Engineered Cas13d protein fused with RNA binding domain and application of engineered Cas13d protein

Abstract

The invention discloses an engineered Cas13d protein and application of the engineered Cas13d protein in CRISPR (clustered regularly interspaced short palindromic repeats) detection. Specifically, the invention provides an engineered Cas13d protein fused with an RNA binding domain, the engineered Cas13d protein comprises: (a) a Cas13d protein, the amino acid sequence of the Cas13d protein is shown as SEQ ID NO: 1, and (b) a Cas13d protein, the amino acid sequence of the Cas13d protein is shown as SEQ ID NO: 2; (b) at least one heterologous RNA binding domain (RBD); wherein the at least one heterologous RNA binding domain is inserted into a site selected from the group consisting of N-terminus, C-terminus, N47 site, Y119 site, or a combination thereof, of the Cas13d protein. The engineered Cas13d protein disclosed by the invention has a stable mutation detection capability during single nucleotide mutation detection.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to an engineered Cas13d protein and its application in CRISPR detection. Background Technology

[0002] Single nucleotide variant (SNV) detection is a fundamental technology in tumor molecular subtyping, targeted therapy decision-making, and drug resistance monitoring. Currently, commonly used SNV detection methods in clinical practice and research fall into two main categories: the first is traditional molecular detection platforms, and the second is CRISPR nucleic acid detection technology.

[0003] CRISPR nucleic acid detection technology utilizes the paracleavage activity triggered by the recognition of target nucleic acids by Cas effector proteins, achieving rapid readout through fluorescent probes, test strips, and other methods. Based on target type, it can be divided into: DNA detection systems represented by Cas12 and RNA detection systems represented by Cas13.

[0004] For SNV identification around Cas13, existing technologies generally employ crRNA mismatch engineering to achieve single-base resolution. The core strategy involves designing artificial mismatches at the mutation site and its vicinity, utilizing the perturbation of the binding stability of the Cas13-crRNA complex by these mismatches to amplify the activation differences between wild-type and mutant strains, thereby creating a recognition difference. However, this identification strategy is often a double-edged sword: while improving specificity, it often significantly sacrifices the overall affinity of the Cas protein for the target, leading to slower reaction kinetics or missed detections of low-abundance samples.

[0005] To overcome the limitations of traditional platforms in terms of timeliness and accessibility, CRISPR nucleic acid detection technology has been widely developed. Among them, the Cas13 system, due to its ability to directly recognize RNA and trigger bypass cleavage signals, has the potential to achieve rapid molecular diagnostics. However, for the specific need of high-resolution identification of SNVs, existing strategies still have core technical bottlenecks, such as the balance between specificity and sensitivity in single-base discrimination, reaction kinetic lag and increased time costs, high dependence on front-end amplification and its side effects, and the influence of flanking sequence constraints or preferences on site designability.

[0006] Therefore, there is an urgent need in this field to develop a new detection method to improve the sensitivity of the CRISPR system in single-base discrimination and overcome technical problems such as amplification dependence and limited site designability. Summary of the Invention

[0007] This invention provides an engineered Cas13d protein and its application in CRISPR detection.

[0008] In a first aspect of the invention, an engineered Cas13d protein fused with an RNA-binding domain is provided, the engineered Cas13d protein comprising:

[0009] (a) A Cas13d protein, the amino acid sequence of which is shown in SEQ ID NO: 1;

[0010] (b) At least one heterologous RNA-binding domain (RBD);

[0011] Wherein, the at least one heterologous RNA-binding domain is inserted into a site selected from the group consisting of the N-terminus, C-terminus, N47 site, Y119 site, or a combination thereof in the Cas13d protein;

[0012] Furthermore, the at least one heterologous RNA-binding domain is inserted into the corresponding site of the Cas13d protein via direct linking or linker peptide; and when at least one heterologous RNA-binding domain is inserted at multiple sites, the insertion method at each site is independently selected from direct linking or linker peptide linking.

[0013] In another preferred embodiment, the N47 or Y119 site corresponding to SEQ ID NO: 1 refers to the amino acid residue site that corresponds to N47 or Y119 when the Cas13d protein is compared with the amino acid sequence shown in SEQ ID NO: 1.

[0014] In another preferred embodiment, the engineered Cas13d protein comprises at least two heterologous RNA-binding domains inserted at at least two different insertion sites in the Cas13d protein.

[0015] In another preferred embodiment, the at least two heterologous RNA-binding domains are different RNA-binding domains.

[0016] In another preferred embodiment, the RNA-binding domain is selected from the group consisting of RNA recognition motifs (RRM), KH domains, C3H1 zinc fingers, or combinations thereof.

[0017] In another preferred embodiment, the amino acid sequence of the RNA-binding domain is selected from the group consisting of any amino acid sequence shown in SEQ ID NO: 2-6, or a combination thereof.

[0018] In another preferred aspect, the present invention provides an engineered Cas13d protein fused with an RNA-binding domain, having the structure shown in formula (I) or formula (II):

[0019] Z - R or R - Z (I)

[0020] Z - (L1) - R - (L2) - Z or Z - (L1) - R1 - (L2) - Z -(L3) - R2 -(L4) - Z (II)

[0021] Wherein, Z is the Cas13d protein or its functional fragment with nuclease activity, or a component thereof;

[0022] R, R1, and R2 are each independently a non- or heterologous RNA-binding domain (RBD);

[0023] L1, L2, L3, and L4 are each independently either non-linked or linked peptides;

[0024] Furthermore, R, R1, and R2 are inserted at sites selected from the group consisting of the N-terminus, C-terminus, N47 site corresponding to SEQ ID NO: 1, Y119 site corresponding to SEQ ID NO: 1, or combinations thereof, of the Cas13d protein or its functional fragment having nuclease activity.

[0025] In another preferred embodiment, the amino acid sequence of the engineered Cas13d protein is selected from the group consisting of:

[0026] (1) N47-RBD7 + Y119-RBD6, whose amino acid sequence is shown in SEQ ID NO: 23;

[0027] (2) Y119-RBD7, whose amino acid sequence is shown in SEQ ID NO: 17;

[0028] (3) Y119-RBD6, whose amino acid sequence is shown in SEQ ID NO: 15;

[0029] (4) N47-RBD7, whose amino acid sequence is shown in SEQ ID NO: 21;

[0030] (5) Y119-RBD3, whose amino acid sequence is shown in SEQ ID NO: 13;

[0031] (6) C-RBD4, whose amino acid sequence is shown in SEQ ID NO: 11;

[0032] (7) N47-RBD3, whose amino acid sequence is shown in SEQ ID NO: 19;

[0033] (8) C-RBD2, whose amino acid sequence is shown in SEQ ID NO: 9;

[0034] (9) N-RBD6, whose amino acid sequence is shown in SEQ ID NO: 7;

[0035] (10) N47-RBD7 + Y119-RBD7, whose amino acid sequence is shown in SEQ ID NO: 25;

[0036] Or a combination thereof.

[0037] In a second aspect of the invention, a polynucleotide is provided that encodes the engineered Cas13d protein described in the first aspect of the invention.

[0038] In another preferred embodiment, the polynucleotide is DNA or cDNA.

[0039] In a third aspect of the invention, an expression vector is provided, the expression vector comprising the polynucleotide described in the second aspect of the invention.

[0040] In another preferred embodiment, the vector includes: bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors.

[0041] In another preferred embodiment, the vector is a eukaryotic expression vector.

[0042] In a fourth aspect of the invention, a host cell is provided, the host cell comprising the expression vector described in the third aspect of the invention, or having the polynucleotides described in the second aspect of the invention integrated into its genome.

[0043] In another preferred embodiment, the cell is a eukaryotic cell or a prokaryotic cell.

[0044] In another preferred embodiment, the host cell includes a prokaryotic cell or a eukaryotic cell.

[0045] In another preferred embodiment, the host cell is selected from the group consisting of Escherichia coli, yeast cells, and mammalian cells.

[0046] In another preferred embodiment, the prokaryotic cell is Escherichia coli.

[0047] In a fifth aspect of the invention, a detection reagent is provided, the detection reagent comprising the engineered Cas13d protein as described in the first aspect of the invention.

[0048] In another preferred embodiment, the detection reagent further includes crRNA for detecting single-base mutant RNA.

[0049] In another preferred embodiment, the site at which the crRNA recognizes the single base mutation is located at any one of positions 1-23 (preferably positions 2-13) at the 5' end of the crRNA spacer region;

[0050] The crRNA also has the following design:

[0051] (a) The following site intervals at the 5' end of the crRNA spacer region contain 0-3 mismatches:

[0052] The 2nd to 7th positions, the 18th to 22nd positions, or a combination thereof.

[0053] In another preferred embodiment, the crRNA includes a repeat region (Handle) and a spacer region (Guide).

[0054] In another preferred embodiment, the single base mutation is an oncogenic mutation or a variant of an oncogenic single nucleotide (SNV).

[0055] In another preferred embodiment, the oncogenic mutation is selected from the group consisting of KRAS G12D, IDH1 R132C, BRAF V600E, or combinations thereof.

[0056] In another preferred embodiment, when the single base mutation is KRAS G12D, the crRNA is selected from the group consisting of:

[0057] (A) No mismatch was introduced:

[0058] (A1) M0, wherein M0 comprises a nucleotide as shown in SEQ ID NO: 160;

[0059] (B) Introduce a mismatch (one mismatch in positions 2-7 and one mismatch in positions 18-22 respectively):

[0060] (B1) M2, wherein M2 comprises a nucleotide as shown in SEQ ID NO: 221;

[0061] (B2) M3, wherein M3 comprises a nucleotide as shown in SEQ ID NO: 222;

[0062] (B3) M4, wherein M4 comprises the nucleotides shown in SEQ ID NO: 223;

[0063] (B4) M5, wherein M5 comprises a nucleotide as shown in SEQ ID NO: 224;

[0064] (B5) M6, wherein M6 comprises a nucleotide as shown in SEQ ID NO: 225;

[0065] (B6) M17, wherein M17 comprises a nucleotide as shown in SEQ ID NO: 236;

[0066] (B7) M18, wherein M18 comprises a nucleotide as shown in SEQ ID NO: 237;

[0067] (B8) M19, wherein M19 comprises a nucleotide as shown in SEQ ID NO: 238;

[0068] (B9) M20, wherein M20 comprises a nucleotide as shown in SEQ ID NO: 239;

[0069] (B10) M21, wherein M21 comprises a nucleotide as shown in SEQ ID NO: 240;

[0070] (B11) M22, wherein M22 comprises a nucleotide as shown in SEQ ID NO: 241;

[0071] (C) Introduce two mismatches:

[0072] (C1) DM1, wherein DM1 comprises the nucleotides shown in SEQ ID NO: 398;

[0073] (C2) DM2, wherein DM2 comprises the nucleotides shown in SEQ ID NO: 399;

[0074] (C3) DM3, wherein the DM3 comprises the nucleotides shown in SEQ ID NO: 400;

[0075] (C4) DM4, wherein the DM4 comprises the nucleotides shown in SEQ ID NO: 401;

[0076] (C5) DM5, wherein the DM5 comprises the nucleotides shown in SEQ ID NO: 402;

[0077] (C6) DM6, wherein the DM6 comprises the nucleotides shown in SEQ ID NO: 403;

[0078] (C7) DM7, wherein DM7 comprises the nucleotide as shown in SEQ ID NO: 404;

[0079] (C8) DM8, wherein the DM8 comprises the nucleotide as shown in SEQ ID NO: 405;

[0080] (C9) DM9, wherein the DM9 comprises the nucleotides shown in SEQ ID NO: 406;

[0081] (C10) DM10, wherein DM10 comprises the nucleotides shown in SEQ ID NO: 407;

[0082] (C11) DM11, wherein DM11 comprises the nucleotides shown in SEQ ID NO: 408;

[0083] (C12) DM12, wherein DM12 comprises nucleotides as shown in SEQ ID NO: 409.

[0084] In another preferred embodiment, when the single base mutation is IDH1 R132C, the crRNA is selected from the group consisting of:

[0085] (A) No mismatch was introduced:

[0086] (A1) M0, wherein M0 comprises a nucleotide as shown in SEQ ID NO: 163;

[0087] (B) Introduce a mismatch (one mismatch in positions 2-7 and one mismatch in positions 18-22 respectively):

[0088] (B1) M2, wherein M2 comprises a nucleotide as shown in SEQ ID NO: 269;

[0089] (B2) M3, wherein M3 comprises a nucleotide as shown in SEQ ID NO: 270;

[0090] (B3) M4, wherein M4 comprises the nucleotides shown in SEQ ID NO: 271;

[0091] (B4) M5, wherein M5 comprises a nucleotide as shown in SEQ ID NO: 272;

[0092] (B5) M6, wherein M6 comprises a nucleotide as shown in SEQ ID NO: 273;

[0093] (B6) M17, wherein M17 comprises a nucleotide as shown in SEQ ID NO: 284;

[0094] (B7) M18, wherein M18 comprises a nucleotide as shown in SEQ ID NO: 285;

[0095] (B8) M19, wherein M19 comprises a nucleotide as shown in SEQ ID NO: 286;

[0096] (B9) M20, wherein M20 comprises a nucleotide as shown in SEQ ID NO: 287;

[0097] (B10) M21, wherein M21 comprises a nucleotide as shown in SEQ ID NO: 288;

[0098] (B11) M22, wherein M22 comprises a nucleotide as shown in SEQ ID NO: 289;

[0099] (C) Introduce two mismatches:

[0100] (C1) DM1, wherein DM1 comprises the nucleotides shown in SEQ ID NO: 349;

[0101] (C2) DM2, wherein DM2 comprises the nucleotides shown in SEQ ID NO: 350;

[0102] (C3) DM3, wherein the DM3 comprises the nucleotides shown in SEQ ID NO: 351;

[0103] (C4) DM4, wherein the DM4 comprises the nucleotides shown in SEQ ID NO: 352;

[0104] (C5) DM5, wherein the DM5 comprises the nucleotides shown in SEQ ID NO: 353;

[0105] (C6) DM6, wherein the DM6 comprises the nucleotides shown in SEQ ID NO: 354;

[0106] (C7) DM7, wherein DM7 comprises the nucleotide as shown in SEQ ID NO: 355;

[0107] (C8) DM8, wherein the DM8 comprises the nucleotides shown in SEQ ID NO: 356;

[0108] (C9) DM9, wherein the DM9 comprises the nucleotides shown in SEQ ID NO: 357;

[0109] (C10) DM10, wherein DM10 comprises the nucleotides shown in SEQ ID NO: 358;

[0110] (C11) DM11, wherein DM11 comprises the nucleotides shown in SEQ ID NO: 359;

[0111] (C12) DM12, wherein DM12 comprises nucleotides as shown in SEQ ID NO: 360.

[0112] In another preferred embodiment, when the single base mutation is BRAF V600E, the crRNA is selected from the group consisting of:

[0113] (A) No mismatch was introduced:

[0114] (A1) M0, wherein M0 comprises a nucleotide as shown in SEQ ID NO: 162;

[0115] (B) Introduce a mismatch (one mismatch in positions 2-7 and one mismatch in positions 18-22 respectively):

[0116] (B1) M2, wherein M2 comprises a nucleotide as shown in SEQ ID NO: 315;

[0117] (B2) M3, wherein M3 comprises a nucleotide as shown in SEQ ID NO: 316;

[0118] (B3) M4, wherein M4 comprises the nucleotides shown in SEQ ID NO: 317;

[0119] (B4) M5, wherein M5 comprises a nucleotide as shown in SEQ ID NO: 318;

[0120] (B5) M6, wherein M6 comprises a nucleotide as shown in SEQ ID NO: 319;

[0121] (B6) M17, wherein M17 comprises a nucleotide as shown in SEQ ID NO: 330;

[0122] (B7) M18, wherein M18 comprises a nucleotide as shown in SEQ ID NO: 331;

[0123] (B8) M19, wherein M19 comprises a nucleotide as shown in SEQ ID NO: 332;

[0124] (B9) M20, wherein M20 comprises a nucleotide as shown in SEQ ID NO: 333;

[0125] (B10) M21, wherein M21 comprises a nucleotide as shown in SEQ ID NO: 334;

[0126] (B11) M22, wherein M22 comprises a nucleotide as shown in SEQ ID NO: 335;

[0127] (C) Introduce two mismatches:

[0128] (C1) DM1, wherein DM1 comprises the nucleotides shown in SEQ ID NO: 373;

[0129] (C2) DM2, wherein DM2 comprises the nucleotides shown in SEQ ID NO: 374;

[0130] (C3) DM3, wherein the DM3 comprises the nucleotides shown in SEQ ID NO: 375;

[0131] (C4) DM4, wherein the DM4 comprises the nucleotides shown in SEQ ID NO: 376;

[0132] (C5) DM5, wherein the DM5 comprises the nucleotides shown in SEQ ID NO: 377;

[0133] (C6) DM6, wherein the DM6 comprises the nucleotides shown in SEQ ID NO: 378;

[0134] (C7) DM7, wherein DM7 comprises the nucleotides shown in SEQ ID NO: 379;

[0135] (C8) DM8, wherein the DM8 comprises the nucleotides shown in SEQ ID NO: 380;

[0136] (C9) DM9, wherein the DM9 comprises the nucleotides shown in SEQ ID NO: 381;

[0137] (C10) DM10, wherein DM10 comprises the nucleotides shown in SEQ ID NO: 382;

[0138] (C11) DM11, wherein DM11 comprises the nucleotides shown in SEQ ID NO: 383;

[0139] (C12) DM12, wherein DM12 comprises nucleotides as shown in SEQ ID NO: 384.

[0140] In a sixth aspect of the invention, a kit is provided, the kit comprising the detection reagents described in the fifth aspect of the invention.

[0141] In another preferred embodiment, the kit also includes an amplification component for reverse transcription loop-mediated isothermal amplification (RT-LAMP).

[0142] In a seventh aspect of the invention, the use of the engineered Cas13d protein as described in the first aspect of the invention is provided for the preparation of a CRISPR detection reagent or kit for detecting single nucleotide variants.

[0143] In an eighth aspect of the present invention, a method for preparing the engineered Cas13d protein described in the first aspect of the present invention is provided, comprising the steps of:

[0144] (a) Under expression conditions, host cells as described in the fourth aspect of the present invention are cultured to express the engineered Cas13d protein;

[0145] (b) Isolate and purify the engineered Cas13d protein described in (a).

[0146] In a ninth aspect of the present invention, a method for detecting carcinogenic mutations in a test sample is provided, comprising the steps of:

[0147] The test sample is tested using the kit described in the sixth aspect of this invention.

[0148] In another preferred embodiment, the method is an in vitro method.

[0149] In another preferred embodiment, the method is non-diagnostic and non-therapeutic.

[0150] In another preferred embodiment, the method further includes performing loop-mediated isothermal amplification (LAMP) on the sample to be tested prior to detection.

[0151] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0152] Figure 1 The AE in the study demonstrates the design and screening of SNV-specific crRNAs using the Cas13design platform (selectivity was assessed using ΔScore = Score_SNV - Score_WT, with priority given to tests with ΔScore ≥ 0.2), and the effectiveness of these crRNAs in distinguishing WT / SNV from KRAS G12D, IDH1 R132C, and BRAF V600E using fluorescence reporter assays.

[0153] Figure 2 The predicted scores of candidate crRNAs and their cleavage kinetics are shown: Figure 2 A in the table lists the candidate crRNAs generated by Cas13design and their Score_SNV, Score_WT, and ΔScore (red indicates SNV bases, and blue indicates crRNAs covering SNV sites). Figure 2The BD curves in the image show the time-process fluorescence curves of KRAS, IDH1, and BRAF under WT (dashed line) and SNV (solid line) templates, which are used to compare the discrimination effects of different crRNAs.

[0154] Figure 3 This demonstrates how introducing single / double mismatches into the crRNA spacer enhances SNV discrimination and allows for systematic screening of mismatch locations: a single mismatch results in an SNV having one mismatch and a WT having two mismatches. Figure 3 In AC), double mismatches result in SNV having 2 mismatches and WT having 3 mismatches. Figure 3 The DF in the model is used to compare the relative cutting activity of each variant on the SNV and WT templates, and to determine the mismatch position combination that is beneficial to suppressing WT while preserving the SNV signal.

[0155] Figure 4 The design of mismatched crRNA sequences for IDH1 R132C and BRAF V600E is shown: Figure 4 In this context, AB represents the M1-M23 series obtained by sequentially introducing single mismatches into the maternal crRNA. Figure 4 CD in the figure represents the double mismatch DM1-DM12 series obtained by combining single mismatch sites (red represents introduced mismatches, and blue represents WT mismatches at mutation sites).

[0156] Figure 5 The sensitivity assessment of miniCas13d to KRAS G12D was shown: SNV was mixed with WT RNA in different proportions to form a VAF gradient of 0.01%–100% ( Figure 5 (A) Compare the fluorescence responses of crRNA M5 and DM10 under LAMP-free and LAMP-free conditions. Figure 5 (BC in the middle), and estimate the functional limit of detection (fLoD) according to EP17-A2 ( Figure 5 (D in the middle).

[0157] Figure 6 The analytical sensitivity indices calculated according to CLSI EP17-A2 are shown, which are used to compare the blank slope statistics, LoB / LoD slope, sensitivity coefficient b^ and final limit of functional detection fLoD (%) of different Cas13d protein systems under crRNA M5 or DM10 and ±LAMP conditions.

[0158] Figure 7 This demonstrates the rational design and structural localization of the RBD fusion site in the engineered miniCas13d: Figure 7 The A section provides four insertion sites (N-terminus, C-terminus, N47, Y119) and 10 human RBD modules; Figure 7 In the diagram, B represents the ternary complex model predicted by AlphaFold3, which indicates the spatial location of the aforementioned sites within the structure.

[0159] Figure 8 The structure prediction and protein preparation of the RBD-fused miniCas13d variant are shown: Figure 8 The AH in the model represents the fusion protein complex model with different insertion sites / different RBDs predicted by AlphaFold3 (and aligned with the miniCas13d complex to assess structural perturbation), and also provides pTM and ipTM scores. Figure 8 The I in the figure represents the SDS-PAGE results of each fusion protein after purification, showing that the major bands were consistent with the expected molecular weight.

[0160] Figure 9 The performance of RBD fusion with miniCas13d in SNV detection is shown: Figure 9 A in the diagram shows the effect of different insertion sites on enzyme digestion activity; Figure 9 The BC in the data indicates that Y119-RBD7 retains a mismatch position dependence similar to that of the parent compound while its activity is enhanced, thus maintaining the single-base distinction rule. Figure 9 The detection performance of DF in the KRAS G12D gradient dilution system was further verified. The signal output of Y119-RBD7 with M5 or DM10 was compared under LAMP conditions with and without, and the corresponding fLoD (%) evaluation results were given.

[0161] Figure 10 The time-course fluorescence activity of the engineered miniCas13d-RBD variant on the KRAS G12D target was compared. Figure 10 (A in the text), and the ability spectrum of Y119-RBD7 to distinguish between SNV and WT in single / double mismatch crRNA libraries at IDH1 R132C and BRAF V600E sites ( Figure 10 (BC in the middle).

[0162] Figure 11 The AD in the figure shows the time-course fluorescence kinetics of engineered miniCas13d variants in the KRAS G12D gradient dilution series when combined with M5 or DM10, and compares the signal decreases with decreasing mutation ratio under LAMP pre-amplification conditions without LAMP and LAMP pre-amplification conditions.

[0163] Figure 12The AC results show the detection performance of the dual RBD insert variant N47-RBD7+Y119-RBD6 in the KRAS G12D gradient dilution system: the fluorescence fold change relative to WT (0% G12D) was compared with M5 or DM10 under LAMP conditions and with / without LAMP conditions, and the corresponding fLoD evaluation results under different conditions were given by CLSI EP17-A2 calculation.

[0164] Figures 13A-13F The analytical performance and clinical validation of the engineered miniCas13d platform under different enzyme backbones, crRNA, and ±LAMP combinations were demonstrated: Figures 13A-13D This demonstrates a unified quantification of the detection threshold and quantitative stability of each combination based on CLSI EP17-A2; Figures 13E-13F The results showed that in 15 PDAC samples (M5+LAMP), the three enzyme backbones could be clearly distinguished between KRAS G12D positive and negative.

[0165] Figure 14 The fit indices (Ri) between the enzyme backbone and the detection mode were shown in the low abundance ranges of 0-5% and 0-10% VAF. 2 WRMSEsd, NRMSE, RMSE, and the low interval / full interval slope ratio are used to compare the fitting stability and sensitivity consistency of the low VAF segment.

[0166] Figure 15 The clinical application scenarios of the amplification-free, ultrasensitive, rapid detection mode and robust clinical detection mode using the crRNA and / or engineered Cas13d protein of the present invention are shown. Detailed Implementation

[0167] Through extensive and in-depth research, the inventors have developed, for the first time, an engineered Cas13d protein fused with an RNA-binding domain (RBD). This protein can be co-designed with crRNA mismatch rules and protein engineering to reliably identify single nucleotide variants in RNA. Experiments show that, compared to the original miniCas13d, the engineered Cas13d protein fused with an RNA-binding domain (RBD) has a higher baseline activity and reduced dependence on amplification; it also exhibits more robust detection capabilities even without pre-amplification. Based on these findings, this invention was completed.

[0168] the term

[0169] To facilitate a clearer understanding of this disclosure, certain terms are first defined. As used herein, unless otherwise expressly specified herein, each of the following terms shall have the meaning given below. Other definitions are set forth throughout the application.

[0170] The term "about" can refer to a value or composition within an acceptable margin of error for a particular value or composition as determined by a person skilled in the art, depending in part on how the value or composition is measured or determined. For example, as used herein, the expression "about 100" includes all values ​​between 99 and 101.

[0171] As used herein, the terms “containing” or “including (comprise)” can be open-ended, semi-closed, or closed. In other words, the terms also include “consistently made of” or “made of”.

[0172] As used herein, unless otherwise stated, any concentration range, percentage range, proportion range, or integer range shall be understood to include any integer value within the range and, where appropriate, its fractional value (e.g., one-tenth and one-hundredth of an integer).

[0173] As used herein, the term “and / or” refers to and covers any and all possible combinations of one or more of the related listed items.

[0174] As used in this article, "Cas13d" refers to an RNA-targeted CRISPR effector protein; "miniCas13d" refers to a miniaturized Cas13d backbone, the specific amino acid sequence of which is shown in SEQ ID NO: 1; "RBD" refers to the RNA-binding domain; "crRNA" refers to the guide RNA required for Cas13d; "SNV" refers to a single nucleotide variant; "WT" refers to the wild type; "Mut" refers to the mutant type; and "RT-LAMP" refers to reverse transcription loop-mediated isothermal amplification.

[0175] As used herein, unless otherwise specified, the crRNA sequences containing T bases listed in this invention refer to the coding sequences of the crRNAs. Furthermore, unless otherwise specified, for visual demonstration of base complementarity, sequences are presented in the 3' to 5' direction, which is equivalent to the complementary sequence of the target sequence.

[0176] In this invention, the crRNA consists of a fixed Handle sequence (i.e., a repeat region) and a variable Guide sequence (i.e., a spacer region). Table 2 lists the complete candidate crRNA sequences (standard 5′-Handle-Guide-3′ orientation); while Figure 1 B in Figure 3 B and E in Figure 4 The AD section lists the guide sequences of each candidate crRNA. To visually demonstrate their recognition and mismatch design at the SNV site, the sequences in these figures are written in reverse from left to right in the 3′ to 5′ direction and are the DNA sequences corresponding to the crRNAs.

[0177] It should be noted that all sequences in this invention are presented in the sequence listing in the direction of 5'→3' or N→C.

[0178] Construction and composition of the engineered Cas13d protein of the present invention

[0179] Cas13d backbone: Based on the Cas13d effector protein (which can be a full-length or miniaturized Cas13d backbone), it serves as the core enzyme element that recognizes crRNA and triggers paracleavage signals upon activation of the target RNA.

[0180] RBD fusion design: Introducing at least one RNA-binding domain (RBD) into the Cas13d protein, wherein the RBD can be a domain type with RNA-binding capability; fusion methods include, but are not limited to:

[0181] Single RBD configuration: Cas13d fuses one RBD;

[0182] Dual RBD configuration: Cas13d fuses two RBDs (homogeneous or heterogeneous RBDs).

[0183] Fusion site and connection method: The RBD can be fused with Cas13d through a linker peptide; the fusion site can be the internal region or the terminal region of the Cas13d protein, and preferably a region that does not disrupt the Cas13d fold and crRNA loading, so as to maintain enzyme stability and reactivity.

[0184] Functional features: The engineered Cas13d fused with RBD is used to improve the effective binding / activation efficiency of target RNA in the case of signal reduction caused by crRNA mismatch design, thereby enhancing the detection signal and detection capability.

[0185] In another preferred embodiment, the present invention provides an engineered Cas13d protein fused with an RNA-binding domain, comprising at least:

[0186] (1) Cas13d protein backbone (full-length or miniaturized backbone);

[0187] (2) At least one RBD structural domain;

[0188] (3) Connecting peptide (optional) for connecting RBD to Cas13d or for flexible connection of internal insertion.

[0189] In another preferred embodiment, the engineered configuration includes, but is not limited to:

[0190] 1) Single RBD configuration: Cas13d fuses one RBD;

[0191] 2) Dual RBD configuration: Cas13d fuses two RBDs (which may be the same or different);

[0192] 3) Multiple RBD configuration: Multiple RBDs are fused without affecting enzyme stability and crRNA loading.

[0193] In another preferred embodiment, the RBD can be fused to the terminal or internal region of Cas13d. Preferably, the fusion site is selected in a region exposed on the protein surface with a certain degree of flexibility to reduce the impact on protein folding, crRNA loading, and catalytic core conformation. The linker peptide can be a flexible linker peptide to reduce steric hindrance and facilitate domain movement.

[0194] In a preferred embodiment, the RBD fusion is used to improve the effective contact and binding of the target RNA, thereby increasing the reaction signal intensity or reducing the detection limit while maintaining the single-base discrimination capability.

[0195] In another preferred embodiment, engineered Cas13d can be prepared using a prokaryotic or eukaryotic expression system. For example, after expression in *E. coli*, the protein can be purified by affinity chromatography combined with ion exchange or gel filtration to obtain a protein formulation suitable for in vitro assays. The resulting protein can then be further subjected to concentration determination and purity assessment.

[0196] The SNV detection system and method based on engineered Cas13d of this invention

[0197] The detection system of the present invention includes at least:

[0198] (1) Engineered Cas13d protein (single RBD or double RBD).

[0199] (2) crRNA corresponding to the target site (including mismatch design);

[0200] (3) Reporter probes (e.g., fluorescence quenching probes that can be side-cut or other readable probes);

[0201] (4) Reaction buffer system and necessary ions or cofactors.

[0202] In optional implementations, isothermal amplification modules (such as RT-LAMP) may also be included, along with their associated enzymes and primer sets.

[0203] In another preferred embodiment, the detection reaction procedure (direct detection without amplification) includes:

[0204] 1) Engineered Cas13d was pre-assembled with crRNA to form an RNP complex;

[0205] 2) Add the RNA sample to be tested;

[0206] 3) Add the reporter probe and react under isothermal conditions;

[0207] 4) Collect fluorescence or other signals;

[0208] 5) Based on the preset threshold or the difference between Mut and WT, SNV classification is achieved.

[0209] In another preferred embodiment, when the target for detection is low abundance or low proportion of variation, the RT-LAMP module can be added before the Cas13d reaction or as part of a predetermined procedure.

[0210] In another preferred embodiment, the detection reaction procedure (RT-LAMP enhanced detection) includes:

[0211] 1) Reverse transcription and isothermal amplification of RNA samples were performed to obtain amplification products;

[0212] 2) Introduce the amplification product into the Cas13d / crRNA detection reaction;

[0213] 3) Acquire signals and perform threshold interpretation.

[0214] RT-LAMP is an optional module used to lower the detection limit; its introduction may change the background signal, so it is preferred to combine it with a mismatch strategy and engineered enzyme configuration.

[0215] The scene-driven preset detection configuration of the present invention

[0216] This invention provides preset detection configurations for different scenarios, specifically as follows: Figure 15 As shown. The following implementation schemes are included:

[0217] Implementation Plan 1: Amplification-Free Rapid Detection Mode

[0218] Applicable scenarios: Rapid screening that emphasizes speed and streamlined processes.

[0219] Module combination: dual RBD engineered Cas13d + dual mismatched crRNA, amplification module not enabled.

[0220] Performance characteristics: Stable SNV discrimination signal and genotyping window are obtained without amplification.

[0221] Implementation Plan 2: Robust Clinical Testing Model

[0222] Applicable scenarios: detection of low abundance or low proportion of variants.

[0223] Module combination: single or dual RBD miniCas13d + single mismatch crRNA + LAMP.

[0224] Performance characteristics: The detection limit is improved by amplification module, while ensuring stable typing in low abundance samples.

[0225] The reagent kit of the present invention

[0226] The engineered Cas13d protein constructed based on this invention can be used in conjunction with crRNA to detect mutations or single nucleotide variants (SNVs) in test samples via the CRISPR method. Therefore, this invention also provides a detection kit, the kit comprising:

[0227] 1) Engineered Cas13d protein components;

[0228] 2) crRNA components (including pre-made mismatched crRNAs targeting different sites);

[0229] 3) Report probe composition;

[0230] 4) Reaction buffer solution and related consumables;

[0231] 5) (Optional) RT-LAMP-related enzymes and primer components;

[0232] 6) Instruction manual, providing recommended configurations, response conditions, and interpretation methods for different scenarios.

[0233] The main advantages of this invention include:

[0234] (a) Alleviating the "specificity-sensitivity" trade-off in SNV identification

[0235] Existing CRISPR SNV systems typically improve discrimination through mismatches, but this can easily lead to signal degradation. This invention improves single-base resolution while maintaining usable signal output through a combined design of "mismatch discrimination + RBD enhancement," thereby improving readability and stability in the detection of low abundance or low proportion variants.

[0236] (b) Reduce the strong dependence on amplification and provide optional sensitivity enhancement pathways.

[0237] Unlike many schemes that rely on amplification, this invention provides an amplification-free mode to simplify the process, while offering an optional amplification module when extreme detection limits are required, taking into account sensitivity requirements in different scenarios, and reducing the impact of background risks introduced by amplification on genotyping through configuration logic.

[0238] (c) Enhance site designability and applicability

[0239] In SNV identification systems where mismatch locations need to be fixed according to rules, flanking sequence constraints often compress the designable window. This invention uses the Cas13d system and its targeting properties to improve the designable coverage of clinically relevant SNV sites in different sequence backgrounds and reduce detection "blind spots".

[0240] (d) Establish reusable and standardized configuration patterns to lower the application threshold.

[0241] Compared to existing solutions that require repeated exploration of multiple parameter combinations, this invention solidifies key elements into a small number of preset configuration modes, allowing users to directly select configurations based on the detection target, thereby improving the repeatability, scalability, and engineering application potential of the method.

[0242] (e) Rational insertion site design based on structure to improve the effectiveness and transferability of fusion engineering

[0243] Traditional fusion strategies are mostly limited to the N-terminus or C-terminus of proteins, which are easily affected by conformational freedom and spatial orientation, resulting in unstable enhancement effects. This invention, based on ternary complex structure prediction and spatial proximity analysis, identifies one or more "structurally permissible sites" suitable for insertion into the RNA-binding domain without significantly affecting the stability of the Cas13d protein backbone. This allows the exogenous RBD to achieve site-specific enhancement in the spatial region related to crRNA / target RNA interaction, thereby improving system activity and signal output while essentially maintaining the mismatch sensitivity characteristics of the parent enzyme.

[0244] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0245] Example 1: Construction and composition of engineered Cas13d protein

[0246] 1.1 Selection of the Cas13d protein backbone

[0247] This invention selects miniCas13d as the basic protein backbone. MiniCas13d is a miniaturized variant of Cas13d based on Ruminococcusflavefaciens XPD3002 (RfxCas13d). The design of miniCas13d is derived from the study "A strategy for Cas13 miniaturization based on the structure and AlphaFold" (Nat Commun. 2023;14:5545). This study successfully designed and optimized the miniCas13d protein using structure prediction and the AlphaFold model. Compared with traditional Cas13d, miniCas13d has a smaller molecular weight but still maintains strong RNA binding and cleavage activity, making it suitable for rapid RNA detection.

[0248] The amino acid sequence of the miniCas13d protein is shown below:

[0249] MIEKKKSFAKGMGVKSTLVSGSKVYMTTTFAEGSDAFSHPKGYAVVANNPLYTGPVQQDMLGLKETLEKRYFGESADGNDNICIQVIHNILDIEKILAEYITNAAYAVNNISGLDEGRNYIINYGNECYDILALLSGLRHWVVHNNEEESRISRTWLYNLDKNLDNEYIST LNYLYDRITNELTNSFSKNSAEQYFRFSIMKEQKNLGFNITKLREVMLDRKDMSEIRKNHKVFDSIRTKVYTMMDFVIYRYYIEEEKDIFVINLRGSFNDDQKDALYYDEANRIWRKLENIMHNIKEFRGNKTREYKKKDAPRLPRILPAGRDVSAFSKLMYALTMFLDGK EINDLLTTLINKFDNIQSFLKVMPLIGVNAKFVEEYAFFKDSAKIADELRLIKSFARMGEPIADARRAMYIDAIRILGTKLKKGKHGMRNFIINNVISNKRFHYLIRYGDPAHLHEIAKNEAVVKFVLGRIADIQKKQGQNGKNQIDRYYETCIQFDKKRSVIEDTGREN AEREKFKKIISLYLTVIYHILKNIVNINARYVIGFHCVERDAQLYKEKGYDINLKKLEEQINREKAKTALNAYLRNTKWNVIIREDLLRIDNKTCTLFRNKAVHLEVARYVHAYINDINDEKKYNDRLLKLLCVPFGYCIPRFKNLSIEALFDRNEAAKFDKEKKKVSGNS (SEQ ID NO: 1)

[0250] 1.2 RNA-binding domain (RBD) fusion design

[0251] To enhance the RNA binding affinity of miniCas13d, this invention involves fusing RBDs at multiple specific sites within the miniCas13d protein structure. The selected RBDs include the following:

[0252] RNA recognition motif (RRM): This is a classic RNA-binding domain widely used in the study of RNA-protein interactions, which can enhance the affinity of RNA binding.

[0253] KH domain: The KH domain is an RNA-binding domain that is often used to recognize specific sequences or structures of RNA.

[0254] C3H1-type zinc finger: This domain can bind to RNA via zinc ions, further stabilizing the RNA-protein complex.

[0255] 1.3 Engineered Cas13d protein with fused RNA-binding domain

[0256] This invention uses the following RBD as an example to construct an engineered Cas13d protein:

[0257] RBD2:

[0258] HILFLTNLPEETNELMLSMLFNQFPGFKEVRLVPGRHDIAFVEFDNEVQAGAARDALQGFKITQNNAMKISFAKK (SEQ ID NO: 2)

[0259] RBD3:

[0260] TNLIVNYLPQNMTQDELRSLFSSIGEVESAKLIRDKVAGHSLGYGFVNYVTAKDAERAINTLNGLRLQSKTIKVSYARP (SEQ ID NO: 3)

[0261] RBD4:

[0262] ANLYISGLPRTMTQKDVEDMFSRFGRIINSRVLVDQTTGLSRGVAFIRFDKRSEAEEAITSFNGHKPPGSSEPITVKFAAN (SEQ ID NO: 4)

[0263] RBD6:

[0264] MVELRILLQSKNAGAVIGKGGKNIKALRTDYNASVSVPDSSGPERILSISADIETIGEILKKI (SEQID NO: 5)

[0265] RBD7:

[0266] DCELRLLIHQSLAGGIIGVKGAKIKELRENTQTTIKLFQECCPHSTDRVVLIGGKPDRVVECIKII(SEQ ID NO: 6)

[0267] These RBDs were designed to fuse with specific sites (N-terminus, C-terminus, N47 site, Y119 site) of the miniCas13d protein, and their structures were predicted using AlphaFold3. The sequences of the engineered Cas13d proteins are shown below (the amino acid or nucleotide sequences of the RBDs are indicated in bold and underlined font):

[0268] (1) The amino acid and nucleotide sequences of mini Cas13d N-RBD6 are shown below:

[0269] amino acid sequence:

[0270] MVELRILLQSKNAGAVIGKGGKNIKALRTDYNASVSVPDSSGPERILSISADIETIGEILKKI MIEKKKSFAKGMGVKSTLVSGSKVYMTTTFAEGSDAFSHPKGYAVVANNPLYTGPVQQDMLGLKETLEKRYFGESADGNDNICIQVIHNILDIEKILAEYITNAAYAVNNISGLDEGRNYIINYGNECYDILALLSGLRHWVVHNNEEESRISRTWLYNLDKNLDNEYIST LNYLYDRITNELTNSFSKNSAEQYFRFSIMKEQKNLGFNITKLREVMLDRKDMSEIRKNHKVFDSIRTKVYTMMDFVIYRYYIEEEKDIFVINLRGSFNDDQKDALYYDEANRIWRKLENIMHNIKEFRGNKTREYKKKDAPRLPRILPAGRDVSAFSKLMYALTMFLDGK EINDLLTTLINKFDNIQSFLKVMPLIGVNAKFVEEYAFFKDSAKIADELRLIKSFARMGEPIADARRAMYIDAIRILGTKLKKGKHGMRNFIINNVISNKRFHYLIRYGDPAHLHEIAKNEAVVKFVLGRIADIQKKQGQNGKNQIDRYYETCIQFDKKRSVIEDTGREN AEREKFKKIISLYLTVIYHILKNIVNINARYVIGFHCVERDAQLYKEKGYDINLKKLEEQINREKAKTALNAYLRNTKWNVIIREDLLRIDNKTCTLFRNKAVHLEVARYVHAYINDINDEKKYNDRLLKLLCVPFGYCIPRFKNLSIEALFDRNEAAKFDKEKKKVSGNS (SEQ IDNO: 7)

[0271] Nucleotide sequence:

[0272] atggttgaattacgcattctgcttcagagcaagaatgctggggcagtgattggaaaaggaggcaagaa tattaaggctctccgtacagactacaatgccagtgtttcagtcccagacagcagtggccccgagcgcatattgagt atcagtgctgatattgaaacaattggagaaattctgaagaaaatc

[0273] (2) The amino acid sequence and nucleotide sequence of mini Cas13d C-RBD2 are shown below respectively:

[0274] Amino acid sequence:

[0275] MIEKKKSFAKGMGVKSTLVSGSKVYMTTFAEGSDAFSHPKGYAVVANNPLYTGPVQQDMLGLKETLEKRYFGESADGNDNICIQVIHNILDIEKILAEYITNAAYAVNNISGLDEGRNYIINYGNECYDILALLSGLRHWVVHNNEEESRISRTWLYNLDKNLDNEYISTLNYLYDRITNELTNSFSKNSAEQYFRFSIMKEQKNLGFNITKLREVMLDRKDMSEIRKNHKVFDSIRTKVYTMMDFVIYRYYIEEEKDIFVINLRGSFNDDQKDALYYDEANRIWRKLENIMHNIKEFRGNKTREYKKKDAPRLPRILPAGRDVSAFSKLMYALTMFLDGKEINDLLTTLINKFDNIQSFLKVMPLIGVNAKFVEEYAFFKDSAKIADELRLIKSFARMGEPIADARRAMYIDAIRILGTKLKKGKHGMRNFIINNVISNKRFHYLIRYGDPAHLHEIAKNEAVVKFVLGRIADIQKKQGQNGKNQIDRYYETCIQFDKKRSVIEDTGRENAEREKFKKIISLYLTVIYHILKNIVNINARYVIGFHCVERDAQLYKEKGYDINLKKLEEQINREKAKTALNAYLRNTKWNVIIREDLLRIDNKTCTLFRNKAVHLEVARYVHAYINDINDEKKYNDRLLKLLCVPFGYCIPRFKNLSIEALFDRNEAAKFDKEKKKVSGNS HILFLTNLPEETNELMLSMLFNQFPGFKEVRLVPGRHDIAFVEFDNEVQAGAARDALQGFKITQNNAMKI SFAKK (SEQ ID NO: 9)

[0276] Nucleotide sequence:

[0277] cacatcttgttcctcaccaacctgccagaggagaccaacgagctcatgctgtccatgcttttcaatcagttccc tggcttcaaggaggtccgtctggtacccgggcggcatgacatcgccttcgtggagtttgacaatgaggtacaggca ggggcagctcgcgatgccctgcagggctttaagatcacgcagaacaacgccatgaagatctcctttgccaagaag taa (SEQ ID NO: 10)

[0278] (3) The amino acid sequence and nucleotide sequence of mini Cas13d C-RBD4 are shown below:

[0279] Amino acid sequence:

[0280] MIEKKKSFAKGMGVKSTLVSGSKVYMTTFAEGSDAFSHPKGYAVVANNPLYTGPVQQDMLGLKETLEKRYFGESADGNDNICIQVIHNILDIEKILAEYITNAAYAVNNISGLDEGRNYIINYGNECYDILALLSGLRHWVVHNNEEESRISRTWLYNLDKNLDNEYISTLNYLYDRITNELTNSFSKNSAEQYFRFSIMKEQKNLGFNITKLREVMLDRKDMSEIRKNHKVFDSIRTKVYTMMDFVIYRYYIEEEKDIFVINLRGSFNDDQKDALYYDEANRIWRKLENIMHNIKEFRGNKTREYKKKDAPRLPRILPAGRDVSAFSKLMYALTMFLDGKEINDLLTTLINKFDNIQSFLKVMPLIGVNAKFVEEYAFFKDSAKIADELRLIKSFARMGEPIADARRAMYIDAIRILGTKLKKGKHGMRNFIINNVISNKRFHYLIRYGDPAHLHEIAKNEAVVKFVLGRIADIQKKQGQNGKNQIDRYYETCIQFDKKRSVIEDTGRENAEREKFKKIISLYLTVIYHILKNIVNINARYVIGFHCVERDAQLYKEKGYDINLKKLEEQINREKAKTALNAYLRNTKWNVIIREDLLRIDNKTCTLFRNKAVHLEVARYVHAYINDINDEKKYNDRLLKLLCVPFGYCIPRFKNLSIEALFDRNEAAKFDKEKKKVSGNS ANLYISGLPRTMTQKDVEDMFSRFGRIINSRVLVDQTTGLSRGVAFIRFDKRSEAEEAITSFNGHKPPGS SEPITVKFAAN (SEQ ID NO: 11)

[0281] Nucleotide sequence:

[0282] gccaacttgtacatcagcgggctcccgcggaccatgacccagaaggacgtagaagacatgttctctcggtttgg gcggatcatcaactcgcgggtcctcgtggatcagactacaggtttgtccagaggggttgcgtttatccggtttgac aaacggtcggaggcagaagaggcaattaccagtttcaatggtcataaacccccaggttcctctgagcccatcacag tgaagtttgcagccaac taa (SEQ ID NO: 12)

[0283] (4) The amino acid sequence and nucleotide sequence of mini Cas13d Y119-RBD3 are shown below:

[0284] Amino acid sequence:

[0285] MIEKKKSFAKGMGVKSTLVSGSKVYMTTFAEGSDAFSHPKGYAVVANNPLYTGPVQQDMLGLKETLEKRYFGESADGNDNICIQVIHNILDIEKILAEYITNAAYAVNNISGLDEGRNY TNLIVNYLPQNMTQDELRSLFSSIG EVESAKLIRDKVAGHSLGYGFVNYVTAKDAERAINTLNGLRLQSKTIKVSYARP IINYGNECYDILALLSGLRHWVVHNNEEESRISRTWLYNLDKNLDNEYISTLNYLYDRITNELTNSFSKNSAEQYFRFSIMKEQKNLGFNITKLREVMLDRKDMSEIRKNHKVFDSIRTKVYTMMDFVIYRYYIEEEKDIFVINLRGSFNDDQKDALYYDEANRIWRKLENIMHNIKEFRGNKTREYKKKDAPRLPRILPAGRDVSAFSKLMYALTMFLDGKEINDLLTTLINKFDNIQSFLKVMPLIGVNAKFVEEYAFFKDSAKIADELRLIKSFARMGEPIADARRAMYIDAIRILGTKLKKGKHGMRNFIINNVISNKRFHYLIRYGDPAHLHEIAKNEAVVKFVLGRIADIQKKQGQNGKNQIDRYYETCIQFDKKRSVIEDTGRENAEREKFKKIISLYLTVIYHILKNIVNINARYVIGFHCVERDAQLYKEKGYDINLKKLEEQINREKAKTALNAYLRNTKWNVIIREDLLRIDNKTCTLFRNKAVHLEVARYVHAYINDINDEKKYNDRLLKLLCVPFGYCIPRFKNLSIEALFDRNEAAKFDKEKKKVSGNS (SEQ ID NO: 13)

[0286] Nucleotide sequence:

[0287] atgatcgaaaagaagaagagctttgcaaaaggcatgggcgttaaaagtaccctggtgagcggcagtaaagtgtatatgaccacctttgccgaaggcagtgatgcattttcacatccgaaaggttatgccgtggtggcaaataatccgctgtataccggcccggttcagcaggatatgctgggcctgaaagaaaccctggaaaaacgctattttggtgaaagcgcagatggtaatgataatatttgcattcaggttatccacaatatcctggatattgaaaaaatcctggcagaatatatcaccaatgcagcctatgcagttaataatattagcggtctggatgaaggtcgcaattat acgaatttgatcgtc aactacctccctcagaacatgacccaggatgagttacgaagcctgttcagcagcattggtgaagttgaatctgcaa aacttattcgggataaagtagcaggacacagcttgggctatggctttgtgaactacgtgaccgcgaaggatgcaga gagagcgatcaacacgctgaacggcttgaggctccagtcaaaaaccattaaggtgtcgtatgctcgcccg

[0288] (5) The amino acid sequence and nucleotide sequence of mini Cas13d Y119-RBD6 are shown below:

[0289] Amino acid sequence:

[0290] MIEKKKSFAKGMGVKSTLVSGSKVYMTTFAEGSDAFSHPKGYAVVANNPLYTGPVQQDMLGLKETLEKRYFGESADGNDNICIQVIHNILDIEKILAEYITNAAYAVNNISGLDEGRNY MVELRILLQSKNAGAVIGKGGKNIK ALRTDYNASVSVPDSSGPERILSISADIETIGEILKKI IINYGNECYDILALLSGLRHWVVHNNEEESRISRTWLYNLDKNLDNEYISTLNYLYDRITNELTNSFSKNSAEQYFRFSIMKEQKNLGFNITKLREVMLDRKDMSEIRKNHKVFDSIRTKVYTMMDFVIYRYYIEEEKDIFVINLRGSFNDDQKDALYYDEANRIWRKLENIMHNIKEFRGNKTREYKKKDAPRLPRILPAGRDVSAFSKLMYALTMFLDGKEINDLLTTLINKFDNIQSFLKVMPLIGVNAKFVEEYAFFKDSAKIADELRLIKSFARMGEPIADARRAMYIDAIRILGTKLKKGKHGMRNFIINNVISNKRFHYLIRYGDPAHLHEIAKNEAVVKFVLGRIADIQKKQGQNGKNQIDRYYETCIQFDKKRSVIEDTGRENAEREKFKKIISLYLTVIYHILKNIVNINARYVIGFHCVERDAQLYKEKGYDINLKKLEEQINREKAKTALNAYLRNTKWNVIIREDLLRIDNKTCTLFRNKAVHLEVARYVHAYINDINDEKKYNDRLLKLLCVPFGYCIPRFKNLSIEALFDRNEAAKFDKEKKKVSGNS (SEQ IDNO: 15)

[0291] Nucleotide sequence:

[0292] atgatcgaaaagaagaagagctttgcaaaaggcatgggcgttaaaagtaccctggtgagcggcagtaaagtgtatatgaccacctttgccgaaggcagtgatgcattttcacatccgaaaggttatgccgtggtggcaaataatccgctgtataccggcccggttcagcaggatatgctgggcctgaaagaaaccctggaaaaacgctattttggtgaaagcgcagatggtaatgataatatttgcattcaggttatccacaatatcctggatattgaaaaaatcctggcagaatatatcaccaatgcagcctatgcagttaataatattagcggtctggatgaaggtcgcaattat atggttgaattacgc attctgcttcagagcaagaatgctggggcagtgattggaaaaggaggcaagaatattaaggctctccgtacagact acaatgccagtgtttcagtcccagacagcagtggccccgagcgcatattgagtatcagtgctgatattgaaacaat tggagaaattctgaagaaaatc

[0293] (6) The amino acid sequence and nucleotide sequence of mini Cas13d Y119-RBD7 are shown below:

[0294] Amino acid sequence:

[0295] MIEKKKSFAKGMGVKSTLVSGSKVYMTTFAEGSDAFSHPKGYAVVANNPLYTGPVQQDMLGLKETLEKRYFGESADGNDNICIQVIHNILDIEKILAEYITNAAYAVNNISGLDEGRNY DCELRLLIHQSLAGGIIGVKGAKIK ELRENTQTTIKLFQECCPHSTDRVVLIGGKPDRVVECIKII IINYGNECYDILALLSGLRHWVVHNNEEESRISRTWLYNLDKNLDNEYISTLNYLYDRITNELTNSFSKNSAEQYFRFSIMKEQKNLGFNITKLREVMLDRKDMSEIRKNHKVFDSIRTKVYTMMDFVIYRYYIEEEKDIFVINLRGSFNDDQKDALYYDEANRIWRKLENIMHNIKEFRGNKTREYKKKDAPRLPRILPAGRDVSAFSKLMYALTMFLDGKEINDLLTTLINKFDNIQSFLKVMPLIGVNAKFVEEYAFFKDSAKIADELRLIKSFARMGEPIADARRAMYIDAIRILGTKLKKGKHGMRNFIINNVISNKRFHYLIRYGDPAHLHEIAKNEAVVKFVLGRIADIQKKQGQNGKNQIDRYYETCIQFDKKRSVIEDTGRENAEREKFKKIISLYLTVIYHILKNIVNINARYVIGFHCVERDAQLYKEKGYDINLKKLEEQINREKAKTALNAYLRNTKWNVIIREDLLRIDNKTCTLFRNKAVHLEVARYVHAYINDINDEKKYNDRLLKLLCVPFGYCIPRFKNLSIEALFDRNEAAKFDKEKKKVSGNS (SEQ ID NO: 17)

[0296] Nucleotide sequence:

[0297] atgatcgaaaagaagaagagctttgcaaaaggcatgggcgttaaaagtaccctggtgagcggcagtaaagtgtatatgaccacctttgccgaaggcagtgatgcattttcacatccgaaaggttatgccgtggtggcaaataatccgctgtataccggcccggttcagcaggatatgctgggcctgaaagaaaccctggaaaaacgctattttggtgaaagcgcagatggtaatgataatatttgcattcaggttatccacaatatcctggatattgaaaaaatcctggcagaatatatcaccaatgcagcctatgcagttaataatattagcggtctggatgaaggtcgcaattat gactgcgagttgagg ctgttgattcatcagagtctagcaggaggaattattggggtcaaaggtgctaaaatcaaagaacttcgagagaaca ctcaaaccaccatcaagcttttccaggaatgctgtcctcattccactgacagagttgttcttattggaggaaaacc cgatagggttgtagagtgcataaagatcatc

[0298] (7) The amino acid sequence and nucleotide sequence of mini Cas13d N47-RBD3 are shown below:

[0299] Amino acid sequence:

[0300] MIEKKKSFAKGMGVKSTLVSGSKVYMTTFAEGSDAFSHPKGYAVVAN TNLIVNYLPQNMTQDELRSLF SSIGEVESAKLIRDKVAGHSLGYGFVNYVTAKDAERAINTLNGLRLQSKTIKVSYARP NPLYTGPVQQDMLGLKETLEKRYFGESADGNDNICIQVIHNILDIEKILAEYITNAAYAVNNISGLDEGRNYIINYGNECYDILALLSGLRHWVVHNNEEESRISRTWLYNLDKNLDNEYISTLNYLYDRITNELTNSFSKNSAEQYFRFSIMKEQKNLGFNITKLREVMLDRKDMSEIRKNHKVFDSIRTKVYTMMDFVIYRYYIEEEKDIFVINLRGSFNDDQKDALYYDEANRIWRKLENIMHNIKEFRGNKTREYKKKDAPRLPRILPAGRDVSAFSKLMYALTMFLDGKEINDLLTTLINKFDNIQSFLKVMPLIGVNAKFVEEYAFFKDSAKIADELRLIKSFARMGEPIADARRAMYIDAIRILGTKLKKGKHGMRNFIINNVISNKRFHYLIRYGDPAHLHEIAKNEAVVKFVLGRIADIQKKQGQNGKNQIDRYYETCIQFDKKRSVIEDTGRENAEREKFKKIISLYLTVIYHILKNIVNINARYVIGFHCVERDAQLYKEKGYDINLKKLEEQINREKAKTALNAYLRNTKWNVIIREDLLRIDNKTCTLFRNKAVHLEVARYVHAYINDINDEKKYNDRLLKLLCVPFGYCIPRFKNLSIEALFDRNEAAKFDKEKKKVSGNS (SEQ ID NO: 19)

[0301] [[ID=​​atgatcgaaaagaagaagagctttgcaaaaggcatgggcgttaaaagtaccctggtgagcggcagtaaagtgtatatgaccacctttgccgaaggcagtgatgcattttcacatccgaaaggttatgccgtggtggcaaat acg aatttgatcgtcaactacctccctcagaacatgacccaggatgagttacgaagcctgttcagcagcattggtgaag ttgaatctgcaaaacttattcgggataaagtagcaggacacagcttgggctatggctttgtgaactacgtgaccgc gaaggatgcagagagagcgatcaacgctgaacggcttgaggctccagtcaaaaccattaaggtgtcgtatgct cgcccg

[0303] (8) The amino acid sequence and nucleotide sequence of mini Cas13d N47-RBD7 are shown below respectively:

[0304] Amino acid sequence:

[0305] MIEKKKSFAKGMGVKSTLVSGSKVYMTTFAEGSDAFSHPKGYAVVAN DCELRLLIHQSLAGGIIGVKG PAYMENTQTTIKLFQECCPHSTDRVVLIGGKPDRVVECIKII NPLYTGPVQQDMLGLKETLEKRYFGESADGNDNICIQVIHNILDIEKILAEYITNAAYAVNNISGLDEGRNYIINYGNECYDILALLSGLRHWVVHNNEEESRISRTWLYNLDKNLDNEYISTLNYLYDRITNELTNSFSKNSAEQYFRFSIMKEQKNLGFNITKLREVMLDRKDMSEIRKNHKVFDSIRTKVYTMMDFVIYRYYIEEEKDIFVINLRGSFNDDQKDALYYDEANRIWRKLENIMHNIKEFRGNKTREYKKKDAPRLPRILPAGRDVSAFSKLMYALTMFLDGKEINDLLTTLINKFDNIQSFLKVMPLIGVNAKFVEEYAFFKDSAKIADELRLIKSFARMGEPIADARRAMYIDAIRILGTKLKKGKHGMRNFIINNVISNKRFHYLIRYGDPAHLHEIAKNEAVVKFVLGRIADIQKKQGQNGKNQIDRYYETCIQFDKKRSVIEDTGRENAEREKFKKIISLYLTVIYHILKNIVNINARYVIGFHCVERDAQLYKEKGYDINLKKLEEQINREKAKTALNAYLRNTKWNVIIREDLLRIDNKTCTLFRNKAVHLEVARYVHAYINDINDEKKYNDRLLKLLCVPFGYCIPRFKNLSIEALFDRNEAAKFDKEKKKVSGNS (SEQ ID NO: 21)

[0306] Nucleotide sequence:

[0307] atgatcgaaaagaagaagagctttgcaaaaggcatgggcgttaaaagtaccctggtgagcggcagtaaagtgtatatgaccacctttgccgaaggcagtgatgcattttcacatccgaaaggttatgccgtggtggcaaat gac tgcgagttgaggctgttgattcatcagagtctagcaggaggaattattggggtcaaaggtgctaaaatcaaagaac ttcgagagaacactcaaccaccatcaagcttttccaggaatgctgtcctcattccactgacagagttgttcttat tggaggaaaacccgatagggttgtagagtgcataaagatcatc

[0308] (9) The amino acid sequence and nucleotide sequence of mini Cas13d N47-RBD7 + Y119-RBD6 are shown below respectively:

[0309] Amino acid sequence:

[0310] MIEKKKSFAKGMGVKSTLVSGSKVYMTTFAEGSDAFSHPKGYAVVAN DCELRLLIHQSLAGGIIGVKG PAYMENTQTTIKLFQECCPHSTDRVVLIGGKPDRVVECIKII NPLYTGPVQQDMLGLKETLEKRYFGESADGNDNICIQVIHNILDIEKILAEYITNAAYAVNNISGLDEGRNY ENVIRONMENTAL SKNAGAVIGKGKNIKALRTDYNASV SVPDSSGPERILSISADIETIGEILKKI IINYGNECYDILALLSGLRHWVVHNNEEESRISRTWLYNLDKNLDNEYISTLNYLYDRITNELTNSFSKNSAEQYFRFSIMKEQKNLGFNITKLREVMLDRKDMSEIRKNHKVFDSIRTKVYTMMDFVIYRYYIEEEKDIFVINLRGSFNDDQKDALYYDEANRIWRKLENIMHNIKEFRGNKTREYKKKDAPRLPRILPAGRDVSAFSKLMYALTMFLDGKEINDLLTTLINKFDNIQSFLKVMPLIGVNAKFVEEYAFFKDSAKIADELRLIKSFARMGEPIADARRAMYIDAIRILGTKLKKGKHGMRNFIINNVISNKRFHYLIRYGDPAHLHEIAKNEAVVKFVLGRIADIQKKQGQNGKNQIDRYYETCIQFDKKRSVIEDTGRENAEREKFKKIISLYLTVIYHILKNIVNINARYVIGFHCVERDAQLYKEKGYDINLKKLEEQINREKAKTALNAYLRNTKWNVIIREDLLRIDNKTCTLFRNKAVHLEVARYVHAYINDINDEKKYNDRLLKLLCVPFGYCIPRFKNLSIEALFDRNEAAKFDKEKKKVSGNS (SEQ ID NO: 23)

[0311] Nucleotide sequence:

[0312] atgatcgaaaagaagaagagctttgcaaaaggcatgggcgttaaaagtaccctggtgagcggcagtaaagtgtatatgaccacctttgccgaaggcagtgatgcattttcacatccgaaaggttatgccgtggtggcaaat gac tgcgagttgaggctgttgattcatcagagtctagcaggaggaattattggggtcaaaggtgctaaaatcaaagaac ttcgagagaacactcaaccaccatcaagcttttccaggaatgctgtcctcattccactgacagagttgttcttat tggaggaaaacccgatagggttgtagagtgcataaagatcatc aatccgctgtataccggcccggttcagcaggatatgctgggcctgaaagaaaccctggaaaaacgctattttggtgaaagcgcagatggtaatgataatatttgcattcaggttatccacaatatcctggatattgaaaaaatcctggcagaatatatcaccaatgcagcctatgcagttaataatattagcggtctggatgaaggtcgcaattat atggttgaattacgcattctgcttcagagcaagaatgctggggca gtgattggaaaaggaggcaagaatattaaggctctccgtacagactacaatgccagtgtttcagtcccagacagca gtggccccgagcgcatattgagtatcagtgctgatattgaaacaattggagaaattctgaagaaaatc

[0313] (10) The amino acid sequence and nucleotide sequence of mini Cas13d N47-RBD7 + Y119-RBD7 are shown below respectively:

[0314] Amino acid sequence:

[0315] MIEKKKSFAKGMGVKSTLVSGSKVYMTTFAEGSDAFSHPKGYAVVAN DCELRLLIHQSLAGGIIGVKG AKIKELRENTQTTIKLFQECCPHSTDRVVLIGGKPDRVVECIKII NPLYTGPVQQDMLGLKETLEKRYFGESADGNDNICIQVIHNILDIEKILAEYITNAAYAVNNISGLDEGRNY DCELRLLIHQSLAGGIIGVKGAKIKELRENTQTTI KLFQECCPHSTDRVVLIGGKPDRVVECIKII IINYGNECYDILALLSGLRHWVVHNNEEESRISRTWLYNLDKNLDNEYISTLNYLYDRITNELTNSFSKNSAEQYFRFSIMKEQKNLGFNITKLREVMLDRKDMSEIRKNHKVFDSIRTKVYTMMDFVIYRYYIEEEKDIFVINLRGSFNDDQKDALYYDEANRIWRKLENIMHNIKEFRGNKTREYKKKDAPRLPRILPAGRDVSAFSKLMYALTMFLDGKEINDLLTTLINKFDNIQSFLKVMPLIGVNAKFVEEYAFFKDSAKIADELRLIKSFARMGEPIADARRAMYIDAIRILGTKLKKGKHGMRNFIINNVISNKRFHYLIRYGDPAHLHEIAKNEAVVKFVLGRIADIQKKQGQNGKNQIDRYYETCIQFDKKRSVIEDTGRENAEREKFKKIISLYLTVIYHILKNIVNINARYVIGFHCVERDAQLYKEKGYDINLKKLEEQINREKAKTALNAYLRNTKWNVIIREDLLRIDNKTCTLFRNKAVHLEVARYVHAYINDINDEKKYNDRLLKLLCVPFGYCIPRFKNLSIEALFDRNEAAKFDKEKKKVSGNS (SEQ ID NO:25)

[0316] Nucleotide sequence:

[0317] atgatcgaaaagaagaagagctttgcaaaaggcatgggcgttaaaagtaccctggtgagcggcagtaaagtgtatatgaccacctttgccgaaggcagtgatgcattttcacatccgaaaggttatgccgtggtggcaaat gac tgcgagttgaggctgttgattcatcagagtctagcaggaggaattattggggtcaaaggtgctaaaatcaaagaac ttcgagagaacactcaaaccaccatcaagcttttccaggaatgctgtcctcattccactgacagagttgttcttat tggaggaaaacccgatagggttgtagagtgcataaagatcatc aatccgctgtataccggcccggttcagcaggatatgctgggcctgaaagaaaccctggaaaaacgctattttggtgaaagcgcagatggtaatgataatatttgcattcaggttatccacaatatcctggatattgaaaaaatcctggcagaatatatcaccaatgcagcctatgcagttaataatattagcggtctggatgaaggtcgcaattat gactgcgagttgaggctgttgattcatcagagtctagcaggagga attattggggtcaaaggtgctaaaaatcaaagaacttcgagagaacactcaaaccaccatcaagcttttccaggaat gctgtcctcattccactgacagagttgttcttattggaggaaaacccgatagggttgtagagtgcataaagatcat c

[0318] Example 2 Expression and purification of engineered Cas13d protein

[0319] The engineered Cas13d protein sequence was cloned into the pET-28b vector, with a 6×His and SUMO tag at the N-terminus. The plasmid was transformed into Rosetta 2(DE3) competent cells, plated on LB agar plates containing antibiotics, and incubated overnight at 37°C. Single colonies were then picked and inoculated into 4 mL of LB medium, and cultured at 37°C with shaking for 10–12 hours until saturation. The culture was then transferred to 1 L of 2×TY medium and cultured at 37°C with shaking until an OD 600 of 0.4–0.5 was achieved. The culture was then cooled to 16°C and continued until an OD 600 of 0.6–0.8 was reached. IPTG was added to a final concentration of 0.1–0.2 mM to induce expression, and the cells were cultured at 16°C for 16–18 hours. The cells were then collected by centrifugation at 8000×g and 4°C.

[0320] The bacterial pellet was resuspended in pre-cooled lysis buffer (50 mM Tris pH 8.0, 800 mM NaCl, 10% glycerol) and the cells were thoroughly lysed by high-pressure homogenization and sonication. The lysate was centrifuged at 12000 rpm and 4℃ for 1 hour to remove cell debris, and the supernatant was collected as the crude extract.

[0321] The crude extract was incubated with Ni-NTA agarose, and after binding, it was eluted by a chromatography column. The SUMO tag was removed by enzymatic digestion with SUMO-specific enzyme (ULP1) and incubated at 16°C for 16 hours.

[0322] After the protein was concentrated to 1.5-2 mL using an ultrafiltration concentrator, it was finely purified using a Superdex 200 Increase 10 / 300GL gel filtration chromatography column. The final target protein was concentrated to 200-250 μL and aliquoted into pre-chilled EP tubes and stored at -80°C.

[0323] Example 3: Target Sequence and crRNA Design

[0324] 3.1 Target Sequence

[0325] Using the NCBI GenBank database, reference coding sequences were obtained for oncogenic mutation sites in the KRAS (NM_033360.4), BRAF (NM_004333.6), and IDH1 (NM_005896.4) genes, and primers were designed using Primer Premier 6.0 software. Based on mutation information from the Cancer Genomics Database (COSMIC), a 150 bp characteristic sequence, including both upstream and downstream mutations, was selected as the target sequence for primer design, centered on the KRAS G12D (c.35G>A), BRAF V600E (c.1799T>A), and IDH1 R132C (c.394C>T) mutation sites. The T7 promoter sequence (TAATACGACTCACTATAGGG (SEQ ID NO: 27)) was introduced at the 5' end of the forward primer, and the reverse primer was designed to ensure complementarity with the target sequence and avoid the formation of secondary structures. Primer parameters were validated using OligoAnalyzer to ensure that the Tm value difference was < 5 ℃, the GC content was 40-60%, and there were no significant hairpin structures.

[0326] The target oncogene sequences are shown in Table 1 below.

[0327] Table 1

[0328]

[0329] Note: Bold and underlined parts are the corresponding sites or mutation sites in the wild type.

[0330] 3.2 crRNA Design

[0331] This invention utilizes the Cas13d Design platform (http: / / cas13design.nygenome.org) to design specific gRNAs targeting KRAS G12D, BRAF V600E, and IDH1 R132C mutations. By inputting a KRAS G12D mutant transcript, the platform generated the top 10 most active gRNAs and assessed their activity using the TIGER score (Score_G12D). Simultaneously, a control library of KRAS wild-type single-base mismatch gRNAs was constructed, and their TIGER scores (Score_WT) were calculated. Through ΔScore (Score_G12D - Score_WT ≥ 0.2) and sequence matching validation, four gRNAs with high activity (Score_G12D = 0.93-0.96) and high specificity (ΔScore = 0.24-0.93) were selected.

[0332] Furthermore, two highly specific gRNAs were also obtained from the BRAF V600E and IDH1 R132C mutation sites using the same screening criteria. The crRNA sequences of the candidate gRNAs include the essential Handle sequence (5'-AACCCCTACCAACTGGTCGGGGTTTGAAAC-3' (SEQ ID NO: 34), derived from Ruminococcusflavefaciens XPD3002) and the gRNA sequence, with the structure 5'-T7 promoter-Handle sequence-gRNA sequence-3'.

[0333] Primers were designed for in vitro transcription. The forward primer was 5'-TAATACGACTCACTATAGGG-[Handle]-3' (SEQ ID NO: 27), and the backward primer was 5'-[Reverse Complement of Guide]-[Reverse Complement of Handle]-3', used to synthesize a double-stranded DNA template containing the T7-Handle-Guide sequence.

[0334] The complete sequences of the candidate crRNAs are shown in Table 2 below.

[0335] Table 2

[0336]

[0337] 3.3 Rational Design of Stepwise Single-Base Mismatch crRNAs Based on Tumor-Driven Mutations:

[0338] (1) Determination of highly active crRNA sequences

[0339] Through preliminary in vitro activity screening experiments, the crRNA sequences with the highest cleavage efficiency against KRAS G12D, BRAF V600E, and IDH1 R132C mutant RNAs (KRAS G12D crRNA4, BRAFV600E crRNA2, and IDH1 R132C crRNA1, respectively) were identified from the candidate crRNAs and used as design templates.

[0340] (2) Targeted introduction strategy for single base mismatch

[0341] In this invention, to meet the requirement of accurate identification of oncogenic single nucleotide variants (SNVs), a rational design strategy is employed to engineer crRNA, thereby optimizing its ability to identify mismatched bases. For mutant RNA (SNV), single-base mutations are introduced one by one into the complementary sequence region of crRNA, constructing a single mismatch library (23nt × 3 mutation types) covering the entire complementary region. This design ensures a single-base mismatch (1 bp mismatch) between crRNA and the single-base mutant RNA, while simultaneously creating a double-base mismatch (2 bp mismatch) between crRNA and wild-type RNA (WT), thus enhancing the ability to distinguish mutant RNA.

[0342] During site-directed mutagenesis, thymine (T) or adenine (A) were preferentially used to replace the original bases. All mutant sequence designs were analyzed and validated using SnapGene software (v6.1.1) to ensure the accuracy of base substitutions and the stability of complementary sequences, thus providing a reliable design basis for subsequent experiments.

[0343] The mismatched crRNA primer sequences are shown in Table 3 below.

[0344] Table 3

[0345]

[0346]

[0347]

[0348]

[0349] (3) Rational design strategies for dibase mismatch

[0350] Based on the recognition results of engineered Cas13d for target RNA under single-base mismatch conditions, a further rational design for double-base mismatches of crRNA was developed. At the M2-M6 and M17-M22 sites (corresponding to sites 2-7 and 18-22 of the crRNA RNA sequence (5'-3')) where the recognition activity of engineered Cas13d was significantly reduced, two sites were selected for pairwise base mutation combinations. A crRNA double-mismatch library was constructed (12 double-base mismatch combinations × 3 mutation types). This design resulted in a double-base mismatch (2 bp mismatch) between crRNA and single-base mutant RNA, while simultaneously creating a triple-base mismatch (3 bp mismatch) between crRNA and wild-type RNA (WT), further enhancing the ability to distinguish mutant RNA.

[0351] The mutagenesis strategy is the same as above, with adenine (A) being the preferred alternative to the original base; if the original base is adenine, it is replaced with cytosine (C) to ensure the diversity and feasibility of the mutation design to the greatest extent.

[0352] The mismatched crRNA primer sequences are shown in Table 4 below.

[0353] Table 4

[0354]

[0355]

[0356] Example 4 Activity Detection

[0357] 4.1 Construction of the in vitro reaction system

[0358] To systematically evaluate the specific recognition activity of engineered Cas13d protein on target RNA, this invention employs an in vitro cleavage reaction system optimized with fluorescent probes.

[0359] First, the purified engineered Cas13d protein (preferably 10-200 nM, more preferably 30-100 nM, and 50 nM is used as an example in this embodiment) and the corresponding crRNA (50 nM) are mixed in a 1:1 molar ratio in a pre-cooled reaction buffer (50 mM Tris-HCl [pH 7.5], 150 mM NaCl) and incubated at room temperature for 10 minutes to form a stable mini Cas13d-crRNA complex with cleavage activity.

[0360] Subsequently, the target RNA (such as KRAS WT / G12D mutant RNA, 50 nM) and a fluorescent reporter probe (50 nM) were added to the system. The probe was designed as a 5' FAM-modified 10-mer poly U sequence coupled to a 3' BHQ1 quencher group (5'-FAM-U10-BHQ1-3', 100 μM stock solution). The release of the probe's fluorescent signal depended on the target RNA recognition activity mediated by the engineered Cas13d protein.

[0361] When the engineered Cas13d protein recognizes and binds to the target RNA, the enzyme cleaves the BHQ1 quencher group at the 3' end of the probe, thereby relieving the quenching effect and releasing a fluorescent signal. The change in the probe's fluorescence signal directly reflects the recognition activity of the engineered Cas13d protein for the target RNA. After the reaction system is mixed by centrifugation at 5000 rpm for 5 seconds, the in vitro cleavage reaction is initiated at room temperature.

[0362] The in vitro cleavage reaction system of the fluorescent probe (10 μL) is shown in Table 5.

[0363] Table 5

[0364]

[0365] 4.2 Experimental Group Design

[0366] The experiment was set up with the following three groups:

[0367] Experimental group: engineered Cas13d protein (50 nM) + G12D specific crRNA (50 nM) + target RNA (50 nM) + fluorescent probe;

[0368] Negative control group: engineered Cas13d protein (50 nM) + NT crRNA (50 nM, non-target sequence) + target RNA (50 nM) + fluorescent probe;

[0369] Blank control group: Contains only reaction buffer and an equal volume of nuclease-free water to replace the protein component; all other conditions are the same as the experimental group. This design effectively eliminates non-specific cleavage and autofluorescence interference.

[0370] 4.3 Real-time fluorescence kinetics detection and data analysis

[0371] Real-time fluorescence kinetics monitoring was performed using a Biotek Synergy H1 multi-functional microplate reader. Parameters were set as follows: excitation wavelength 488 nm, emission wavelength 520 nm, and gain value automatically optimized and calibrated in pre-experimentation. Fluorescence signals (RFU) were continuously acquired at 15, 30, 45, 60, 75, and 90 minutes of reaction.

[0372] To eliminate background interference, the relative fluorescence change (ΔRFU) was calculated using the initial time point (15 minutes) as a baseline, where ΔRFU(t) = RFU(t) - RFU(t15min). This value was used as an indicator to assess recognition activity. RFU represents the fluorescence value of the experimental group at different time points, and RFU(t15min) represents the fluorescence value at the initial stage of the reaction (15 minutes). This change reflects the degree to which the fluorescent probe is cleaved after the target RNA is recognized.

[0373] To quantify the specificity of the mini Cas13d-crRNA system for single-base mutation detection, ΔΔRFU (G12D ΔRFU - WT ΔRFU) was used as the evaluation criterion to further compare the specific recognition of G12D by crRNA.

[0374] All experiments were independently repeated three times (n=3), and data are expressed as mean ± standard deviation. One-way ANOVA was performed using GraphPad Prism 9.0, and differences between groups were assessed using Tukey's multiple comparison test with a significance threshold of α=0.05.

[0375] The activity detection methods used in this embodiment are all performed in accordance with the methods described in this embodiment.

[0376] Example 5: Design and Validation of SNV-Targeting crRNA

[0377] To establish an allele-specific detection framework, this invention first used the Cas13design platform (http: / / cas13design.nygenome.org) to design candidate crRNAs targeting three oncogenic hotspots: KRAS G12D, IDH1 R132C, and BRAF V600E. Figure 1 (A) For each target site, an 80 nt region centered on the variant site was selected as input. The platform ranks primers based on the predicted activity of crRNA against the SNV sequence (Score_SNV), and then extracts the top ten candidate primers for each target site. Figure 2 (A in the middle).

[0378] Next, this invention calculated the predicted activity (Score_WT) for wild-type alleles, and used ΔScore = Score_SNV - Score_WT as the specificity metric. crRNAs with ΔScore ≥ 0.2 were preferentially selected for testing. Figure 1 A in Figure 2 (A in the middle).

[0379] Ultimately, this invention screened four candidate crRNAs for KRAS G12D, while IDH1 R132C and BRAF V600E each screened two candidate crRNAs. Figure 1 B in Figure 2 (A in the middle).

[0380] Figure 1 The sequence (DNA) information (3'-5') of B in the diagram and its corresponding 5'-3' direction RNA sequence are shown in Table 6 below:

[0381] Table 6

[0382]

[0383] The corresponding sequence containing KRAS G12D is shown below:

[0384] ACTTGTGGTAGTTGGAGCTGATGGCGTAGGCAAGAGTGCCTTG (SEQ ID NO: 165).

[0385] Figure 2 The sequence (DNA) information of each part of A is shown in Table 7 below:

[0386] Table 7

[0387]

[0388]

[0389]

[0390] Next, the cleavage activity of these candidate crRNAs was evaluated using a fluorescence reporter assay. In the KRASG12D primers, crRNA4 showed the strongest signal separation between the mutant and wild-type transcripts, consistent with its high predicted ΔScore (0.93). Conversely, although crRNA1 exhibited strong activity, its allele discrimination was poor, reflecting a lower ΔScore (0.24). Figure 1 (C in the original text). For IDH1 R132C, crRNA1 can effectively distinguish between mutant and wild-type RNA, while crRNA2 has poor specificity, which is consistent with their predicted ΔScores (0.86 and 0.45, respectively). Figure 1 In the case of BRAF V600E, both crRNA1 and crRNA2 showed detectable allele bias, with crRNA2 producing the strongest signal separation, again consistent with its higher ΔScore (0.58). Figure 1 (E in the text).

[0391] However, even the top-ranked primers at all target sites retained measurable activity on the wild-type template, resulting in only moderate separation between mutant and wild-type signals. Consistent with these endpoint measurements, full time-course data showed that discriminative crRNAs produced a steeper increase in fluorescence on mutant RNA, while non-discriminatory primers produced overlapping curves. Figure 2 (BD in the middle).

[0392] In summary, these results demonstrate that rationally designed computational methods can generate crRNAs with varying degrees of allele specificity. However, the residual wild-type activity and limited signal separation capabilities observed here are insufficient to accurately distinguish between SNVs and wild-type transcripts, thus requiring further engineering modifications to improve selectivity.

[0393] Example 6: Rationally engineered mismatched crRNA can enhance allele discrimination ability

[0394] To improve allele specificity, this invention engineered crRNA by systematically introducing artificial mismatches into the spacer region. Figure 3 (AB in the original text). For each SNV site, this invention selects the crRNA with the strongest distinguishing ability from the initial screening as the maternal reference (M0): crRNA4 is selected for KRAS G12D, crRNA1 is selected for IDH1 R132C, and crRNA2 is selected for BRAF V600E.

[0395] Figure 3 The information of the crRNA sequences (corresponding DNA) after each missense mutation in B and their corresponding RNA sequences in the 5'-3' direction are shown in Table 8 below.

[0396] The sequence containing the KRAS G12D mutation that is complementary to crRNA is:

[0397] GATGGCGTAGGCAAGAGTGCCTT (SEQ ID NO: 196).

[0398] Table 8

[0399]

[0400] Based on this template, 23 single-mismatch variants (M1–M23) were constructed by replacing each base in the 23 nt spacer one by one. These designs introduce one mismatch between the crRNA and the mutant template, and two mismatches with the wild-type template—one at the engineered mismatch site and the other at the mutation site itself. Figure 3 B in the middle; Figure 4 (AB in the middle).

[0401] Figure 4 The sequence information (DNA) and corresponding RNA sequences in the AD are shown in Tables 9-12 below. Among them, the sequence containing the IDH1 R132C mutation that is complementary to the crRNA is: AGGTTGTCATGCTTATGGGGATC (SEQ ID NO:243); the sequence containing the BRAF V600E mutation that is complementary to the crRNA is: GTCTAGCTACAGAGAAATCTCGA (SEQ ID NO: 244).

[0402] Table 9

[0403]

[0404] Table 10

[0405]

[0406] Table 11

[0407]

[0408] Table 12

[0409]

[0410] Fluorescence assay results were normalized based on "perfectly matched mutant crRNA-target pairs". For all three mutation sites, miniCas13d exhibited significant "mismatch-dependent" sensitivity: the M2–M6 and M17–M22 regions (corresponding to sites 2–7 and 18–22 of the crRNA RNA sequence (5'–3')) produced the most pronounced signal separation, showing strong inhibition of wild-type activity while mutant activity was partially preserved (approximately 0.5–0.8 of the reference level). Figure 3 (C in the above region). Conversely, mismatches located outside the aforementioned region tend to reduce the activity of both mutant and wild-type templates by a similar magnitude, or have almost no effect, thus providing limited discriminative gain.

[0411] These results suggest that M2-M6 and M17-M22 (i.e., positions 2-7 and 18-22 of the RNA sequence (5'-3') corresponding to crRNA) are key sensitive positions for achieving allelic selective engineering.

[0412] Subsequently, this invention further constructed a set of "double mismatch (DM)" crRNAs, and obtained 12 cross-region combinations (DM1–DM12) by randomly pairing mismatch positions between two mismatch-sensitive regions (M2–M6 and M17–M22). Figure 3 DE in Figure 4 (CD in the text). Compared to single mismatches, these designs generally further improve the ability to distinguish between mutants and wild types, but often at the cost of sacrificing catalytic activity. Figure 3 (F in the text). For example, DM10 (M3M18) retains approximately 45% activity on the mutant target but reduces wild-type recognition to below 10%. Similarly, variants such as DM5 (M3M6), DM9 (M3M19), and DM11 (M6M17) almost completely eliminate wild-type cleavage but also reduce mutant activity to 20–40% of baseline.

[0413] Figure 3The sequence information (DNA) and corresponding RNA sequences in E are shown in Table 13 below. Among them, the sequence containing the KRAS G12D mutation that is complementary to crRNA is: GATGGCGTAGGCAAGAGTGCCTT (SEQ ID NO: 385).

[0414] Table 13

[0415]

[0416] Therefore, double mismatches can significantly weaken wild-type recognition, but are accompanied by a decrease in catalytic efficiency, indicating that while mismatch engineering can improve allele discrimination, it is still difficult to completely overcome the trade-off between specificity and activity. This limitation also suggests the need to introduce additional strategies to achieve reliable single-base resolution detection.

[0417] Example 7: Sensitivity Analysis of miniCas13d for SNV Detection

[0418] To evaluate the sensitivity of miniCas13d in SNV detection, this invention used KRAS G12D as a representative mutation and formulated RNA mixtures with well-defined mutant allele frequencies (VAFs), ranging from 100% to 0.01%. Figure 5 (A) Two crRNA guides were tested: M5 (introducing a single mismatch at position 5) and DM10 (introducing a double mismatch at positions 3 and 18).

[0419] Each guide was evaluated under the condition of "whether loop-mediated isothermal amplification (LAMP) was used for pre-amplification". Without amplification, the M5-guided miniCas13d system showed a strong response in samples with a high mutation rate, with a fluorescence signal approximately 8 times that of WT at 100% SNV. Figure 5 (B in the middle).

[0420] As the mutation rate decreased, the signal intensity gradually decreased; when the mutant RNA content was 5%, the signal was still statistically significantly higher than that of WT, but then a plateau occurred, and no significant difference was detected between 5% and 1%, suggesting that its resolution at low allele frequencies is limited.

[0421] LAMP preamplification significantly improves fluorescence output in all VAF groups, up to approximately 20-fold at 100% SNV, but the resolution improvement between 5% and 1% is still limited, indicating that the sensitivity gain has a limited marginal effect in low-frequency detection scenarios. DM10 guidance shows a similar trend.

[0422] Under direct detection conditions, the DM10 also produces a strong signal for samples with a high SNV ratio and maintains a clear distinction from WT within a range of 95% to 5%. Figure 5 (C in the original text). However, its distinguishing ability gradually weakened when the mutation rate decreased to 1% or lower. After the introduction of LAMP, the overall fluorescence output further increased, reaching a maximum of about 14 times that of the WT baseline at 100% SNV. During the amplification reaction, the fluorescence fold change showed a smooth and almost monotonic decreasing curve as the mutation rate decreased from 95% to 5%, and the signal could still be well distinguished within this range; however, at 1% and lower frequencies, the signal separation weakened significantly, and no statistically significant difference was observed between the 1% and 0.5% VAF groups.

[0423] To more intuitively and quantitatively characterize the detection sensitivity at different mutation frequencies, this invention plots the fluorescence fold change relative to WT as a function of the KRAS G12D mutation ratio. Figure 5 (d in the text), and calculate the functional limit of detection (fLoD) according to the CLSI EP17-A2 guideline ( Figure 6 Without pre-amplification, the miniCas13d system showed limited response to low mutation abundance: regardless of whether M5 or DM10 was used, the signal enhancement was minimal when SNV was below 5%, with only significant fold changes occurring above this threshold. Isothermal amplification significantly improved overall signal intensity and broadened the dynamic range, especially at mutation rates ≥10%, but the difference between WT and 1% mutant RNA remained limited. Figure 5 (C in the middle).

[0424] Consistent with the above findings, fLoD results showed that amplification provided a stable but limited improvement in the detection threshold for both guides, with the resolution gain being most significant in high SNV ratio scenarios: for M5 crRNA, fLoD decreased from 2.67% (95% CI: 0.29–3.20%) to 1.53% (95% CI: 0.27–1.72%); for DM10 crRNA, fLoD decreased from 4.11% (95% CI: 0.22–4.94%) to 1.16% (95% CI: 0.17–1.35%). Figure 6 ).

[0425] These results indicate that isothermal amplification can consistently reduce the detection threshold in both types of guided designs, but its most significant resolution improvement is mainly reflected in scenarios with a high SNV ratio.

[0426] Example 8: Structure-guided engineering and rational localization of the miniCas13d RNA-binding domain

[0427] Given that introducing target mismatches to enhance specificity in crRNA design often leads to a decrease in detection activity, this invention attempts to restore the system's functional performance through rational protein engineering.

[0428] To this end, this invention establishes a systematic strategy: fusing heterologous RNA-binding domains (RBDs) at structurally permissible sites to enhance the RNA-binding ability of miniCas13d. Figure 7 (A) Since experimental structures of the miniCas13d complex formed with crRNA and target RNA are currently lacking, this invention first uses AlphaFold3 to predict the structure of the ternary complex (A). Figure 7 (B in the middle).

[0429] The model shows that a flexible, disordered segment spanning A30 to Q57 exists in the region adjacent to the direct repeat (DR) sequence of crRNA. The N47 residue is located near the crRNA-protein interaction interface and spatially far from the catalytic core, thus it is considered a promising RBD insertion site that could enhance RNA binding without perturbing catalytic activity. Figure 7 (B in the middle).

[0430] Simultaneously, this invention compares the AlphaFold3 model of miniCas13d with the cryo-electron microscopy structure of EsCas13d, thereby locating the predicted catalytic residue regions R138–H143 and R610–H615 in miniCas13d. Based on spatial conformational analysis, this invention further identifies Y119 as another insertion site, located near the predicted active site. This invention hypothesizes that fusing an RBD at this site can enhance target recognition and improve cleavage efficiency by locally stabilizing the substrate-enzyme interface.

[0431] Combining the relatively flexible N-terminal and C-terminal positions, the four sites mentioned above—N47, Y119, N-terminus, and C-terminus—constitute the structural basis for domain insertion. This invention constructs a modular library containing 10 human RBDs, covering RRM, KH domains, and C3H1-type motifs (…). Figure 7 A in the example, and thus 40 fusion constructs were generated, covering all combinations: N-RBD1 to 10, C-RBD1 to 10, N47-RBD1 to 10 and Y119-RBD1 to 10.

[0432] AlphaFold3 was then used to predict the structures of all 40 fusion proteins and their corresponding ternary complexes with crRNA and target RNA. The structural reliability was assessed using the predicted template modeling score (pTM) and the interface pTM score (ipTM). All fusion proteins exhibited high overall stability, while the ipTM scores of the ternary complexes ranged from 0.50 to 0.71, and the pTM scores ranged from 0.65 to 0.76.

[0433] These results suggest that most fusion designs can maintain the relative spatial relationships of crRNA, target RNA, and protein scaffold with good fidelity. Notably, some constructs exhibited low ipTM values, suggesting that domain insertion may alter the RNA-protein interaction interface.

[0434] Through comprehensive structural modeling, RNA spatial proximity analysis, and ipTM-guided interface evaluation, this invention ultimately screened eight miniCas13d-RBD fusion constructs for experimental validation: N-RBD6, C-RBD2, C-RBD4, Y119-RBD3, Y119-RBD6, Y119-RBD7, N47-RBD3, and N47-RBD7. Figure 8 (AH in the middle).

[0435] These variants performed well in terms of fold stability (pTM: 0.66–0.76) and had medium to high levels of interface confidence (ipTM: 0.56–0.71). Figure 8 (AH in the middle).

[0436] To further combine functional advantage modules and evaluate potential synergies, this invention also constructs a dual RBD fusion: RBD7 is fused in N47 and RBD6 is fused in Y119 (N47-RBD7+Y119-RBD6).

[0437] All eight single RBD variants were successfully expressed and purified in E. coli, as confirmed by SDS-PAGE results. Figure 8 (I) The molecular weight of the purified protein was consistent with expectations (approximately 75 kDa), indicating that it folded correctly and the construct was intact. The dual RBD fusion was also successfully expressed and purified, with yields and integrity comparable to the single RBD variant.

[0438] Example 9: Engineered miniCas13d-RBD variant enhances SNV recognition activity and specificity

[0439] To assess the functional impact of RBD insertion, this invention used KRAS G12D-targeted, mismatch-optimized crRNAM0 as a guide to quantitatively determine the cleavage activity of each miniCas13d-RBD variant via fluorescence assays. In the terminal fusion constructs, the activity of miniCas13d N-RBD6 was only about half that of the wild type, while miniCas13d C-RBD2 and C-RBD4 maintained or slightly increased their activity levels. Figure 9 A in the image above; Figure 10 (A in the middle).

[0440] Comprehensive structural modeling shows that both the N-terminus and C-terminus of miniCas13d are located near the 3′ end of the crRNA. This region is itself stabilized by extensive native protein-RNA interactions, therefore the functional gain that terminal RBD fusion can provide may be limited. Figure 7 (B in the middle).

[0441] In contrast, inserting RBDs into internal sites significantly enhances target cleavage. Y119-RBD7 is the best-performing single-insertion-site variant, exhibiting a 12.6-fold increase in cleavage activity compared to wild-type miniCas13d. Y119-RBD6, N47-RBD7, and Y119-RBD3 also show significant enhancements, with activity increases ranging from approximately 4.5 to 9.5 times; while the enhancement effect of N47-RBD3 is relatively moderate. The dual-RBD construct N47-RBD7+Y119-RBD6 produces the strongest enhancement, increasing cleavage activity by up to 17.4 times, suggesting a possible synergistic effect between distal RBD domains. Figure 9 A in the diagram below; Figure 10 (A in the middle).

[0442] To assess whether RBD fusion alters sequence specificity, this invention uses a comprehensive crRNA mismatch library to perform specificity profiling analysis on Y119-RBD7. The library targets KRAS G12D, IDH1 R132C, and BRAF V600E, and includes single-base mismatches (M1–M23) covering all spacer positions, as well as some double-mismatch combinations (DM1–DM12). Figure 9 BC in the middle; Figure 10 Despite a significant increase in overall activity, Y119-RBD7 maintained a mismatch sensitivity pattern highly similar to that of wild-type miniCas13d, and the position effect was largely preserved. Figure 9 (B in the middle).

[0443] Consistent with the parent enzyme, Y119-RBD7 exhibits a clear mismatch tolerance gradient, with the M2–M6 and M17–M22 regions producing the strongest mutant / wild-type signal separation: wild-type cleavage is strongly inhibited, while mutant activity is only partially reduced, typically remaining at 50–70% of the perfect match reference. Mismatches on either side of these regions result in a moderate activity penalty.

[0444] Conversely, in the central region (M8–M16), the mutant and wild-type target pairs generally exhibit high tolerance to mismatches; although mutant activity remains high, wild-type signaling increases synchronously, thus limiting the discriminative ability at these locations. Figure 9 (B in the original text). Further analysis of the double mismatch profile of Y119-RBD7 confirmed this positional dependence: specificity was optimal when both mismatches fell simultaneously within the M2–M6 and M17–M22 positions, corresponding to positions 2–7 and 18–22 of the crRNA sequence (5'–3'), respectively—these two "separation bands." Mutant cleavage was still detectable, while wild-type activity was suppressed to background levels. Figure 9 (C in the middle).

[0445] For example, DM10 (M3M18) retains approximately 47% of its matching activity on mutant targets while producing almost no wild-type cleavage. Figure 9 The position rule also applies to IDH1 R132C and BRAF V600E, indicating its broad applicability in different SNV scenarios. Figure 10 (BC in the middle).

[0446] In summary, these results demonstrate that Y119-RBD7 significantly raises the baseline activity while retaining the inherent "position-selective architecture" of wild-type miniCas13d.

[0447] Under the condition of mismatch-optimized guides M5 and DM10, this invention further evaluates the detection performance of Y119-RBD7 in the KRASG12D dilution series (mutation ratio from 100% to 0%), and compares the results under no-amplification and LAMP pre-amplification conditions. For both guides, the fold change relative to the WT control decreased in a dose-dependent manner as the mutation ratio decreased. Figure 9 DE in the middle.

[0448] Time-resolved curves showed that both the initial reaction rate and the endpoint ΔRFU increased with increasing mutant input, and LAMP improved both parameters while maintaining the ordination relationship between dilution series. Figure 11(AB in the original text). The fractional detection limits (fLoD, %) estimated according to EP17-A2 show: for M5, 1.57% without LAMP and 1.56% with LAMP; for DM10, 0.61% without LAMP and 1.66% with LAMP. Figure 9 (F in the middle).

[0449] Therefore, although LAMP can broaden the dynamic range, it cannot consistently improve sensitivity, which is consistent with the phenomenon of "simultaneous amplification of signal and background" in guide sequence-dependent systems.

[0450] The present invention then evaluated the dual-insertion construct N47-RBD7+Y119-RBD6. Compared with Y119-RBD7, this dual-RBD variant exhibited a higher ΔRFU and a steeper kinetic slope in the medium-to-high mutation ratio range. Figure 11 In the CD); under amplification-free and LAMP-free conditions, dose-dependent fold-change responses were observed for both M5 and DM10 (in the CD); Figure 12 (AB in the text). The fLoD values ​​given by EP17-A2 analysis are: M5 2.55% without LAMP and 1.49% with LAMP; DM10 0.59% without LAMP and 2.30% with LAMP. Figure 12 The C in the figure further illustrates that the benefit of pre-amplification is guide-dependent. Compared with the original miniCas13d, the engineered variant has a higher baseline activity and reduced dependence on amplification; the difference in fluorescence signal before and after LAMP is no longer so obvious, suggesting that it has more robust detection capability even without pre-amplification. Figure 9 F in the middle; Figure 12 (C in the middle).

[0451] It is worth noting that, under LAMP-free conditions, the fractional detection limit can reach a mutant allele frequency of approximately 1%, specifically ranging from about 0.6% to 1.6%, depending on the type of guide and construct used. Without sacrificing the inherent position-specific framework of the parent enzyme, the above-mentioned engineering modifications achieved an overall improvement in sensitivity.

[0452] Example 10 Performance evaluation of the modular miniCas13d-RBD platform under different amplification conditions

[0453] This invention provides a quantitative evaluation of platform performance within a unified analytical framework to characterize how the crRNA architecture and isothermal pre-amplification jointly shape single nucleotide detection performance.

[0454] This invention compares three Cas13d configurations: the unmodified miniCas13d, a single RBD fusion (Y119-RBD7), and a dual RBD construct (N47-RBD7+Y119-RBD6). Each configuration was paired with either a single mismatch guide M5 or a dual mismatch guide DM10 for KRAS G12D and tested in a direct detection and LAMP coupled process. Analytical sensitivity was evaluated according to CLSIEP17-A2, and the fractional limit of detection (fLoD), LoD slope, and sensitivity coefficient b^ were reported (Figure 13A). Quantitative behavior in the low VAF range (0–5%, 0–10%) was primarily evaluated by the deviation from the calibration curve relative to the full range (0–100%), specifically expressed as normalized root mean square error (NRMSE), combined with the "local / global slope ratio" to directly characterize the dispersion and fit fidelity in the low range (Figure 13B-). Figure 13C Furthermore, this invention further summarizes the precision and dispersion, as well as the absolute percentage error, using weighted root mean square error (WRMSE) and root mean square error percentage (RMSE%) (Figure 13 C-). Figure 13D , Figure 14 ).

[0455] Amplification does not always yield benefits; its net effect depends on the intrinsic activity of the enzyme-guided combination. For the unmodified miniCas13d, LAMP significantly improved performance: fLoD decreased from 2.67% to 1.53% under M5 conditions and from 4.11% to 1.16% under DM10 conditions. Low-range fidelity also improved simultaneously—the NRMSE of M5 in the 0–10% range decreased from 43.3% to 13.7% of that of M5+LAMP, and the local / global slope ratio (0–10% / 0–100%) approached 1.05 from 2.59, closer to the ideal value of 1, indicating that the low-range response better conforms to the full-range calibration relationship. Figures 13A-13C Conversely, the dual RBD construct already possesses high intrinsic sensitivity; amplification in this case actually increases background and worsens low-end performance: under direct detection conditions, DM10 achieves the lowest fLoD (0.59%), but after adding LAMP, the threshold worsens to 2.30%, while the deviation from global calibration increases, and the slope ratio deviates further from 1 ( Figures 13A-13D In the 0–10% range, NRMSE increased from 18.6% to 31.5%, and WRMSE and RMSE% also increased accordingly. For single RBD enzymes, direct detection with DM10 yielded approximately 0.61% fLoD; with the addition of LAMP, the threshold increased to approximately 1.66%, but the fidelity in the low range improved, with WRMSE in the 0–10% range decreasing from approximately 40.9% to approximately 13.4%, and NRMSE decreasing from 30.5% to 21.1%. Figure 13A – Figure 13D ).

[0456] After considering different enzyme configurations, a simple pairing rule emerges: "DM10+ direct detection" performs best in settings where the lowest detection threshold is most easily achieved; while for applications requiring stable low VAF quantification (especially 0–5% or 0–10%), "M5+LAMP" often provides more reliable low-range fitting and smaller dispersion. For unmodified enzymes, M5+LAMP significantly reduces low-range dispersion and brings the 0–10% / 0–100% slope ratio close to 1, reducing NRMSE to approximately 13.7%; on single-RBD enzymes, this combination also tightens the bias within the 0–5% window. Conversely, on dual-RBD enzymes, amplification increases background and weakens low-end fidelity, therefore "DM10+ direct detection" is more recommended. Figure 13D The radar chart further illustrates this consistent trend: the recommended pairings are closer to the central region and the slope ratio is closer to 1, reflecting the trade-off between sensitivity and fidelity under different skeletons.

[0457] The optimized pairing strategy depends on the specific application objective. For the highest sensitivity and simplest workflow, "dual RBD fusion miniCas13d + dual mismatch guide (DM10)" can achieve a detection threshold of approximately 0.59% without amplification. For stable and accurate quantification in the low-range (0–5%), "unmodified enzyme + M5 + LAMP" minimizes dispersion and better aligns the low-range response with full-range calibration. When prioritizing workflow simplification and rapid results, "direct detection with single RBD enzyme + DM10" is more suitable: it maintains single-base discrimination capability, avoids background increase introduced by amplification, and still achieves sub-percentage detection thresholds. Overall, these results reveal the core principle of the modular platform of this invention: RBD fusion and guide mismatch engineering each provide different performance advantages, and their benefits depend on the diagnostic scenario. Pre-amplification improves the detection of low-abundance targets by amplifying the signal, while RBD insertion enhances the system response by strengthening target binding and transactivation capabilities; however, when the enzyme itself is already highly sensitive, it is necessary to balance these mechanisms to avoid simultaneous background amplification. The ability to flexibly combine crRNA design and RBD configuration according to different clinical applications (e.g., favoring ultrasensitivity or high specificity / robust quantification) demonstrates the predictability, adjustability, and modularity of this engineered Cas13d platform.

[0458] To evaluate the translational application performance of this system, RNA from 15 pancreatic tumor samples was analyzed: 5 were KRAS G12D positive and 10 were G12D negative (wild-type or other non-G12D KRAS mutations, such as G12V). Under a uniform detection configuration (using M5 guidance), all three enzyme backbones were able to clearly separate G12D positive and G12D negative samples, with no overlap between the two groups of signals.

[0459] Unmodified miniCas13d provides basic discrimination capability, with a signal separation of 3.1 times ((4.7-9.6)×10⁻⁶) between the lowest positive sample and the highest negative sample. 3 Compare (1.1-1.5)×10 3 ΔRFU)( Figure 13E The single RBD variant Y119-RBD7 further broadened the dynamic range and improved detection robustness, increasing the separation between positive and negative signals by 7.4 times, while the negative control remained close to baseline (8.1 × 10⁻⁶). 4 –1.47×10 5 Comparison 2.5×10 3 –1.1×10 4 ΔRFU)( Figure 13E The dual RBD enzyme N47-RBD7+Y119-RBD6 produced the highest absolute signal in positive samples, but the negative baseline was also slightly elevated, with a final resolution of 4.8-fold (1.74 × 10⁻⁶). 5 –3.42×10 5 Comparison 1.1×10 4 –3.6×10 4 ΔRFU)( Figure 13E ).

[0460] Under all configurations, every sample was correctly classified, consistent with routine clinical typing results. Consistent with the above comparisons, under the Y119-RBD7 condition, G12D positive cases showed a signal enhancement of 18–37 times relative to the 0% VAF control, while all G12D negative samples clustered near the background. Figure 13F ).

[0461] In summary, these results demonstrate that the present invention enables direct, accurate, and high-contrast detection of oncogenic SNVs in clinical tumor RNA samples.

[0462] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. An engineered Cas13d protein fused with an RNA-binding domain, characterized in that, The engineered Cas13d protein includes: (a) A Cas13d protein, the amino acid sequence of which is shown in SEQ ID NO: 1; (b) At least one heterologous RNA-binding domain; Wherein, the at least one heterologous RNA-binding domain is inserted into a site selected from the group consisting of the N-terminus, C-terminus, N47 site, Y119 site, or a combination thereof in the Cas13d protein; Furthermore, the at least one heterologous RNA-binding domain is inserted into the corresponding site of the Cas13d protein via direct linking or linker peptide; and when at least one heterologous RNA-binding domain is inserted into multiple sites, the insertion method at each site is independently selected from direct linking or linker peptide linking.

2. The engineered Cas13d protein as described in claim 1, characterized in that, The amino acid sequence of the RNA-binding domain is selected from the group consisting of any amino acid sequence shown in SEQ ID NO: 2-6, or a combination thereof.

3. The engineered Cas13d protein as described in claim 1, characterized in that, The amino acid sequence of the engineered Cas13d protein is selected from the following group: (1) N47-RBD7 + Y119-RBD6, whose amino acid sequence is shown in SEQ ID NO: 23; (2) Y119-RBD7, whose amino acid sequence is shown in SEQ ID NO: 17; (3) Y119-RBD6, whose amino acid sequence is shown in SEQ ID NO: 15; (4) N47-RBD7, whose amino acid sequence is shown in SEQ ID NO: 21; (5) Y119-RBD3, whose amino acid sequence is shown in SEQ ID NO: 13; (6) C-RBD4, whose amino acid sequence is shown in SEQ ID NO: 11; (7) N47-RBD3, whose amino acid sequence is shown in SEQ ID NO: 19; (8) C-RBD2, whose amino acid sequence is shown in SEQ ID NO: 9; (9) N-RBD6, whose amino acid sequence is shown in SEQ ID NO: 7; (10) N47-RBD7 + Y119-RBD7, whose amino acid sequence is shown in SEQ ID NO: 25; Or a combination thereof.

4. A polynucleotide, characterized in that, The polynucleotide encodes the engineered Cas13d protein of claim 1.

5. An expression carrier, characterized in that, The expression vector comprises the polynucleotide of claim 4.

6. A host cell, characterized in that, The host cell contains the expression vector of claim 5, or has the polynucleotide of claim 4 integrated into its genome.

7. A detection reagent, characterized in that, The detection reagent contains the engineered Cas13d protein as described in claim 1.

8. A reagent kit, characterized in that, The kit comprises the detection reagent as described in claim 7.

9. The use of the engineered Cas13d protein as described in claim 1, characterized in that, Used to prepare CRISPR detection reagents or kits for detecting single nucleotide variants.

10. A method for preparing the engineered Cas13d protein of claim 1, characterized in that, Including the following steps: (a) Under expression conditions, the host cells of claim 6 are cultured to express the engineered Cas13d protein; (b) Isolate and purify the engineered Cas13d protein described in (a).

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