Small CRISPR-Cas gene editing system and application thereof

Abstract

The invention discloses a small CRISPR (clustered regularly interspaced short palindromic repeats)-Cas gene editing system and application thereof. According to the invention, based on microbial genome and metagenome data, a class of CRISPR-Cas family protein is mined through a biological information method, and is named as Cas12r. A CRISPR-Cas12r editing tool constructed on the basis of the gene can realize gene editing in prokaryotic or eukaryotic cells. The CRISPR-Cas12r gene editing system obtained by the invention has the characteristics of miniaturization and various PAM types.

Description

Technical Field

[0001] This invention relates to the field of gene editing, specifically to a small CRISPR-Cas gene editing system and its applications. Background Technology

[0002] The CRISPR-Cas system is a defense system widely found in bacteria and archaea. It consists of clustered regularly interspaced short palindromic repeats (CRISPR) and their associated proteins (Cas). The CRISPR structure comprises highly conserved repeat sequences and spacers derived from mobile elements such as viruses. In 2012, CRISPR-Cas-based genome editing technology was developed. This technology uses guide RNA (gRNA) to guide Cas nucleases to cut target sites on the genome, activating intracellular repair pathways to achieve genome editing. It offers advantages such as ease of programming (utilizing base matching between the spacer in the gRNA and DNA for specific targeting), high efficiency, and specificity. Genome editing tools developed based on this system have achieved targeted editing in numerous species. Related applications have also expanded to multiple fields, including agricultural breeding, disease treatment, and industrial production.

[0003] Currently, the most widely used CRISPR-Cas9 and CRISPR-Cas12a editing technologies employ nucleases larger than 1000 aa, and vector delivery requires the integration of regulatory sequences such as promoters and termination signals, which is detrimental to delivery. Furthermore, developing single-base editing or lead editing tools based on the CRISPR-Cas system requires the fusion of different functional elements such as deaminases, reverse transcriptases, or glycosylation enzymes, further increasing the size of the editing tools. Commonly used adeno-associated virus (AAV) delivery tools have a payload of only about 4.7 kb. When using a dual AAV delivery strategy, the editing efficiency is generally lower than that of single AAV delivery. In addition, the limitations of PAM (Protein Acid Mold) are also one of the problems that editing tools need to address. Therefore, developing miniaturized gene editing tools with diverse PAM preferences is of great significance. Summary of the Invention

[0004] This invention, through in-depth research and bioinformatics mining based on microbial genome and metagenomic data, identifies a class of CRISPR-Cas family proteins, named Cas12r. A CRISPR-Cas12r editing tool constructed based on this protein can achieve gene editing in eukaryotic cells. The CRISPR-Cas12r gene editing system obtained in this invention is characterized by miniaturization and a diverse range of PAMs (Protein Acids).

[0005] Therefore, this invention provides a small Cas protein, wherein the amino acid sequence of the Cas12r protein is as shown in any one of SEQ ID NO. 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, and 23. These small Cas proteins are homologous proteins and cluster in one branch on the phylogenetic tree. Similar proteins were observed in 10 systems in the sRNA-seq results. The presence of a long RNA transcript of approximately 200 nt and short transcripts similar to mature crRNA and tracrRNA supports the view that this system represents an important intermediate type in the evolution of the TnpB transposon into the Cas12 protein. All 12 editing systems constituted are active in *E. coli*, with eight also active in eukaryotic cells.

[0006] The present invention provides a small Cas protein, the amino acid sequence of which is shown in any one of SEQ ID NO. 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23.

[0007] The present invention further provides a nucleotide molecule encoding the Cas protein. Specifically, its nucleotide sequence is shown in any one of SEQ ID NO. 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24.

[0008] The present invention also provides an expression vector containing the aforementioned nucleotide molecules.

[0009] The present invention further provides a gene editing system CRISPR-Cas12r, which includes: 1) the Cas protein and 2) guide RNA.

[0010] Specifically, a gene editing system CRISPR-Cas12r is provided, the system comprising an expression vector containing and capable of expressing the nucleotide molecule and guide RNA;

[0011] As an example, the expression vector is selected from pCDFDuet-1, pcDNA3.1(+) and pET-28a.

[0012] More specifically, the system includes expression vector 1 and expression vector 2, wherein expression vector 1 contains and is capable of expressing the nucleotide molecule, and expression vector 2 is capable of expressing guide RNA.

[0013] In a specific embodiment, the guide RNA is selected from any of the sequences shown in SEQ ID NO.25-36 and SEQ ID NO.39-58.

[0014] The present invention also provides a guide RNA, the nucleotide sequence of which is shown in any one of SEQ ID NO.25-36 and SEQ ID NO.39-58.

[0015] The present invention further provides the use of the Cas protein and / or the nucleotide molecules and / or the expression vector described herein in the preparation of reagents and / or kits for editing target DNA in cells.

[0016] Specifically, the target DNA sequence is adjacent to the original spacer sequence and the motif sequence; more specifically, the cell is a eukaryotic cell or a prokaryotic cell;

[0017] In specific implementations, the neighboring motifs of the original spacer sequence identified by each system are as follows: Cas12r.01 system identifies 5'-WWRCCH-3'; Cas12r.04 system identifies 5'-RDHCCC-3'; Cas12r.05 system identifies 5'-WWRWSWY-3'; Cas12r.06 system identifies 5'-YGTY-3'; Cas12r.08 system identifies 5'-RKTC-3'; Cas12r.09... The system identifies 5'-WATWN-3'; the Cas12r.10 system identifies 5'-VTYN-3'; the Cas12r.11 system identifies 5'-RTTN-3'; the Cas12r.12 and Cas12r.13 systems identify 5'-YTC-3'; the Cas12r.14 and Cas12r.15 systems identify 5'-TTC-3', where W represents A / T, R represents A / G, H represents A / C / T, D represents A / G / T, Y represents C / T, S represents C / G, K represents G / T, V represents A / C / G, and N represents A / G / C / T.

[0018] This invention presents a CRISPR-Cas12r editing tool based on a miniature Cas protein, enabling gene editing in both prokaryotic and eukaryotic cells. The CRISPR-Cas12r gene editing system obtained in this invention features miniaturization and a diverse range of PAMs. Attached Figure Description

[0019] Figure 1 The Cas12r system exhibits diverse PAM preferences.

[0020] Figure 2 The Cas12r system has plasmid interference capabilities.

[0021] Figure 3 sRNA-seq results of the Cas12r.01-Cas12r.09 system.

[0022] Figure 4 sRNA-seq results of the Cas12r.10-Cas12r.15 system.

[0023] Figure 5 The modified Cas12r system still retains plasmid interference functionality based on sRNA-seq results.

[0024] Figure 6 The Cas12r system enables eukaryotic cell editing. Detailed Implementation

[0025] The present invention will be described below through specific embodiments in order to better understand the present invention, but this does not constitute a limitation on the present invention.

[0026] Example 1: Obtaining the Cas12r gene and system

[0027] 1. Data source: Data is sourced from NCBI’s microbial genome and publicly available metagenomic data from published articles.

[0028] 2. CRISPR and Gene Annotation: The Prodigal (Version 2.6.3) tool was used to predict open reading frames (ORFs). Domain annotation was performed using the Pfam database, with an E-value threshold of 1E-5. The minced (version 0.2.0) tool was used to annotate the CRISPR array with the following parameters: -searchWL 6 -minNR 2.

[0029] 3. RuvC domain protein identification: Profiles of known Cas proteins were obtained, covering Cas12a, Cas12b, Cas12c, Cas12d, Cas12e, Cas12g, Cas12h, Cas12i, and V-U1-5. Simultaneously, profiles of the known TnpB protein were constructed. hmmsearch (version 3.4) was used to align all ORFs with the TnpB and Cas12 profiles, with a threshold of 1E-3.

[0030] 4. CRISPR array-related protein analysis: Search for CRISPR array structures within a 10kb range upstream and downstream of candidate proteins. Select candidate proteins containing CRISPR arrays within a 10kb range upstream and downstream, and remove sequences containing X or unconventional amino acids, as well as sequences shorter than 200 amino acids. Further confirmation is made regarding the presence of RuvC-I, RuvC-II, and RuvC-III domains.

[0031] 5. Clustering and Phylogenetic Tree Construction of CRISPR-Related Proteins: CRISPR-coupled proteins were clustered using the MMseqs2 tool (Version: 12.113e3) with the following parameters: -c 0.3 --min-seq-id 0.7 -e 0.01 --cluster-mode 1. Representative sequences were selected from the clustering results, and known Cas proteins were added. A phylogenetic tree was constructed using IQ-TREE (version 2.3.0). Phylogenetic analysis revealed a new CRISPR-Cas protein family, named Cas12r. Furthermore, more Cas12r proteins were obtained through homology screening. Ultimately, 12 active homologous proteins (Cas12r.01, Cas12r.04, Cas12r.05, Cas12r.06, Cas12r.08, Cas12r.09, Cas12r.10, Cas12r.11, Cas12r.12, Cas12r.13, Cas12r.14, Cas12r.15) were obtained, all of which were coupled to a CRISPR array.

[0032] The amino acid and base sequence numbers of the 12 active proteins are as follows: Cas12r.01 amino acid sequence (SEQ ID No:1), Cas12r.01 nucleotide sequence (SEQ ID No:2), Cas12r.04 amino acid sequence (SEQ ID No:3), Cas12r.04 nucleotide sequence (SEQ ID No:4), Cas12r.05 amino acid sequence (SEQ ID No:5), Cas12r.05 nucleotide sequence (SEQ ID No:6), Cas12r.06 amino acid sequence (SEQ ID No:7), Cas12r.06 nucleotide sequence (SEQ ID No:8), Cas12r.08 amino acid sequence (SEQ ID No:9), Cas12r.08 nucleotide sequence (SEQ ID No:10), Cas12r.09 amino acid sequence (SEQ ID No:11), Cas12r.09 nucleotide sequence (SEQ ID No:12), Cas12r.10 amino acid sequence (SEQ ID No:13), Cas12r.10 nucleotide sequence (SEQ ID No:14), Cas12r.05 amino acid sequence (SEQ ID No:5), Cas12r.05 nucleotide sequence (SEQ ID No:6), Cas12r.06 amino acid sequence (SEQ ID No:7), Cas12r.06 nucleotide sequence (SEQ ID No:8), Cas12r.08 amino acid sequence (SEQ ID No:9), Cas12r.08 nucleotide sequence (SEQ ID No:10), Cas12r.09 nucleotide sequence (SEQ ID No:11), Cas12r.09 nucleotide sequence (SEQ ID No:12), Cas12r.10 amino acid sequence (SEQ ID No:13), Cas12r.10 nucleotide sequence (SEQ ID No:14), Cas12r.10 nucleotide sequence (SEQ ID No:15), Cas12r.06 amino acid sequence (SEQ ID No:11), Cas12r.09 nucleotide sequence (SEQ SEQ ID No:14), Cas12r.11 amino acid sequence (SEQ ID No:15), Cas12r.11 nucleotide sequence (SEQ ID No:16), Cas12r.12 amino acid sequence (SEQ ID No:17), Cas12r.12 nucleotide sequence (SEQ ID No:18), Cas12r.13 amino acid sequence (SEQ ID No:19), Cas12r.13 nucleotide sequence (SEQ ID No:20), Cas12r.14 amino acid sequence (SEQ ID No:21), Cas12r.14 nucleotide sequence (SEQ ID No:22), Cas12r.15 amino acid sequence (SEQ ID No:23), Cas12r.15 nucleotide sequence (SEQ ID No:24).

[0033] Example 2: Identification of PAM Preferences and Interference Functions

[0034] 1. Construction of expression plasmids: First, the expression plasmid for Cas12r was constructed. The DNA sequence encoding the full-length Cas12r protein (preserving the original mined base sequence without codon optimization) was constructed between the XbaI and BlpI restriction sites in the pET-28a vector (GenScript Biotechnology Co., Ltd.) to obtain the protein expression plasmid. The intergenic region near the Cas12r system, the protein terminal sequence (possibly expressing tracrRNA), and the mini-CRISPR (repeat-spacer-repeat) sequence were tandemly constructed between the XbaI and AvrII restriction sites in the pCDFDuet-1 vector (GenScript Biotechnology Co., Ltd.) to obtain the gRNA expression plasmid.

[0035] The constructed gRNA expression sequences are numbered as follows: Cas12r.01-gRNA1 (SEQ ID No:25), Cas12r.04-gRNA1 (SEQ ID No:26), Cas12r.05-gRNA1 (SEQ ID No:27), Cas12r.06-gRNA1 (SEQ ID No:28), Cas12r.08-gRNA1 (SEQ ID No:29), Cas12r.09-gRNA1 (SEQ ID No:30), Cas12r.10-gRNA1 (SEQ ID No:31), Cas12r.11-gRNA1 (SEQ ID No:32), Cas12r.12-gRNA1 (SEQ ID No:33), Cas12r.13-gRNA1 (SEQ ID No:34), Cas12r.14-gRNA1 (SEQ ID No:35), and Cas12r.15-gRNA1 (SEQ ID No:36).

[0036] 2. Preparation of dual-plasmid electroporation competent cells: Plasmids expressing Cas12r protein and gRNA were transformed into *E. coli* BL21(DE3) competent cells and plated on LB agar plates containing 50 µg / mL kanamycin sulfate and 50 µg / mL spectinomycin resistance. The plates were incubated overnight at 37°C and 200 rpm with shaking. Single colonies were picked and inoculated into 3 mL of LB liquid medium containing kanamycin sulfate and spectinomycin, and incubated overnight at 37°C and 200 rpm with shaking. 500 µL of the bacterial culture was then transferred to 50 mL of LB liquid medium containing kanamycin sulfate and spectinomycin. OD600 was measured using a UV spectrophotometer. When the OD600 reached approximately 0.3 at 37°C and 200 rpm with shaking, 0.1 mM IPTG was added. Incubate at 25°C and 200 rpm on a shaker until the bacterial OD600 reaches 0.6-0.8. Collect the bacteria into 50 mL centrifuge tubes, place on ice for 30 min, then centrifuge at 4°C and 6000 rpm for 10 min. Discard the supernatant, add 40 mL of ice-treated sterile ddH2O, gently mix with a pipette, and centrifuge at 4°C and 6000 rpm for 10 min. Repeat the above steps. Discard the supernatant, add 40 mL of ice-treated sterile 10% glycerol, gently mix with a pipette, and centrifuge at 4°C and 6000 rpm for 20 min. Discard the supernatant. Add an appropriate amount of 10% glycerol and mix well. Aliquot into sterile 1.5 mL centrifuge tubes, 100 µL per tube, and store at -80°C for later use.

[0037] 3. Construction of PAM library plasmid: Based on the vector pACYC184 (from ATCC, number 37033), a fragment with a 7N random sequence was obtained by introducing a random sequence into the primers and constructing it between the restriction sites XbaI / ScaI to obtain the pAYAC-7N (PAM+ target sequence: NNNNNNNAAGCTTTACTCTGACCTAGGTGGGAAGCTGCTAATA, SEQ ID No:37) plasmid.

[0038] The nucleotide fragment between the XbaI / ScaI restriction sites in the pAYAC-7N target plasmid is shown in SEQ ID No:38.

[0039] 4. PAM Elimination Assay: 200 ng of the PAM library plasmid (pAYAC-7N) was added to the electroporated competent cells. After gentle mixing with a pipette, the cells were quickly transferred to a pre-chilled electroporation cuvette and incubated on ice for 10 min. The electroporator was turned on, the voltage was set to 2.5 kV, and 900 µL of LB liquid medium was added immediately after electroporation. The cells were incubated at 37°C and 200 rpm for 1 h. After recovery, approximately 200 µL of the bacterial culture was inoculated into 10 mL of LB medium containing kanamycin sulfate, spectinomycin, and chloramphenicol, and 0.05 mM IPTG was added. The cells were then incubated at 25°C and 200 rpm for 48 h. The plasmid was extracted, and the target plasmid amplicon sequencing was used to identify preferentially depleted PAMs. The PAM region sequence was amplified by PCR and sequenced. The concentration of the target fragment recovered from the gel was determined using Nanodrop, and the sequence was then performed at Suzhou Kingwiz Biotechnology Co., Ltd. Sequencing data were mapped onto corresponding plasmids, and sequences containing random PAM regions were extracted. After alignment, the number of each possible nucleotide combination was calculated (total reads for each sample were normalized). Enriched PAM was calculated by calculating the logarithmic ratio of its abundance to that in the control group (pAYAC-7N plasmid as the PCR template), and sequence markers were generated using the WebLogo website (https: / / weblogo.berkeley.edu / logo.cgi). Experimental results are shown below. Figure 1All 12 systems exhibited PAM elimination activity and diverse PAM preferences. The Cas12r.01 system recognized 5'-WWRCCH-3' and showed a specific preference for the 5'-TGCCC-3' sequence; the Cas12r.04 system recognized 5'-RDHCCC-3' and showed a specific preference for the 5'-GTCCC-3' sequence. The Cas12r.05 system used the 5'-WWRWSWY-3' PAM sequence and showed a strong preference for the 5'-GTC-3' or 5'-GTT-3' PAM sequences; the Cas12r.06 system used the 5'-YGTY-3' PAM sequence. The PAM preferences of the CRISPR-Cas12r.08–q15 systems showed some similarities. The CRISPR-Cas12r.08 system uses the 5'-RKTC-3' PAM sequence and exhibits a strong preference for the 5'-GTTC-3' PAM sequence; the CRISPR-Cas12r.09 system shows a preference for the 5'-WATWN-3' PAM sequence and a strong preference for the 5'-TTN-3' PAM sequence; the Cas12r.10 system uses the 5'-VTYN-3' PAM sequence and exhibits a strong preference for the 5'-GTTN-3' PAM sequence; the CRISPR-Cas12r.11 system uses the 5'-RTTN-3' PAM sequence and exhibits a strong preference for the 5'-TTN-3' PAM sequence; the CRISPR-Cas12r.12 system shows a preference for the 5'-YTC-3' PAM sequence and also shows preference for the 5'-TTC-3' and 5'-CTC-3' PAM sequences. The sequences all exhibit strong bias; the CRISPR-Cas12r.13 system has the same PAM characteristics as the CRISPR-Cas12r.12 system; the CRISPR-Cas12r.14 and CRISPR-Cas12r.15 systems exhibit typical 5'-TTC-3' PAM sequence bias.

[0040] 5. Plasmid Interference Validation of PAM Preference: Based on the above PAM elimination results, plasmid interference experiments were conducted to further validate PAM preference. 200 ng of the target plasmid pAYAC-CCC (replacing the 7N sequence of pAYAC-7N with CCC) was used in the Cas12r.01 / Cas12r.04 system for plasmid interference validation of PAM preference; pAYAC-GTT (replacing the 7N sequence of pAYAC-7N with GTT) was used in the Cas12r.05 / Cas12r.06 system for plasmid interference validation of PAM preference; and pAYAC-TTC (replacing the 7N sequence of pAYAC-7N with TTC) was used in the Cas12r.08 to Cas12r.15 systems for plasmid interference validation of PAM preference. All systems used plasmid pAYAC-NT (based on pAYAC-TTC, with the first 20 bp of TTC and the target sequence deleted) as a non-target control NT. The target plasmid was transferred into dual-plasmid electroporation competent cells. After gently mixing with a pipette, the cells were quickly transferred to pre-chilled electroporation cuvettes and incubated on ice for 10 min. The electroporator was turned on, the voltage was set to 2.5 kV, and 900 µL of LB broth was added immediately after electroporation. The cells were incubated at 37°C and 200 rpm for 1 h in a shaker. Then, approximately 200 µL of the bacterial culture was inoculated into 10 mL of LB broth containing kanamycin sulfate, spectinomycin, and chloramphenicol, and 0.05 mM IPTG was added. The cells were incubated at 200 rpm for 2 h. After centrifugation at 5000 rpm for 2 min at room temperature, the supernatant was discarded, and the cells were resuspended in 100 µL of LB broth. The cells were then irrigated with LB broth according to a 10:1 ratio. -1 10 -2 10 -3 10 -4 10 -5 The stock solution was serially diluted. Using a 1-10 μL pipette, 3 µL of each of the different dilutions was added to an LB agar plate containing kanamycin sulfate, spectinomycin, and chloramphenicol, and incubated overnight at 37°C. Colony growth was observed. The experimental results are as follows: Figure 2 Compared to the NT control, the experimental groups showed a reduction in the number of colonies formed by gradient dilutions, demonstrating that each system can recognize the corresponding PAM and exert plasmid interference function. These results further prove that the Cas12r system can target and cleave double-stranded DNA in prokaryotic cells, showing its potential for development into a gene editing tool.

[0041] Example 3: gRNA Identification

[0042] 1. Small RNA sequencing (sRNA-seq) for gRNA transcript identification: E. coli BL21(DE3) transformed with Cas protein and gRNA expression plasmid were cultured to the logarithmic growth phase. 4 mL of the logarithmic-phase bacterial culture was collected in a 1.5 mL centrifuge tube without RNase, centrifuged at 12000 rpm for 1 min at room temperature, and the supernatant was discarded. 1 mL of TRlZol reagent was added to each tube, and the mixture was pipetted to thoroughly lyse the bacteria until the liquid became clear. After standing at room temperature for 5 min, the mixture was centrifuged at 12000 rpm for 5 min at 4°C, and the supernatant was transferred to a new centrifuge tube. 200 μL of chloroform was added, and the mixture was inverted and vigorously shaken. After standing at room temperature for 3 min, the mixture was centrifuged at 12000 rpm for 10 min at 4°C. Carefully transfer the upper aqueous phase to a new 1.5 mL centrifuge tube (approximately 500 μL). Add phenol / chloroform and mix well. Centrifuge at 12000 rpm for 10 min at 4°C. Transfer the supernatant to a new 1.5 mL centrifuge tube. Add 500 μL of isopropanol and invert to mix. Incubate at room temperature for 10 min. Centrifuge at 12000 rpm for 10 min at 4°C. After centrifugation, place the centrifuge tube on ice. Subsequent operations should be performed on ice whenever possible. The white precipitate at the bottom of the tube is RNA. Aspirate the supernatant, being careful not to lose the precipitate. Add 1 mL of 75% ethanol to each tube to resuspend the precipitate and wash away the salts. A gentle vortex can be used. Centrifuge at 7500 rpm for 5 min at 4°C. Aspirate the liquid. Briefly centrifuge the sample, aspirate the supernatant with a pipette, and place the tube open on ice for approximately 5-10 min to allow the alcohol to evaporate. However, be careful not to let the RNA dry out too much and become difficult to dissolve. Dissolve the RNA precipitate in approximately 20-30 μL of DEPC water. After the RNA was completely dissolved, its concentration was measured using NanoDrop and stored at -80°C. 3 μg of extracted RNA was used to construct a library, selecting RNA sequences ranging from 30 to 300 nt. Sequencing was performed using Illumina HiSeq sequencing (paired-end, 150-bp reads), and adapters were removed from the raw data using Cutadapt (version 3.5) software. The obtained sequences were aligned with the target fragment using BLASTN. Preliminary analysis revealed that the transcripts contained similar... The RNA transcripts range from approximately 200 nt long transcripts to short transcripts similar to mature crRNA and tracrRNA. It is speculated that the Cas12r system may be an early product of TnpB coupled with a CRISPR array, capable of utilizing both long transcripts and dual-RNA hybrid transcripts (tracrRNA:crRNA). Therefore, from sRNA-seq paired-end sequencing data, we first precisely determined the start and end positions of all transcripts. To identify long transcripts, we screened for those that started in the intergenic region and extended to the first repeat sequence (…). Figure 3 and Figure 4 The pink region in the middle represents long RNA, corresponding to the left Y-axis. For short transcripts, we screened reads that met the following two conditions ( Figure 3 and Figure 4 The gray area, i.e., shortRNA, corresponds to the right Y-axis: (1) Termination occurs upstream of the first direct repeat (DR) sequence (possible tracrRNA). (2) Initiation occurs downstream of the DR start site (possible mature crRNA). Finally, the reads selected by these criteria were used to calculate the coverage of each genomic location using a custom Perl script, and a coverage map was generated using GraphPad software. The analysis results of 11 systems are as follows: Figure 3 and Figure 4 (The sequencing data from the Cas12r.11 system was too small to be further analyzed). Except for Cas12r.06, which did not show obvious tracrRNA-like transcripts, the other 10 systems all showed similar transcripts. The presence of a long RNA transcript of approximately 200 nt and short transcripts similar to mature crRNA and tracrRNA suggests that the Cas12r system may employ a dual-mode RNA—retaining characteristics of the TnpB system. The RNA pathway has evolved into a typical dual RNA guidance system of tracrRNA and crRNA.

[0043] 2. Modify the gRNA expression plasmid: It is speculated that the long RNA transcript contains all the sequences required for gRNA to function. Based on sRNA-seq or sequence alignment, other interstitial regions were deleted from the gRNA1 expression plasmid, and the long RNA region of Cas12r was expressed only using the J23100 constitutive promoter.

[0044] The modified gRNA backbone expression sequence numbers are as follows (excluding the spacer sequence):

[0045] Cas12r.01-seq(SEQ ID No:39), Cas12r.04-seq(SEQ ID No:40), Cas12r.05-seq(SEQ ID No:41), Cas12r.06-seq(SEQ ID No:42), Cas12r.08-seq(SEQ ID No:43), Cas12r.09-seq(SEQ ID No:44), Cas12r.10-seq (SEQ ID No:45), Cas12r.11-seq (SEQ ID No:46), Cas12r.12-seq (SEQ ID No:47), Cas12r.13-seq (SEQ ID No:48), Cas12r.14-seq (SEQ ID No:49), Cas12r.15-seq (SEQ ID No:50).

[0046] like Figure 5 Compared to the NT control, the number of colonies formed in the experimental groups using gradient dilutions was reduced, demonstrating that when expressing gRNA using the above sequences, each system can recognize the corresponding PAM and exert plasmid interference function. These results indicate that the truncated sequence contains the core sequence of the Cas12r system gRNA sequence, and expression of this sequence can target and cleave double-stranded DNA in prokaryotic cells, showing potential for development into a gene editing tool.

[0047] Example 4: Cas12r implementation of eukaryotic editing

[0048] 1. Construction of expression plasmids: Using the Gibson assembly method, all Cas12r protein expression plasmids and gRNA expression plasmids (target gene CA2, target sequence AAGTTAGCGAGAAGAGT) were constructed based on the pcDNA3.1(+) vector (GenScript Biotechnology Co., Ltd.). Cas12r protein was expressed using the CMV promoter, with 3xFLAG added to the N-terminus and NLS added to both the N-terminus and C-terminus. Furthermore, EGFP fluorescent protein was linked to the C-terminus of the protein using T2A for fluorescence sorting. gRNA (using the approximately 200 nt sequence described above) was expressed using the human U6 promoter. Transformants with correct sequencing were inoculated into 10 mL of LB liquid medium containing 100 µg / mL Amp antibiotic and cultured at 37°C and 200 rpm for 15 h. Plasmids were extracted using an endotoxin-free plasmid extraction kit (Tiangen Biotech Co., Ltd.) according to the manufacturer's instructions, and the plasmid concentration was determined.

[0049] 2. Cell Transfection: Under a microscope, HEK293T cells in 12-well plates were observed to be in good growth condition, with cells covering 70-90% of the culture dish area. 2 µg of plasmid (1 µg protein expression plasmid and 1 µg RNA expression plasmid) was added to 50 µL of transformation medium and gently mixed. 2 µL of Lipofectamine 2000 transfection reagent (Thermo Fisher Scientific) was added to 50 µL of transformation medium and gently mixed. The transformation media containing the plasmid and transfection reagent were then thoroughly mixed, and after standing for 15-20 min, 500 µL of transformation medium was added to the mixture. The medium in each well of the 12-well plate was discarded, and the mixture was gently added along the sidewall. The plates were then incubated at 37°C and 5% CO2 for 42-48 h.

[0050] 3. Fluorescent Cell Sorting: Cells in a 12-well plate are considered healthy under a microscope, with cells covering approximately 100% of the culture dish. Discard the culture medium from the cell culture dish. Using a dropper, gently add 5 mL of PBS buffer along the sidewall of the cell culture dish to wash away any remaining culture medium and dead / shedded cells. Discard the PBS, add 3 mL of trypsin, and gently mix for 45-60 seconds. Terminate the trypsin reaction with 5 mL of culture medium, and collect any cells shed after trypsin treatment. Centrifuge at 1000 rpm for 5 minutes, discard the supernatant, and resuspend the pellet in 1 mL of PBS. Store the cells on ice. Use flow cytometry to screen and collect cells expressing green fluorescence. Select 10,000 cells expressing green fluorescence for subsequent experiments.

[0051] 4. Amplicon Sequencing: Cells sorted by flow cytometry were lysed using cell lysis buffer. The collected cells were centrifuged at 12000 rpm for 10 min, the supernatant was discarded, and the cell pellet was collected and lysed using 30 µL of cell lysis buffer. The lysed cells were used as templates for PCR amplification. The target bands (containing the target sequence) obtained from PCR amplification were purified using gel extraction. The purified DNA sequences were then subjected to next-generation sequencing. No significant editing efficiency was observed in any system.

[0052] 5. gRNA Optimization and Modification: gRNA has a significant impact on editing efficiency. Based on RNA secondary structure prediction and the group's previous experience, gRNAs for different systems were optimized accordingly. Eukaryotic cell editing was successfully achieved in the Cas12r.08–15 systems. Figure 6 The editing efficiency was 5%-6%, indicating the effectiveness of the small Cas12r gene editing tool.

[0053] The eukaryotic expression sequences of the constructed gRNA backbone are numbered as follows (excluding promoter and spacer sequences):

[0054] Cas12r.08-mut(SEQ ID No:51), Cas12r.09-mut(SEQ ID No:52), Cas12r.10-mut(SEQ ID No:53), Cas12r.11-mut(SEQ ID No:54), Cas12r.12-mut(SEQ ID No:55), Cas12r.13-mut(SEQ ID No: 56), Cas12r.14-mut (SEQ ID No: 57), Cas12r.15-mut (SEQ ID No: 58).

[0055] The protein expression sequence is taken as an example of the Cas12r.08 system, and its nucleotide sequence is as follows (SEQ ID No:59) (from the CMV promoter sequence to the bGH poly(A) signal).

[0056] In summary, the Cas12r system can achieve targeted cleavage in both prokaryotic and eukaryotic cells, enabling gene editing in eukaryotic cells. The Cas12r gene editing tool offers the advantage of miniaturization. Furthermore, editing efficiency can be further improved through system modifications.

Claims

1. A small Cas protein, characterized in that, The amino acid sequence of the Cas protein is shown in any one of SEQ ID NO. 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23.

2. A nucleotide molecule, characterized in that, The nucleotide molecule encodes the Cas protein of claim 1, specifically, its nucleotide sequence is shown in any one of SEQ ID NO. 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24.

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

4. A gene editing system, CRISPR-Cas12r, characterized in that, The system comprises: 1) the Cas protein of claim 1; and 2) a guide RNA.

5. A gene editing system, CRISPR-Cas12r, characterized in that, The system includes an expression vector that expresses the nucleotide molecule of claim 2 and the guide RNA; As an example, the expression vector is selected from pCDFDuet-1, pcDNA3.1(+) and pET-28a.

6. The gene editing system CRISPR-Cas12r as described in claim 5, characterized in that, The system includes expression vector 1 and expression vector 2, wherein expression vector 1 expresses the nucleotide molecule of claim 2 and expression vector 2 expresses guide RNA.

7. The system according to any one of claims 4-6, characterized in that, The guide RNA is selected from any of the sequences shown in SEQ ID NO.25-36 and SEQ ID NO.39-58.

8. A guide RNA, characterized in that, Its nucleotide sequence is shown in any one of SEQ ID NO. 25-36 or SEQ ID NO. 39-58.

9. Use of the Cas protein of claim 1 and / or the nucleotide molecule of claim 2 and / or the expression vector of claim 3 in the preparation of reagents and / or kits for editing target DNA in cells.

10. The use according to claim 9, characterized in that, The target DNA sequence is adjacent to the original spacer sequence and the motif sequence; specifically, the cell is a eukaryotic cell or a prokaryotic cell; More specifically, the nearest motifs of the original spacer sequence identified by each system are as follows: Cas12r.01 system identifies 5'-WWRCCH-3'; Cas12r.04 system identifies 5'-RDHCCC-3'; Cas12r.05 system identifies 5'-WWRWSWY-3'; Cas12r.06 system identifies 5'-YGTY-3'; Cas12r.08 system identifies 5'-RKTC-3'; Cas12r.09 system identifies 5'-WATWN-3'; Cas12r.05 system identifies 5'-WATWN-3'. The Cas12r.10 system identifies 5'-VTYN-3'; the Cas12r.11 system identifies 5'-RTTN-3'; the Cas12r.12 and Cas12r.13 systems identify 5'-YTC-3'; the Cas12r.14 and Cas12r.15 systems identify 5'-TTC-3', where W represents A / T, R represents A / G, H represents A / C / T, D represents A / G / T, Y represents C / T, S represents C / G, K represents G / T, V represents A / C / G, and N represents A / G / C / T.

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