A cas12a protein mutant, a base editor containing the same and application

Abstract

The application discloses a Cas12a protein mutant, a base editor containing the same and application. The Cas12a protein mutant is based on dLbCas12a protein and is subjected to any one or a combination of at least two of the following mutations: D156R, G532R and K538R combination mutation, G532R and K595R combination mutation or G532R, K538V and Y542R combination mutation. The application is based on dLbCas12a and is reformed to obtain a Cas12a protein mutant with a widened PAM recognition range, and a high-efficiency base editor capable of recognizing a widened PAM range is constructed. The base editor can realize high-efficiency C>T or A>G editing on a classic PAM and a non-classic PAM at a cell and embryo level, can realize simultaneous multi-site high-efficiency C>T or A>G editing at a cell and embryo level, and successfully enriches an existing base editing toolbox.

Description

Technical Field

[0001] This invention belongs to the field of gene editing technology, and relates to a Cas12a protein mutant, a base editor containing it, and its applications. Background Technology

[0002] Thanks to the emergence and development of gene-editing technology, we now live in an era of flexible and precise manipulation of genes. We can modify genes to study gene function, conduct gene therapy, and improve the traits of plants and animals. The advent of gene-editing technology has greatly promoted innovative development in the study of human genetic diseases, from symptoms and genetic basis research to disease treatment.

[0003] With the discovery and development of the CRISPR / Cas system, it has become one of the most commonly used novel gene editing technologies by researchers due to its advantages such as simple design, ease of operation, high editing activity, and low cost. According to surveys, the largest category of human genetic diseases is caused by gene point mutations (also known as single nucleotide polymorphisms, SNPs), accounting for approximately 58% of pathogenic genetic variations. Among these, C>T mutations account for 47% and A>G mutations account for 14% of diseases caused by gene point mutations. Before the advent of base editing tools, base substitution was mainly performed through homology-directed repair (HDR). Although the CRISPR / Cas system has significantly improved HDR efficiency, its efficiency remains relatively low. Therefore, developing novel gene editing methods that can effectively introduce or correct single-base mutations in the genome is of great significance for the study of the mechanisms and treatment of point mutation-related diseases.

[0004] Base editing technology is a newly developed gene editing technology that can meet the needs of the aforementioned single-base mutations. By fusing deaminases with the CRISPR / Cas system, cytosine base editors (CBEs) that can induce the conversion of base C to T and adenine base editors (ABEs) that can induce the conversion of base A to G have been successfully constructed (Komor AC, Kim YB, Packer MS, et al. Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage[J]. Nature, 2016, 533(7603):420-424; Gaudelli NM, Komor AC, Rees HA, et al. Programmable base editing of A·T to G·C in genomic DNA without DNA cleavage[J]. Nature, 2017, 551(7681):464-471). However, most commonly used ABEs and CBEs are based on Cas9, which recognizes C / G-rich PAM (Protospacer adjacent motif) sequences, thus limiting the targeting range of these base editors. Cas12a (formerly known as Cpf1) can recognize A / T-rich PAM sequences, thus complementing existing Cas9-based base editors and broadening the target range of base editing systems. Furthermore, Cas12a has other advantages: it produces sticky ends after cleaving the DNA double strand; it possesses RNase activity, capable of cleaving pre-crRNA to form multiple mature guide RNAs (gRNAs); and it has higher specificity than Cas9. Among these, Cas12a's RNase activity is particularly important, as it can cleave pre-crRNA to form multiple mature guide RNAs, simultaneously targeting multiple gene sites and enabling simultaneous multi-gene editing. This makes Cas12a a valuable gene editing tool with unique applications. Currently, while reported natural Cas9 and Cas12a homologs can recognize different PAM sequences, their target range remains limited, especially since some natural proteins have stronger PAM restrictions or poorer activity in mammalian cells. Therefore, modifying existing Cas9 and Cas12a proteins to obtain protein variants with fewer PAM restrictions is an effective way to overcome this limitation.

[0005] Currently, researchers have fused different Cas9 variants with deaminases to develop a series of PAM sequence-extended base editors, but studies on expanding the targeting range of Cas12a-derived base editors are still scarce. Furthermore, attempts to use Cas12a-derived base editors for simultaneous multi-site editing have encountered extremely low efficiency issues. In conclusion, developing base editors based on Cas12a with a broader targeting range and the ability to efficiently edit multiple gene sites simultaneously is of great significance to the field of gene editing. Summary of the Invention

[0006] To address the shortcomings of existing technologies and practical needs, this invention provides a Cas12a protein mutant, a base editor containing it, and its applications. Based on dLbCas12a, this invention introduces mutations to obtain a Cas12a protein mutant with an expanded PAM recognition range, which can be effectively applied to gene editing, expanding the gene editing target range. At the same time, by utilizing its RNase activity, a highly efficient base editor can be obtained that can simultaneously perform base editing on multiple gene sites.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] In a first aspect, the present invention provides a Cas12a protein mutant, wherein the Cas12a protein mutant is based on the dLbCas12a protein and undergoes any one or at least two of the following combinations of mutations: D156R, G532R and K538R, G532R and K595R, or G532R, K538V and Y542R. The amino acid sequence of the dLbCas12a protein includes the sequence shown in SEQ ID NO.1.

[0009] In this invention, addressing the current limitation of the Cas12a protein's targeting range, a modified version of the dLbCas12a protein (Catalytically dead Lachnospiraceae bacterium Cas12a) is obtained by introducing combined mutations of D156R, G532R, and K538R (D156R / G532R / K538R), G532R and K595R (G532R / K595R), or G532R, K538V, and Y542R (G532R / K538V / Y542R). This results in a Cas12a protein mutant with an expanded PAM (Physical Targeting Aspect) range, which can be effectively applied in gene editing and is of great significance for broadening the genome targeting range of existing gene editing systems.

[0010] SEQ ID NO.1:

[0011]

[0012] In a second aspect, the present invention provides a nucleic acid molecule containing a nucleic acid sequence encoding the Cas12a protein mutant described in the first aspect.

[0013] Thirdly, the present invention provides an expression vector containing the nucleic acid molecule described in the second aspect.

[0014] Fourthly, the present invention provides a recombinant cell containing the nucleic acid molecules described in the second aspect.

[0015] Fifthly, the present invention provides the application of the Cas12a protein mutant described in the first aspect in the preparation of gene editing products.

[0016] In a sixth aspect, the present invention provides a base editor, wherein the base editor is a fusion protein formed by fusing a deaminase and the Cas12a protein mutant described in the first aspect.

[0017] In this invention, a Cas12a protein mutant with an expanded PAM recognition range is obtained through genetic modification. This mutant can be fused with a deaminase to further construct a highly efficient base editor with an expanded PAM recognition range.

[0018] In this invention, the deaminase may be fused to the N-terminus, C-terminus, or internal fusion site of the Cas12a protein mutant.

[0019] In this invention, any deaminase used in the art to construct a base editor is applicable to this invention.

[0020] In this invention, the deaminase may be selected from cytosine deaminase or adenine deaminase.

[0021] In this invention, the cytosine deaminase is selected from human cytosine deaminase (hAPOBEC3A, hA3A).

[0022] This invention has found that, compared with a base editor constructed using rat-derived cytosine deaminase (rAPOBEC1), a base editor obtained by fusing cytosine deaminase hA3A and the Cas12a protein mutant of this invention has higher editing efficiency and is not affected by the sequence environment.

[0023] In this invention, the amino acid sequence of hA3A includes the sequence shown in SEQ ID NO.2 or SEQ ID NO.3.

[0024] SEQ ID NO.2:

[0025] MEASPASGPRHLMDPHIFTSNFNNGIGRHKTYLCYEVERLDNGTSVKMDQHRGFLHNQAKNLLCGFYGRHAELRFLDLVPSLQLDPAQIYRVTWFISWSPCFSWGCAGEVRAFLQENTHVRLRIFAARIFDYDPLYKEALQMLRDAGAQVSIMTYDEFKHCWDTFVDHQGCPFQPWDGLDEHSQALSGRLRAILQNQGN.

[0026] SEQ ID NO.3:

[0027] MEASPASGPRHLMDPHIFTSNFNNGIGRHKTYLCYEVERLDNGTSVKMDQHRGFLHGQAKNLLCGFYGRHAELRFLDLVPSLQLDPAQIYRVTWFISWSPCFSWGCAGEVRAFLQENTHVRLRIFAARIYDYDPLYKEALQMLRDAGAQVSIMTYDEFKHCWDTFVDHQGCPFQPWDGLDEHSQALSGRLRAILQNQGN.

[0028] In this invention, the adenine deaminase may be selected from TadA-8e (TadA*8e).

[0029] In this invention, the amino acid sequence of TadA-8e includes the sequence shown in SEQ ID NO.4.

[0030] SEQ ID NO.4:

[0031] MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGWRNSKRGAAGSLMNVLNYPGMNHRVEITEGILADECAALLCDFYRMPRQVFNAQKKAQSSIN.

[0032] In this invention, a cytosine base editor (named dLbCas12a-CBE) is prepared by fusing cytosine deaminase with a mutant of the dLbCas12a protein of this invention, and an adenine base editor (named dLbCas12a-ABE) is prepared by fusing adenine deaminase with a mutant of the Cas12a protein of this invention. The schematic diagrams of their mechanisms of action are shown below. Figure 1 and Figure 2As shown, mutants of the dLbCas12a protein form a complex by binding to guide RNA, then search for target sites on the genome. After the guide RNA binds to the genome, it uses the deamination activity of deaminase to deaminate bases within a window range, thereby achieving base editing.

[0033] Furthermore, the Cas12a protein possesses RNase properties, enabling it to cleave tandem pre-crRNA to form multiple mature guide RNAs. These guide RNAs can then form different base editor complexes, allowing for simultaneous and efficient editing of multiple gene sites. Specifically, for example... Figure 9 As shown, multiple guide RNAs can be tandemly downstream of the promoter. After transcription to form pre-crRNA, the RNase properties of Cas12a can be used to process it into multiple mature guide RNAs. After Cas12a and guide RNA form a complex, they can search for target sites in the genome to achieve gene editing. Simultaneous editing of multiple genomic sites can be achieved at the cellular and embryonic levels, and the editing efficiency is no different from that of single-site editing.

[0034] In a seventh aspect, the present invention provides the application of the Cas12a protein mutant described in the first aspect or the base editor described in the sixth aspect in gene editing.

[0035] Preferably, the gene editing can be performed simultaneously at multiple gene sites.

[0036] Eighthly, the present invention provides a gene editing method, the gene editing method comprising:

[0037] Gene editing can be performed using a CRISPR-Cas system composed of the Cas12a protein mutant and guide RNA as described in the first aspect, or...

[0038] The CRISPR-Cas system, composed of the Cas12 protein mutant described in the first aspect and guide RNA formed by processing tandem guide RNA, is used for simultaneous gene editing at multiple gene sites.

[0039] Ninthly, the present invention provides a base editing method, the base editing method comprising:

[0040] Base editing is performed using the base editor described in the sixth aspect.

[0041] Preferably, the base editing method includes:

[0042] Simultaneous base editing at multiple gene sites using the base editor and tandem guide RNA described in the sixth aspect.

[0043] Preferably, the base editing method includes the following steps:

[0044] A Cas12a mutant capable of recognizing non-classical PAMs was obtained by introducing any of the following mutants into the dLbCas12a protein: D156R / G532R / K538R, G532R / K595R, or G532R / K538V / Y542R. This mutant was then fused with a deaminase to prepare a base editor with a broadened targeting range. This editor, along with a target site guide RNA, was then transferred into the cells to be edited, and the protein-RNA complex formed was used to edit the target site.

[0045] Compared with the prior art, the present invention has the following beneficial effects:

[0046] Based on dLbCas12a, this invention introduces mutants D156R / G532R / K538R, G532R / K595R, and G532R / K538V / Y542R to obtain Cas12a protein mutants with an expanded PAM recognition range: enCas12a, RR, and RVR. Subsequently, cytosine deaminase hA3A or adenine deaminase TadA-8e are fused with these Cas12a protein mutants to construct a high-performance protein capable of recognizing a broadened PAM range. This efficient base editor enables highly efficient C>T or A>G editing of both classical PAM (TTTV) and non-classical PAM at both the cellular and embryonic levels. Furthermore, leveraging the RNase activity of Cas12a itself, the system can simultaneously and efficiently edit multiple gene sites. It has been demonstrated that this system can simultaneously and efficiently edit multiple genomic sites at both the cellular and embryonic levels using C>T or A>G techniques, with editing efficiency comparable to single-site editing, successfully enriching the existing base editing toolkit. Attached Figure Description

[0047] Figure 1 This is a schematic diagram illustrating the principle of the cytosine base editor of the present invention;

[0048] Figure 2 This is a schematic diagram illustrating the principle of the adenine base editor of the present invention;

[0049] Figure 3 This is a schematic diagram of the cytosine base editor structure of the present invention;

[0050] Figure 4 This is a schematic diagram of the adenine base editor structure of the present invention;

[0051] Figure 5 A graph showing the efficiency of cytosine base editing in HEK293T cells;

[0052] Figure 6 A graph showing the efficiency of adenine base editing in HEK293T cells;

[0053] Figure 7 A graph showing the efficiency of cytosine base editing in embryos;

[0054] Figure 8 A graph showing the efficiency of adenine base editing in embryos;

[0055] Figure 9 A schematic diagram illustrating the use of Cas12a's RNase function to achieve simultaneous editing at multiple sites;

[0056] Figure 10A The image shows the results of simultaneous editing of multiple sites using a cytosine base editor constructed on HEK293T cells using a Cas12a protein mutant.

[0057] Figure 10B Image showing the results of simultaneous editing of multiple sites using an adenine base editor constructed on HEK293T cells using a Cas12a protein mutant;

[0058] Figure 11 The image shows the results of simultaneous editing of multiple sites using a cytosine base editor constructed on porcine embryos using a Cas12a protein mutant. Detailed Implementation

[0059] To further illustrate the technical means and effects of this invention, the following description, in conjunction with embodiments and accompanying drawings, provides a further explanation of the invention. It is understood that the specific embodiments described herein are merely illustrative of the invention and not intended to limit it.

[0060] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0061] Example 1

[0062] In this embodiment, Cas12a protein mutants were constructed. Based on dLbCas12a (amino acid sequence shown in SEQ ID NO.1), mutations D156R / G532R / K538R, G532R / K595R, and G532R / K538V / Y542R were introduced to obtain three Cas12a protein mutants, which were named enCas12a, RR, and RVR, respectively.

[0063] Example 2

[0064] This embodiment constructs a base editor, taking the N-terminus as an example. The three mutants of dLbCas12a from Example 1—enCas12a, RR, and RVR—are fused at their N-termini with cytosine deaminase hA3A and adenine deaminase TadA-8e to construct the relevant base editor. A schematic diagram is shown below. Figure 3 and Figure 4 As shown. Figure 3 The diagram shows the structure of the cytosine base editor. Two hA3A proteins were used (one containing the Y130F mutation, sequence SEQ ID NO.2; the other containing the N57G mutation, sequence SEQ ID NO.3), where UGI is a uracil glycosylase inhibitor, NLS is a nuclear localization signal, and dCas12a represents the wild-type dLbCas12a protein as a control. Figure 4 This is a schematic diagram of the adenine base editor structure. The TadA-8e used contains the V106W mutation and its sequence is SEQ ID NO.4. Here, dCas12a represents the wild-type dLbCas12a protein. As a control, bqNLS is the bipartite SV40 NLS.

[0065] Example 3

[0066] This embodiment analyzes the editing efficiency of the base editor constructed in Example 2 at both the cellular and embryonic levels.

[0067] Specific methods include:

[0068] At the cellular level, the coding plasmids for the base editor and guide RNA were transfected into cells with appropriate confluence. After 72 hours, the cellular genome was extracted, and the target editing sites were amplified by PCR using specific primers, followed by Sanger sequencing. The sequencing results could be analyzed using the EditR online tool to assess editing efficiency.

[0069] At the embryonic level, RNA was obtained through in vitro transcription using a base editor and guide RNA plasmid. The RNA was then injected into animal embryos using a micromanipulator, cultured in vitro to the blastocyst stage, and individual blastocysts were collected for genome acquisition. PCR amplification of the intended editing sites was then performed using specific primers, followed by Sanger sequencing. Sequencing results were analyzed using the EditR online tool to assess editing efficiency.

[0070] The results are as follows Figures 5-8 As shown, Figure 5 The graph shows the cytosine base editing efficiency in HEK293T cells. The dLbCas12a-CBE constructed with enCas12a and RR can improve the editing efficiency of the non-classical PAM TCCC from less than 10% to 40%, an 8-fold increase. The dLbCas12a-CBE constructed with RR can improve the editing efficiency of the non-classical PAM TCCA from 2% to nearly 20%, an approximately 10-fold increase. The dLbCas12a-CBE constructed with enCas12a and RVR can improve the editing efficiency of the non-classical PAM TATG from almost none to more than 30%. Figure 6The diagram shows the efficiency of adenine base editing in HEK293T cells, indicating that dLbCas12a-ABE constructed with the Cas12a mutant can also effectively deaminate non-classical PAMs. For example, for the non-classical PAM TTCC, dLbCas12a-ABE constructed with enCas12a and RR can increase the efficiency from 10% to over 20%, an increase of about 2 times; for TATAPAM, dLbCas12a-ABE constructed with RVR can increase the efficiency from less than 5% to 10%.

[0071] At the embryonic level, Figure 7 This diagram illustrates the efficiency of cytosine base editing in embryos. The dLbCas12a-CBE system constructed with the Cas12a mutant exhibits similar activity to the original dLbCas12a-CBE in the classic PAM TTTV, but shows significant improvements for some non-classical PAMs. For example, for the non-classical PAM TTCA, the dLbCas12a-CBE constructed with enCas12a and RR increases efficiency from less than 5% to approximately 50%, a tenfold increase. For the non-classical PAMs TTCT, TCCC, and TCCA, efficiency is increased from nearly ineffective to almost 40%. The dLbCas12a-CBE constructed with RR shows improvements for ATTC, TATG, and TGTA. Figure 8 As shown, the dLbCas12a-ABE constructed with RR can improve the efficiency at the PUM1-16 (TCCA) site from less than 5% to more than 35%, a 7-fold increase; while for IGF1-41 (CTTC), the dLbCas12a-ABE constructed with en-Cas12a can improve the efficiency from almost none to more than 40%.

[0072] Example 4

[0073] This embodiment utilizes the base editor constructed in Example 2 to achieve simultaneous editing of multiple genes in HEK293T cells and porcine embryos.

[0074] like Figure 10A and Figure 10B As shown, at the cellular level, guide RNAs targeting DNMT3B, KLF4, TET1, PRR5L, and CFTR were directed according to... Figure 9The guide RNAs were tandemly transfected into HEK293T cells using either CBE or ABE constructed from the Cas12a protein mutant in Example 2. After 72 hours, genomic DNA was extracted, and Sanger sequencing was performed following PCR with specific primers. Editing efficiency was evaluated using the EditR online tool. Cells transfected with multiple guide RNAs and those transfected with a single guide RNA served as control groups. The efficiency statistics showed that the editing efficiency was not significantly different regardless of whether single guide RNA, multiple guide RNA, or multiple guide RNAs were transfected in tandem. Furthermore, the results were similar for both CBE and ABE.

[0075] like Figure 11 As shown, at the embryonic level, guide RNAs targeting porcine PUM1, GHR, HMGA2, and PUM2 were administered according to... Figure 9 The guide RNA was tandemly injected with various Cas12a variants to construct CBEs or ABEs, transcribed into RNA in vitro, and then injected into parthenogenetic pig embryos via microinjection. After development to the blastocyst stage, the genome was extracted, and PCR amplification was performed using specific primers, followed by Sanger sequencing. The sequencing results were evaluated for editing efficiency using the EditR online tool. Multiple guide RNAs were injected together as a control group. The efficiency statistics showed that for the PUM1, GHR, and HMGA2 sites, there was no difference in editing efficiency between tandem guide RNAs and multiple guide RNAs; however, for the PUM2 site, the editing efficiency of tandem guide RNAs was significantly improved compared to multiple guide RNAs.

[0076] In summary, this invention modifies dLbCas12a to obtain a Cas12a protein mutant with an expanded PAM recognition range. Furthermore, by fusing a deaminase with the Cas12a protein mutant, a highly efficient base editor capable of recognizing a broadened PAM range is constructed. This editor can simultaneously and efficiently edit multiple sites at both the cellular and embryonic levels, effectively complementing existing base editing toolkits and successfully expanding the targetable genome range. It can also be applied to disease animal model construction, gene therapy, and breed improvement.

[0077] The applicant declares that the detailed method of the present invention is illustrated by the above embodiments, but the present invention is not limited to the above detailed method, that is, it does not mean that the present invention must rely on the above detailed method to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. A Cas12a protein mutant, characterized in that, The Cas12a protein mutant is a combination of D156R, G532R and K538R mutations on the basis of dLbCas12a protein. The amino acid sequence of the dLbCas12a protein is shown in SEQ ID NO.

1.

2. A nucleic acid molecule, characterized in that, The nucleic acid molecule contains a nucleic acid sequence encoding the Cas12a protein mutant of claim 1.

3. An expression carrier, characterized in that, The expression vector contains the nucleic acid molecule as described in claim 2.

4. A base editor, characterized in that, The base editor is a fusion protein formed by fusing a deaminase and the Cas12a protein mutant of claim 1; The deaminase is fused to the N-terminus of the Cas12a protein mutant; The deaminase is selected from cytosine deaminase or adenine deaminase; The cytosine deaminase is selected from hA3A; The amino acid sequence of hA3A is shown in SEQ ID NO.2; The adenine deaminase is selected from TadA-8e; The amino acid sequence of TadA-8e is shown in SEQ ID NO.

4.

5. The use of the Cas12a protein mutant of claim 1 or the base editor of claim 4 in gene editing for purposes other than disease diagnosis and / or treatment.

6. The application of the base editor according to claim 5 in gene editing for non-disease diagnosis and / or treatment purposes, characterized in that, The gene editing includes simultaneous gene editing at multiple gene sites.

7. A gene editing method for purposes other than disease diagnosis and / or treatment, characterized in that, The gene editing method includes: Gene editing can be performed using a CRISPR-Cas system composed of the Cas12a protein mutant and guide RNA as described in claim 1, or... The CRISPR-Cas system, composed of the Cas12 protein mutant described in claim 1 and guide RNA formed by processing tandem guide RNA, is used for simultaneous gene editing at multiple gene sites.

8. A base editing method for purposes other than disease diagnosis and / or treatment, characterized in that, The base editing method includes: Base editing is performed using the base editor described in claim 4.

9. The base editing method according to claim 8 for purposes other than disease diagnosis and / or treatment, characterized in that, The base editing method includes: Simultaneous base editing at multiple gene sites using the base editor and tandem guide RNA as described in claim 4.

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