升了近3倍，在293T细胞中的最高编辑效率可达78%。当然，由于使用了两条sgRNA，PE3的随机插入缺失（Indels）风险也随之提高，这是PE3未来需要加以改进的不足之处。
图3 PE3和PE3b的原理图：PE3增加的sgRNA识别位点是未编辑的基因组DNA，PE3b增加的sgRNA只识别编辑后的基因组DNA。
研究者对不同的工具进行比较后发现，与单碱基编辑工具CBE、ABE相比，PE3/3b的单碱基编辑在效率上略有不如，但能实现更精准的编辑；而与传统的HDR相比，PE3/3b有着更高的编辑效率和更低的Indels风险。此外，PE3/3b系统在U2OS、K562、HeLa三种细胞系以及小鼠皮层原代神经元中均能发挥精准编辑效果，这表明新系统有着广泛的适用性。
总体而言，本研究开发的新工具PE是精准基因编辑领域的重大突破，在单碱基随意转换和小片段多碱基的增删方面潜力巨大，这将极大的推动生物医学的基础研究和临床基因治疗研究。
专家点评：
大约有75000人类基因组位点和遗传疾病有关【4】。随着基因编辑快速发展，直接修改基因组治疗遗传疾病带来了可能性。使用CRISPR/Cas9基因编辑系统在致病位点产生DNA双链断裂,利用细胞的NHEJ或者HDR DNA修复通路，已经在动物模型上实现了治疗的人类遗传疾病的目的【5】。但是在大量细胞中同时诱导产生DNA双链断裂，有可能导致基因组的异位，倒位【6】，激活p53信号【7】等潜在问题，损伤基因组。为了消除这些不利因素，基因编辑大牛David liu与同事将失去催化活性的Cas9与脱氨酶融合，在sgRNA指导下，实现了对相应位点的碱基替换，建立了单碱基编辑系统【8,9】。根据融合的脱氨酶不同分为胞嘧啶单碱基编辑系统和腺嘌呤单碱基编辑系统，可以不产生DNA双链断裂的情况下将基因组中C转变成T, 或者将A转变成G。较高安全性和高效性促使单碱基编辑系统问世以来迅速在应用于动物植物以及人类基因组的编辑研究，并且利用动物模型研究治疗遗传疾病的探索。然而，最近多个实验室报道，单碱编辑工具会导致严重的基因组【10,11】和转录组【12,13】
范围内的脱靶，虽有一些改良版本，可以消除转录组范围内的脱靶【12,14】，但是单碱基编辑系统编辑窗口比较窄，而且仅能编辑C-T, T-C, A-G, G-C，不能做C-A, C-G, G-C, G-T, A-C, A-T, T-A和T-G，也不能做插入替换等编辑，这些问题大大局限了单碱基编辑工具在遗传疾病治疗的应用范围。
近日，David liu 团队在Nature杂志发表了题为“Search-and-replace genome editing without double-strand breaks or donor DNA ”的研究论文，建立了一种被称为Prime editing的基因编辑新系统。在不产生DNA双链断裂，不使用DNA模版的情况下，可以在酵母和哺乳动物细胞靶位点高效产生DNA插入、删除和任意单碱基的替换。
他们将nCas9(H840A)与M-MLV逆转录酶形成融合蛋白，基因组结合序列、骨架序列、新的遗传信息以及单链DNA结合序列共同组建了pegRNA。在pegRNA指导下融合蛋白结合到基因组特定序列，nCas9使用单链切割活性，切开pegRNA非互补链。pegRNA携带的单链DNA结合序列与被切开的非互补链按照碱基匹配规律结合，单链DNA暴露一个3’-自由羟基，逆转录酶与之结合，按照碱基匹配规律以RNA为模版合成DNA。随后，合成后的DNA整合到基因组中，实现了靶向位点高效产生DNA的插入、删除和单碱基替换完成基因编辑的过程，构建了被称为PE1的系统。在PE1的基础上David liu团队又修改了M-MLV逆转录氨基酸序列（D200N+L603W+T330P+T306K+W313F），改善了热稳定性，逆转录连续性以及DNA：RNA结合紧密性，提高了编辑效率，建立了PE2系统。在PE2基础上，在pegRNA下游50bp附近，引入另一条与pegRNA方向相反的sgRNA，达到分别切割非互补链的目的，建立PE3系统，进一步提高了编辑效率。
Prime editing基因编辑系统与以往基因编辑系统相比避免了DNA双链断裂的产生、提高了基因编辑效率，拓展适用范围。然而Prime editing组成构件太大也限制了其在体内的临床应用。此外，逆转录酶作为主要构成要件，在细胞中过量表达，其安全性仍然是一个需要考虑的问题.
1. Rees, H.A. & Liu, D.R. Base editing: precision chemistry on the genome and transcriptome of living cells. Nat Rev Genet (2018).
2. Komor AC, Kim YB, Packer MS, Zuris JA, Liu DR. Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. Nature 533, 420-424 (2016).
3. Gaudelli, N.M. et al. Programmable base editing of A*T to G*C in genomic DNA without DNA cleavage. Nature 551, 464-471 (2017).
4. Landrum, M. J. et al. ClinVar: public archive of interpretations of clinically relevant variants. Nucleic Acids Res
44, D862-D868, doi:10.1093/nar/gkv1222 (2016).
5. Jinek, M. et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816-821, doi:10.1126/science.1225829 science.1225829 [pii] (2012).
6. Kosicki, M., Tomberg, K. & Bradley, A. Repair of double-strand breaks induced by CRISPR-Cas9 leads to large deletions and complex rearrangements. Nat Biotechnol 36, 765-771, doi:10.1038/nbt.4192nbt.4192 [pii] (2018).
7. Haapaniemi, E., Botla, S., Persson, J., Schmierer, B. & Taipale, J. CRISPR-Cas9 genome editing induces a p53-mediated DNA damage response. Nat Med 24, 927-930, doi:10.1038/s41591-018-0049-z10.1038/s41591-018-0049-z [pii] (2018).
8. Gaudelli, N. M. et al. Programmable base editing of A*T to G*C in genomic DNA without DNA cleavage. Nature 551, 464-471, doi:10.1038/nature24644 nature24644 [pii] (2017).
9. Komor, A. C., Kim, Y. B., Packer, M. S., Zuris, J. A. & Liu, D. R. Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage.Nature 533, 420-424, doi:10.1038/nature17946nature17946 [pii] (2016).
10. Zuo, E. et al. Cytosine base editor generates substantial off-target single-nucleotide variants in mouse embryos.Science 364, 289-292, doi:10.1126/science.aav9973 science.aav9973 [pii] (2019).
11. Jin, S. et al. Cytosine, but not adenine, base editors induce genome-wide off-target mutations in rice. Science 364, 292-+, doi:10.1126/science.aaw7166 (2019).
12. Zhou, C. et al. Off-target RNA mutation induced by DNA base editing and its elimination by mutagenesis. Nature 571, 275-278, doi:10.1038/s41586-019-1314-0 10.1038/s41586-019-1314-0 [pii] (2019).
13. Grunewald, J. et al. Transcriptome-wide off-target RNA editing induced by CRISPR-guided DNA base editors. Nature 569, 433-437, doi:10.1038/s41586-019-1161-z10.1038/s41586-019-1161-z [pii] (2019).
14. Grunewald, J. et al. CRISPR DNA base editors with reduced RNA off-target and self-editing activities.Nat Biotechnol 37, 1041-1048, doi:10.1038/s41587-019-0236-610.1038/s41587-019-0236-6 [pii] (2019).
子夜星河 