KR102586501B1 - Composition for inducing disturbance of circadian clock and model for disturbing circadian clock made using same - Google Patents
Composition for inducing disturbance of circadian clock and model for disturbing circadian clock made using same Download PDFInfo
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Abstract
본 발명은 생체시계(circadian clock) 조절용 조성물 및 이를 이용한 생체시계 조절 방법에 관한 것으로서, 보다 구체적으로 생체시계 관련 유전자인 Cry1 및 Cry2를 동시에 녹아웃 시키기 위해 직접 설계한 sgRNA를 포함하는 CRISPR/Cas9 시스템, 이를 포함하는 생체시계 교란 유도용 조성물, 이를 활용해 제조한 생체시계 교란 모델 및 상기 모델을 활용한 생체시계 교란 치료물질의 스크리닝 방법에 관한 것이다.
본 발명에 따르면 생체시계와 관련된 유전자들을 타겟으로 하는 sgRNA를 활용하는 CRISPR/Cas9 기술을 통해 Cry1 및 Cry2를 동시에 녹아웃 시킨 생체시계 교란 돌연변이 모델을 제작할 수 있으며, 타겟 유전자 녹아웃 외의 효과가 미미하여 생체시계만 교란된 돌연변이 모델을 제작할 수 있고, 이렇게 제작된 돌연변이 모델을 활용하여 생체시계 교정물질을 스크리닝을 통해 발굴해낼 수 있다.The present invention relates to a composition for controlling the circadian clock and a method for controlling the circadian clock using the same, and more specifically, to a CRISPR/Cas9 system comprising sgRNA designed to simultaneously knock out the circadian clock-related genes Cry1 and Cry2; It relates to a composition for inducing biological clock disruption containing the same, a biological clock disruption model manufactured using the same, and a screening method for biological clock disruption treatment substances using the model.
According to the present invention, it is possible to create a biological clock-disrupting mutation model in which Cry1 and Cry2 are simultaneously knocked out through CRISPR/Cas9 technology using sgRNA targeting genes related to the biological clock, and since the effects other than target gene knockout are minimal, only the biological clock is affected. A perturbed mutation model can be created, and biological clock correction materials can be discovered through screening using the created mutation model.
Description
본 발명은 생체시계(circadian clock) 조절용 조성물 및 이를 이용한 생체시계 조절 방법에 관한 것으로서, 보다 구체적으로 생체시계 관련 유전자인 Cry1, Cry2를 동시에 녹아웃 시키기 위해 직접 설계한 sgRNA를 포함하는 CRISPR/Cas9 시스템, 이를 포함하는 생체시계 교란 유도용 조성물, 이를 활용해 제조한 생체시계 교란 모델 및 상기 모델을 활용한 생체시계 교란 치료물질의 스크리닝 방법에 관한 것이다.The present invention relates to a composition for controlling the circadian clock and a method for controlling the circadian clock using the same. More specifically, the CRISPR/Cas9 system includes a sgRNA designed to simultaneously knock out the circadian clock-related genes Cry1 and Cry2; It relates to a composition for inducing biological clock disruption containing the same, a biological clock disruption model manufactured using the same, and a screening method for biological clock disruption treatment substances using the model.
포유동물에서 빛에 의한 정보는 망막시상하부 경로(retino-hypothalamic tract, RHT)를 통하여 시교차상핵(suprachiasmatic nucleus, SCN)으로 전달된다. SCN은 일련의 생체시계 유전자의 전사-번역 피드백 루프로 구성된 내재적 분자 시계 메커니즘을 갖는다. CLOCK(circadian locomotor output cycles kaput)은 E-box(CACGTG)에 결합하여 Cry1/2 유전자의 발현을 활성화 시킨다. 외부 환경의 명-암(Light-dark, LD) 주기에 의해 결정되는 외부시계에 의한 자극과 생체내부 시계의 정보를 바탕으로 SCN은 통합된 시간의 산출 신호를 생산하고, 각 신체부위에 존재하는 말초시계를 조절한다. 따라서, 생리적, 활동적 주기는 생체시계 및 외부 환경의 명-암주기(LD 사이클)에 의한 종합적인 결과물이다. In mammals, information from light is transmitted to the suprachiasmatic nucleus (SCN) through the retino-hypothalamic tract (RHT). The SCN has an intrinsic molecular clock mechanism consisting of a transcription-translation feedback loop of a series of clock genes. CLOCK (circadian locomotor output cycles kaput) binds to the E-box (CACGTG) and activates the expression of Cry1/2 genes. Based on stimulation by the external clock determined by the light-dark (LD) cycle of the external environment and information from the internal biological clock, the SCN produces an integrated time calculation signal and monitors the signals present in each body part. Regulates peripheral clock. Therefore, the physiological and activity cycle is a comprehensive result of the biological clock and the light-dark cycle (LD cycle) of the external environment.
생체 내부 시계와 환경적인 LD 사이클은 정상적인 경우 같은 주기로 나타나지만 이 주기가 흐트러지면 사람 및 쥐에서 다양한 병리학적 결과가 나타나는 것으로 보고되어 왔다. 또한 최근 연구에서는 LD 사이클의 특정 조건에서는 대사 및 비만에 중요한 역할을 하는 것으로 밝혀졌다. 그럼에도 불구하고, 생체시계 교란 그 자체가 건강에 해가 되는지, 혹은 특정 LD 사이클이 특정 질병에 대해서 취약하게 만드는지에 대해서는 밝혀진 바가 거의 없다. 생체시계 교란의 생리학적 의미는 유전학적 변이체 연구를 통하여 유추될 수 있는데, Clock와 같은 생체시계 유전자가 결여된 변이체는 리듬을 잃는 것뿐 아니라 조기 노화, 비만 및 암에 대한 민감성이 증가된다는 것이 보고되었다. 그러나 이러한 변화가 생체시계의 조절에 따른 것인지에 대한 구체적인 연구는 미진한 실정이기에, 이러한 돌연변이 모델의 제작을 위한 기술의 개발이 필요한 실정이다.Normally, the internal biological clock and the environmental LD cycle appear in the same cycle, but it has been reported that when this cycle is disturbed, various pathological outcomes occur in humans and mice. Additionally, recent studies have shown that it plays an important role in metabolism and obesity under certain conditions of the LD cycle. Nevertheless, little is known about whether circadian clock disruption itself is detrimental to health, or whether specific LD cycles predispose people to specific diseases. The physiological meaning of biological clock disruption can be inferred through studies of genetic variants. It has been reported that mutants lacking biological clock genes such as Clock not only lose rhythm but also have increased susceptibility to premature aging, obesity, and cancer. It has been done. However, since detailed research on whether these changes are due to regulation of the biological clock is insufficient, there is a need to develop technology for producing such mutation models.
상기와 같은 배경하에, 본 발명자들은 효율적으로 생체시계를 교란시킬 수 있는 조성물에 대한 연구를 수행한 결과, 직접 설계한 본 발명의 sgRNA를 포함하는 CRISPR/Cas9 시스템을 사용하는 경우 생체시계 관련 유전자를 동시에 적중함으로써 생체시계 교란 모델을 제작할 수 있음을 확인함으로써 본 발명을 완성하였다.Against the above background, the present inventors conducted research on a composition that can efficiently disrupt the biological clock, and as a result, when using the CRISPR/Cas9 system containing the sgRNA of the present invention designed by the present invention, biological clock-related genes can be disrupted. The present invention was completed by confirming that a biological clock disturbance model can be created by simultaneously hitting the target.
이에, 본 발명은 서열번호 1, 서열번호 2 및 서열번호 3으로 구성된 군으로부터 선택되는 염기서열로 이루어진, 생체시계 관련 유전자를 표적하는, 가이드 RNA(guide RNA)를 제공하는 것을 목적으로 한다.Accordingly, the purpose of the present invention is to provide a guide RNA that targets biological clock-related genes and consists of a base sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3.
또한, 본 발명은 상기 가이드 RNA를 포함하는, 생체시계 관련 유전자를 표적하는 재조합 가이드 RNA 벡터를 제공하는 것을 다른 목적으로 한다.Another object of the present invention is to provide a recombinant guide RNA vector targeting a circadian clock-related gene, including the guide RNA.
또한, 본 발명은 상기 가이드 RNA 벡터; 및In addition, the present invention provides the guide RNA vector; and
Cas9(CRISPR associated protein 9) 핵산분해효소를 포함하는, 생체시계 교란 유도용 CRISPR/Cas9 복합체를 제공하는 것을 또 다른 목적으로 한다.Another purpose is to provide a CRISPR/Cas9 complex for inducing circadian clock disruption, including Cas9 (CRISPR associated protein 9) nuclease.
또한, 본 발명은 상기 CRISPR/Cas9 복합체를 유효성분으로 포함하는, 생체시계 교란 유도용 조성물을 제공하는 것을 또 다른 목적으로 한다.Another object of the present invention is to provide a composition for inducing biological clock disruption, comprising the CRISPR/Cas9 complex as an active ingredient.
또한, 본 발명은 야생형의 시교차상핵(suprachiasmatic nucleus)에서 생체시계 관련 유전자가 녹아웃된 생체시계 교란 유도된 동물 모델을 제공하는 것을 또 다른 목적으로 한다.Another object of the present invention is to provide an animal model induced by circadian clock disruption in which circadian clock-related genes are knocked out in the suprachiasmatic nucleus of wild type.
또한, 본 발명은 하기의 단계를 포함하는, 생체시계 교정 물질의 스크리닝 방법을 제공하는 것을 또 다른 목적으로 한다.Another object of the present invention is to provide a screening method for biological clock correction materials, comprising the following steps.
(a) 상기 생체시계 교란 유도된 동물모델에 후보물질을 처리하는 단계; 및(a) treating the biological clock disruption-induced animal model with a candidate material; and
(b) 후보물질이 처리된 생체시계 교란 유도된 동물모델을 무처리 대조군과 비교하여 주기적 특성의 회복 여부를 확인하는 단계.(b) Comparing the biological clock disruption-induced animal model treated with the candidate substance with the untreated control group to determine whether cyclical characteristics are recovered.
그러나 본 발명이 이루고자 하는 기술적 과제는 이상에서 언급한 과제에 제한되지 않으며, 언급되지 않은 또 다른 과제들은 아래의 기재로부터 당업자에게 명확하게 이해될 수 있을 것이다.However, the technical problem to be achieved by the present invention is not limited to the problems mentioned above, and other problems not mentioned will be clearly understood by those skilled in the art from the description below.
상기와 같은 본 발명의 목적을 달성하기 위하여, 본 발명은 서열번호 1, 서열번호 2 및 서열번호 3으로 구성된 군으로부터 선택되는 염기서열로 이루어진, 생체시계 관련 유전자를 표적하는, 가이드 RNA(guide RNA)를 제공한다.In order to achieve the object of the present invention as described above, the present invention provides a guide RNA targeting a biological clock-related gene, consisting of a base sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3. ) is provided.
본 발명의 일구현예로, 상기 생체시계 관련 유전자는 Cry1(Cryptochrome Circadian Regulator 1) 또는 Cry2(Cryptochrome Circadian Regulator 2)일 수 있다.In one embodiment of the present invention, the biological clock-related gene may be Cry1 (Cryptochrome Circadian Regulator 1) or Cry2 (Cryptochrome Circadian Regulator 2).
본 발명의 다른 구현예로, 상기 서열번호 1로 이루어진 가이드 RNA는 Cry1 유전자를 표적하는 것일 수 있다.In another embodiment of the present invention, the guide RNA consisting of SEQ ID NO: 1 may target the Cry1 gene.
본 발명의 또 다른 구현예로, 상기 서열번호 2 또는 3으로 이루어진 가이드 RNA는 Cry2 유전자를 표적하는 것일 수 있다.In another embodiment of the present invention, the guide RNA consisting of SEQ ID NO: 2 or 3 may target the Cry2 gene.
또한, 본 발명은 상기 가이드 RNA를 포함하는, 생체시계 관련 유전자를 표적하는 재조합 가이드 RNA 벡터를 제공한다.Additionally, the present invention provides a recombinant guide RNA vector targeting a circadian clock-related gene, including the guide RNA.
본 발명의 일구현예로, 상기 생체시계 관련 유전자는 Cry1(Cryptochrome Circadian Regulator 1) 또는 Cry2(Cryptochrome Circadian Regulator 2)일 수 있다.In one embodiment of the present invention, the biological clock-related gene may be Cry1 (Cryptochrome Circadian Regulator 1) or Cry2 (Cryptochrome Circadian Regulator 2).
본 발명의 다른 구현예로, 상기 벡터는 아데노연관바이러스(Adeno-associated virus; AAV) 벡터일 수 있다.In another embodiment of the present invention, the vector may be an Adeno-associated virus (AAV) vector.
본 발명의 또 다른 구현예로, 상기 가이드 RNA 벡터는 Cry1 및 Cry2를 동시에 표적할 수 있다.In another embodiment of the present invention, the guide RNA vector can simultaneously target Cry1 and Cry2.
또한, 본 발명은 상기 가이드 RNA 벡터; 및In addition, the present invention provides the guide RNA vector; and
Cas9(CRISPR associated protein 9) 핵산분해효소를 포함하는, 생체시계 교란 유도용 CRISPR/Cas9 복합체를 제공한다.Provided is a CRISPR/Cas9 complex for inducing circadian clock disruption, including Cas9 (CRISPR associated protein 9) nuclease.
또한, 본 발명은 상기 CRISPR/Cas9 복합체를 유효성분으로 포함하는, 생체시계 교란 유도용 조성물을 제공한다.Additionally, the present invention provides a composition for inducing circadian clock disturbance, comprising the CRISPR/Cas9 complex as an active ingredient.
또한, 본 발명은 야생형의 시교차상핵(suprachiasmatic nucleus)에서 생체시계 관련 유전자가 녹아웃된 생체시계 교란 유도된 동물 모델을 제공한다.Additionally, the present invention provides an animal model induced by circadian clock disruption in which circadian clock-related genes are knocked out in the suprachiasmatic nucleus of wild type.
본 발명의 일구현예로, 상기 생체시계 관련 유전자는 Cry1(Cryptochrome Circadian Regulator 1) 또는 Cry2(Cryptochrome Circadian Regulator 2) 일 수 있다.In one embodiment of the present invention, the biological clock-related gene may be Cry1 (Cryptochrome Circadian Regulator 1) or Cry2 (Cryptochrome Circadian Regulator 2).
본 발명의 다른 구현예로, 상기 생체시계 교란 유도된 동물 모델은 생체시계 관련 유전자가 발현하는 단백질의 수준이 감소하는 것일 수 있다.In another embodiment of the present invention, the animal model induced by circadian clock disruption may have a decrease in the level of proteins expressed by circadian clock-related genes.
또한, 본 발명은 하기의 단계를 포함하는, 생체시계 교정 물질의 스크리닝 방법을 제공한다.Additionally, the present invention provides a method for screening biological clock correction materials, comprising the following steps.
(a) 상기 생체시계 교란 유도된 동물모델에 후보물질을 처리하는 단계; 및(a) treating the biological clock disruption-induced animal model with a candidate material; and
(b) 후보물질이 처리된 생체시계 교란 유도된 동물모델을 무처리 대조군과 비교하여 주기적 특성의 회복 여부를 확인하는 단계.(b) Comparing the biological clock disruption-induced animal model treated with the candidate substance with the untreated control group to determine whether cyclical characteristics are recovered.
본 발명은 생체시계 관련 유전자를 대상으로 하는 직접 설계한 sgRNA를 활용하는 CRISPR/Cas9 기법을 활용하는 경우 생체시계의 교란을 위해 필요한 유전자를 동시에 적중함으로써 효과적으로 생체시계를 교란시킬 수 있음을 확인하였는바, 생체시계를 위한 임상적 연구 등에 유용하게 사용될 수 있을 것으로 기대된다. The present invention has confirmed that when using the CRISPR/Cas9 technique that utilizes a directly designed sgRNA targeting biological clock-related genes, it is possible to effectively disrupt the biological clock by simultaneously hitting the genes necessary for disrupting the biological clock. , it is expected to be useful for clinical research on biological clocks.
단, 본 발명의 효과는 상기 효과로 한정되는 것은 아니며, 본 발명의 상세한 설명 또는 청구범위에 기재된 발명의 구성으로부터 추론 가능한 모든 효과를 포함하는 것으로 이해되어야 한다.However, the effects of the present invention are not limited to the above effects, and should be understood to include all effects that can be inferred from the configuration of the invention described in the detailed description or claims of the present invention.
도 1은 설계한 본 발명의 sgRNA에 대한 것으로서, 도 1a는 CHOPCHOP을 활용한 sgRNA 제작 모식도를 나타낸 것이고, 도 1b는 설계한 sgRNA를 U6 프로모터 구동 벡터에 클로닝한 모식도이며, 도 1c는 도 1b에서 제작한 벡터를 투여한 다음, mutation mismatch cleavage assay를 수행한 결과이다.
도 2는 본 발명의 sgRNA를 2종 이상 클로닝한 벡터에 대한 것으로서, 도 2a는 3가지의 sgRNA가 클로닝된 벡터의 모식도를 나타낸 것이고, 도 2b는 Cry1 또는 Cry2를 표적하는 상이한 sgRNA를 탑재한 벡터를 처리했을 때, 단백질의 발현 변화를 확인한 결과이다.
도 3은 본 발명의 sgRNA를 포함하는 AAV 벡터가 시교차상핵에 미치는 영향을 루시퍼레이즈 리포터를 이용한 생체발광량으로 확인한 결과로서, 도 3a는 생체발광량 측정을 위한 실험 계획이고, 도 3b는 AAV를 투여한 다음 DAPI 염색을 통해 sgRNA의 수준을 확인한 결과이며, 도 3c(sgLacZ 투여군, 대조군) 및 도 3d(CSAC-Crys 투여군)는 각각의 AAV를 투여한 다음, 생체발광량을 확인한 결과이고, 도 3e는 확인한 생체발광 패턴의 진폭을 나타낸 결과이며, 도 3f는 확인한 생체발광 패턴의 강인성을 나타낸 결과이고, 도 3g는 확인한 생체발광의 주기를 나타낸 결과이다.
도 4는 본 발명의 sgRNA를 포함하는 AAV 벡터를 시교차상핵에 투여한 결과로서, 도 4a는 시교차상핵에 AAV 벡터를 투여하는 모식도이고, 도 4b는 DAPI 염색을 통해 AAV 벡터가 잘 투여되었는지 확인한 결과이며, 도 4c(대조군) 및 도 4d(CSAC-Crys 투여군)는 AAV 투여에 따른 감염 부위를 나타낸 결과이다.
도 5는 본 발명의 sgRNA를 포함하는 AAV 벡터를 시교차상핵에 주입한 마우스의 일반운동활동을 확인한 결과로서, 도 5a(대조군) 및 도 5b(CSAC-Crys 투여군)는 명, 암 조건에 따른 일반운동활성을 관찰한 결과이며, 도 5c(대조군) 및 도 5d(CSAC-Crys 투여군)는 상기에서 얻은 일반운동활동 데이터에 카이-제곱 주기도 분석을 수행한 결과이다.
도 6은 본 발명의 sgRNA를 포함하는 AAV 벡터를 시교차상핵에 주입한 마우스의 온도를 확인한 결과로서, 도 6a(대조군) 및 도 6b(CSAC-Crys 투여군)는 명, 암 조건에 따른 온도를 관찰한 결과이며, 도 6c(대조군) 및 도 6d(CSAC-Crys 투여군)는 상기에서 얻은 온도 데이터에 카이-제곱 주기도 분석을 수행한 결과이다.
도 7은 상기 도 5 및 도 6에서 얻은 운동활동 및 온도 데이터의 평균값을 나타낸 결과로서, 도 7a는 명, 암 조건에서 운동활동의 평균값을 나타낸 결과이고, 도 7b는 명, 암 조건에서 온도의 평균값을 나타낸 결과이며, 도 7c는 일정한 암 조건에서 운동활동의 평균값을 나타낸 결과이고, 도 7d는 일정한 암 조건에서 온도의 평균값을 나타낸 결과이다.Figure 1 shows the designed sgRNA of the present invention, Figure 1a shows a schematic diagram of sgRNA production using CHOPCHOP, Figure 1b shows a schematic diagram of the designed sgRNA cloned into a U6 promoter driving vector, and Figure 1c shows a schematic diagram of sgRNA production using CHOPCHOP. This is the result of performing a mutation mismatch cleavage assay after administering the constructed vector.
Figure 2 shows a vector in which two or more types of sgRNAs of the present invention were cloned. Figure 2a shows a schematic diagram of a vector in which three types of sgRNAs were cloned, and Figure 2b shows a vector carrying different sgRNAs targeting Cry1 or Cry2. This is the result of confirming changes in protein expression when treated.
Figure 3 shows the results of confirming the effect of the AAV vector containing the sgRNA of the present invention on the suprachiasmatic nucleus by measuring bioluminescence using a luciferase reporter. Figure 3a is an experimental plan for measuring bioluminescence, and Figure 3b shows the effect of AAV upon administration of AAV. This is the result of confirming the level of sgRNA through DAPI staining, and Figure 3c (sgLacZ administered group, control group) and Figure 3d (CSAC-Crys administered group) are the results of confirming the amount of bioluminescence after administering each AAV, and Figure 3e is the result. This is a result showing the amplitude of the confirmed bioluminescence pattern, Figure 3f is a result showing the robustness of the confirmed bioluminescence pattern, and Figure 3g is a result showing the cycle of the confirmed bioluminescence pattern.
Figure 4 shows the results of administering an AAV vector containing the sgRNA of the present invention to the suprachiasmatic nucleus. Figure 4a is a schematic diagram of administering the AAV vector to the suprachiasmatic nucleus, and Figure 4b shows whether the AAV vector was successfully administered through DAPI staining. This is a confirmed result, and Figures 4c (control group) and Figure 4d (CSAC-Crys administered group) show the results showing the infection site according to AAV administration.
Figure 5 shows the results of confirming the general motor activity of mice injected with the AAV vector containing the sgRNA of the present invention into the suprachiasmatic nucleus, and Figures 5a (control group) and Figure 5b (CSAC-Crys administration group) show the results according to light and dark conditions. This is the result of observing general motor activity, and Figures 5c (control group) and Figure 5d (CSAC-Crys administered group) are the results of chi-square periodogram analysis performed on the general motor activity data obtained above.
Figure 6 shows the results of confirming the temperature of mice injected into the suprachiasmatic nucleus with an AAV vector containing the sgRNA of the present invention. Figures 6a (control group) and Figure 6b (CSAC-Crys administered group) show the temperature according to light and dark conditions. These are the observed results, and Figures 6c (control group) and Figure 6d (CSAC-Crys administered group) are the results of chi-square periodogram analysis performed on the temperature data obtained above.
Figure 7 is a result showing the average value of the exercise activity and temperature data obtained in Figures 5 and 6, Figure 7a is a result showing the average value of exercise activity in light and dark conditions, and Figure 7b is a result showing the temperature in light and dark conditions. This is a result showing the average value. Figure 7c is a result showing the average value of motor activity under constant dark conditions, and Figure 7d is a result showing the average value of temperature under constant dark conditions.
본 발명자들은 생체시계 관련 유전자를 대상으로 하는 직접 설계한 sgRNA를 포함하는 CRISPR/Cas9 시스템을 활용하여 유전자를 조작하는 경우 생체시계의 교란을 위해 필요한 유전자를 동시에 적중함으로써 효과적으로 생체시계를 교란시킬 수 있음을 확인 확인하였는바, 이로써 본 발명을 완성하게 되었다.When the present inventors manipulate genes using the CRISPR/Cas9 system, which includes a directly designed sgRNA targeting biological clock-related genes, they can effectively disrupt the biological clock by simultaneously hitting the genes necessary for disrupting the biological clock. was confirmed, and thus the present invention was completed.
이하, 본 발명을 자세히 설명한다.Hereinafter, the present invention will be described in detail.
이에, 본 발명은 서열번호 1, 서열번호 2 및 서열번호 3으로 구성된 군으로부터 선택되는 염기서열로 이루어진, 생체시계 관련 유전자를 표적하는, 가이드 RNA(guide RNA)를 제공한다.Accordingly, the present invention provides a guide RNA that targets biological clock-related genes and consists of a base sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3.
본 발명에서 사용되는 용어, "가이드 RNA(guide RNA; gRNA)"란 편집하고자 하는 표적하는 특정 DNA의 위치를 찾아내어 Cas 단백질을 표적 DNA로 인도하는 역할을 하는 단일 가닥의 RNA로서, 상기 가이드 RNA는 PAM(protospacer adjacent motif) 자리와 인접하며, 편집하려는 DNA의 10~25bp의 염기 서열과 상보적인 서열을 포함하는 것일 수 있다.As used in the present invention, the term "guide RNA (gRNA)" refers to a single-stranded RNA that finds the location of a specific DNA target to be edited and guides the Cas protein to the target DNA. The guide RNA is adjacent to the PAM (protospacer adjacent motif) site and may contain a sequence complementary to the 10 to 25 bp nucleotide sequence of the DNA to be edited.
상기 생체시계 관련 유전자는 Cry1(Cryptochrome Circadian Regulator 1) 또는 Cry2(Cryptochrome Circadian Regulator 2)일 수 있다.The biological clock-related gene may be Cry1 (Cryptochrome Circadian Regulator 1) or Cry2 (Cryptochrome Circadian Regulator 2).
본 발명에 있어서, 가이드 RNA는 상기 생체시계 관련 유전자를 표적하는 것으로 보다 구체적으로, 서열번호 1로 이루어진 가이드 RNA는 Cry1 유전자를 표적하는 것일 수 있으며, 상기 서열번호 2 또는 3으로 이루어진 가이드 RNA는 Cry2 유전자를 표적하는 것일 수 있다.In the present invention, the guide RNA targets the biological clock-related gene. More specifically, the guide RNA consisting of SEQ ID NO: 1 may target the Cry1 gene, and the guide RNA consisting of SEQ ID NO: 2 or 3 may target the Cry2 gene. It may be targeting genes.
본 발명에 있어서, 상기 Cry1 유전자를 표적하는 폴리뉴클레오티드는 서열번호 1로 표시되는 염기서열로 이루어진 것일 수 있으며, 이 때 상기 서열번호 1로 표시되는 염기서열과 70% 이상, 바람직하게는 80% 이상, 더욱 바람직하게는 90% 이상, 가장 바람직하게는 95%, 96%, 97%, 98% 또는 99% 이상의 서열 상동성을 가지는 염기 서열을 포함할 수 있다.In the present invention, the polynucleotide targeting the Cry1 gene may be composed of the base sequence shown in SEQ ID NO: 1, and at least 70%, preferably 80% or more, of the base sequence shown in SEQ ID NO: 1. , more preferably at least 90%, most preferably at least 95%, 96%, 97%, 98%, or 99%.
본 발명에 있어서, 상기 Cry2 유전자를 표적하는 폴리뉴클레오티드는 서열번호 2 또는 서열번호 3으로 표시되는 염기서열로 이루어진 것일 수 있으며, 이 때 상기 서열번호 2 또는 3으로 표시되는 염기서열과 70% 이상, 바람직하게는 80% 이상, 더욱 바람직하게는 90% 이상, 가장 바람직하게는 95%, 96%, 97%, 98% 또는 99% 이상의 서열 상동성을 가지는 염기 서열을 포함할 수 있다.In the present invention, the polynucleotide targeting the Cry2 gene may be composed of the base sequence shown in SEQ ID NO: 2 or SEQ ID NO: 3, where the base sequence shown in SEQ ID NO: 2 or 3 and 70% or more, It may include a base sequence having sequence homology of preferably at least 80%, more preferably at least 90%, and most preferably at least 95%, 96%, 97%, 98%, or 99%.
또한, 본 발명의 다른 양태로서, 본 발명은 상기 가이드 RNA를 포함하는, 생체시계 관련 유전자를 표적하는 재조합 가이드 RNA 벡터를 제공한다.Additionally, in another aspect of the present invention, the present invention provides a recombinant guide RNA vector targeting a circadian clock-related gene, including the guide RNA.
본 발명에서 사용되는 용어, "벡터(vector)"란 목적하는 DNA 단편을 숙주 세포에 도입시켜 증식할 수 있는 DNA로써 클로닝 운반체(cloning vehicle)라고도 한다. "발현 벡터"는 목적하는 코딩서열과, 특정 숙주생물에서 작동가능하게 연결된 코딩 서열을 발현하는데 필수적인 적정 핵산 서열을 포함하는 재조합 DNA 분자를 의미한다. 본 발명에서 상기 벡터는 발현벡터와 동일한 의미로 사용될 수 있다.As used in the present invention, the term "vector" refers to DNA that can proliferate by introducing a desired DNA fragment into a host cell, and is also called a cloning vehicle. “Expression vector” refers to a recombinant DNA molecule containing a desired coding sequence and an appropriate nucleic acid sequence essential for expressing the operably linked coding sequence in a specific host organism. In the present invention, the vector may be used in the same sense as an expression vector.
본 발명에 있어서, 상기 벡터는 동등한 기능을 제공하는 바이러스 벡터, 예컨대 아데노-연관 바이러스(AAV) 벡터, 아데노바이러스 벡터, 헤르페스 바이러스 벡터, 레트로바이러스 벡터, 렌티바이러스 벡터, 백시니아바이러스 벡터, 폭스바이러스 벡터, 단순포진바이러스 벡터 등의 다른 형태의 발현 벡터를 이용할 수 있고, 바람직하게는 아데노-연관 바이러스 벡터를 이용할 수 있다.In the present invention, the vector is a viral vector that provides equivalent functions, such as adeno-associated virus (AAV) vector, adenovirus vector, herpes virus vector, retrovirus vector, lentivirus vector, vaccinia virus vector, poxvirus vector. , other types of expression vectors such as herpes simplex virus vectors can be used, and adeno-associated virus vectors are preferably used.
또한, 본 발명의 또 다른 양태로서, 본 발명은 상기 가이드 RNA 벡터; 및 Cas9(CRISPR associated protein 9) 핵산분해효소를 포함하는, 생체시계 교란 유도용 CRISPR/Cas9 복합체를 제공한다.In addition, as another aspect of the present invention, the present invention provides the guide RNA vector; and Cas9 (CRISPR associated protein 9) nuclease, and provides a CRISPR/Cas9 complex for inducing circadian clock disruption.
본 발명에 있어서 사용되는 용어, "생체시계(Circadian clock)"란 식물, 동물, 균루, 박테리아 등을 포함하는 지구상의 생명체들에서 생화학적, 생리학적 또는 행동학적 흐름이 거의 24시간의 주기에 따라 나타나는 현상을 의미한다.As used in the present invention, the term "circadian clock" refers to the biochemical, physiological or behavioral flow of living things on Earth, including plants, animals, fungi, bacteria, etc., according to a cycle of approximately 24 hours. It means a phenomenon that appears.
본 발명에서 사용되는 용어, "생체시계 교란"이란 교대근무 수면 손실 등의 이유로 인해 생체시계에 따른 주기적인 특성(주기에 따른 활동 및 온도의 변화)이 망가진 상태를 의미한다.As used in the present invention, the term "biological clock disturbance" refers to a state in which the periodic characteristics of the biological clock (changes in activity and temperature according to the cycle) are disrupted due to reasons such as sleep loss during shift work.
본 발명에서, 상기 "Cas9"이란 "CRISPR-Cas9"을 지칭하며, CRISPR-Cas9는 3세대 유전자가위로써 이용하고자 하는 특정 염기서열을 인식하여 절단하고 편집하며, 게놈의 목적 장소에 특정 유전자를 삽입하거나 특정 유전자의 활동을 정지시키는 조작을 간단하고 신속하고 효율성 있게 실시하는데 유용하다.In the present invention, "Cas9" refers to "CRISPR-Cas9", and CRISPR-Cas9 is a third-generation gene scissors that recognizes, cuts and edits a specific base sequence to be used, and inserts a specific gene into the target site of the genome. It is useful for simply, quickly, and efficiently carrying out operations to stop the activity of a specific gene.
Cas9 단백질 또는 유전자 정보는 NCBI(National Center for Biotechnology Information)의 GenBank와 같은 공지의 데이터 베이스에서 얻을 수 있으나, 이에 제한되는 것은 아니다. 또한, Cas9 단백질은 그 목적에 따라 당업자가 추가적인 도메인을 적절하게 연결할 수 있다. Cas9 protein or gene information can be obtained from known databases such as GenBank of the National Center for Biotechnology Information (NCBI), but is not limited thereto. Additionally, a person skilled in the art can appropriately connect additional domains to the Cas9 protein depending on its purpose.
본 발명에 있어서, Cas9 단백질은 야생형 Cas9 뿐만아니라, 유전자 편집을 위한 핵산분해효소의 기능을 갖는 것이라면 Cas9의 변이체를 모두 포함할 수 있다.In the present invention, the Cas9 protein may include not only wild-type Cas9 but also all variants of Cas9 as long as it has the function of a nucleic acid decomposition enzyme for gene editing.
또한, 본 발명에서 Cas9 단백질은 그 유래가 제한되지 않으며, 비제한적인 예시로써 스트렙토코커스 피요제네스(Streptococcus pyogenes), 프란시셀라 노비시다(Francisella novicida), 스트렙토코커스 써모필러스(Streptococcus thermophilus), 레지오넬라 뉴모필라 (Legionella pneumophila), 리스테리아 이노큐아(Listeria innocua), 또는 스트렙토코커스 뮤탄스(Streptococcus mutans) 유래일 수 있고, 당업자가 적절히 선택하여 이용할 수 있다.Additionally, in the present invention, the origin of the Cas9 protein is not limited, and non-limiting examples include Streptococcus pyogenes, Francisella novicida, Streptococcus thermophilus, and Legionella. It may be derived from Legionella pneumophila, Listeria innocua, or Streptococcus mutans, and can be appropriately selected and used by those skilled in the art.
또한, 본 발명의 또 다른 양태로서, 본 발명은 상기 CRISPR/Cas9 복합체를 유효성분으로 포함하는, 생체시계 교란 유도용 조성물을 제공한다.In addition, in another aspect of the present invention, the present invention provides a composition for inducing biological clock disruption, comprising the CRISPR/Cas9 complex as an active ingredient.
본 발명에서 사용되는 용어, "유전자의 발현 억제"는 유전자의 발현을 감소시키는 모든 행위를 의미하며, 구체적으로 유전자 넉아웃(knock-out), 넉다운(knock-down), 유전자 DNA 서열에 결실, 중복, 역위, 또는 교체 등의 변이를 도입함으로써 이루어질 수 있다.As used in the present invention, the term "inhibition of gene expression" refers to all actions that reduce the expression of a gene, specifically gene knock-out, knock-down, deletion in the gene DNA sequence, This can be done by introducing mutations such as duplications, inversions, or substitutions.
본 발명자들은 구체적인 실시예를 통해 본 발명에서 직접설계한 sgRNA를 활용한 CRISPR/Cas9 시스템을 사용하는 경우 생체시계 관련 유전자를 동시에 녹아웃 함으로써 효율적으로 생체시계를 교란할 수 있다는 사실을 확인하였다.Through specific examples, the present inventors have confirmed that when using the CRISPR/Cas9 system using the sgRNA designed in the present invention, the biological clock can be efficiently disrupted by simultaneously knocking out biological clock-related genes.
본 발명의 일실시예에서는, 직접 설계하여 제작한 본 발명의 sgRNA가 생체시계 관련 유전자인 Cry1, Cry2의 발현을 효과적으로 억제한다는 사실을 확인하였다(실시예 2 참조).In one example of the present invention, it was confirmed that the sgRNA of the present invention, which was designed and produced, effectively inhibits the expression of Cry1 and Cry2, which are biological clock-related genes (see Example 2).
본 발명의 다른 실시예에서는, 시교차상핵 부위에 본 발명의 sgRNA를 포함하는 바이러스 벡터를 투여한 다음, 주기적인 특성인 활동 및 온도 변화를 관찰한 결과, 대조군에 비해 본 발명의 sgRNA를 포함하는 바이러스를 투여한 마우스에서 생체시계의 교란이 일어남을 확인하였다(실시예 4 참조).In another embodiment of the present invention, a viral vector containing the sgRNA of the present invention was administered to the suprachiasmatic nucleus region, and then cyclical characteristics of activity and temperature changes were observed. As a result, compared to the control group, the viral vector containing the sgRNA of the present invention was observed. It was confirmed that the biological clock was disturbed in mice administered the virus (see Example 4).
상기와 같은 결과를 통해 본 발명자들은 본 발명의 sgRNA를 포함하는 바이러스 벡터가 효과적으로 생체시계 관련 유전자를 녹아웃하여 생체시계를 교란시킬 수 있다는 사실을 확인하였다.Through the above results, the present inventors confirmed that the viral vector containing the sgRNA of the present invention can disrupt the biological clock by effectively knocking out biological clock-related genes.
또한, 본 발명의 또 다른 양태로서, 본 발명은 야생형의 시교차상핵(suprachiasmatic nucleus)에서 생체시계 관련 유전자가 녹아웃된 생체시계 교란 유도된 동물 모델을 제공한다. In addition, in another aspect of the present invention, the present invention provides an animal model induced by circadian clock disruption in which circadian clock-related genes are knocked out in the suprachiasmatic nucleus of wild type.
본 발명에 있어서, 상기 생체시계 교란 유도된 동물 모델은 상기 생체시계 관련 유전자가 발현하는 단백질의 수준이 감소된 것일 수 있다.In the present invention, the animal model induced by circadian clock disruption may have a reduced level of protein expressed by the circadian clock-related gene.
또한, 본 발명의 또 다른 양태로서, 본 발명은 하기의 단계를 포함하는, 생체시계 교정 물질의 스크리닝 방법을 제공한다.In addition, as another aspect of the present invention, the present invention provides a method for screening a biological clock correction material, comprising the following steps.
(a) 상기 생체시계 교란 동물모델에 후보물질을 처리하는 단계; 및(a) treating the biological clock disruption animal model with a candidate material; and
(b) 후보물질이 처리된 생체시계 교란 동물모델을 무처리 대조군과 비교하여 생체 주기적 특성의 회복 여부를 확인하는 단계.(b) Comparing the circadian clock disruption animal model treated with the candidate substance with the untreated control group to determine whether circadian rhythm characteristics are restored.
본 발명에서 사용되는 용어 "생체 주기적 특성"은 약 24시간이 경과하면서 발생하는 명 및 암조건의 변화에 의한 활동 수준 및 체내 온도의 주기적 변화를 의미한다.The term “biological cyclical characteristics” used in the present invention refers to periodic changes in activity level and body temperature due to changes in light and dark conditions that occur over about 24 hours.
이하, 본 발명의 이해를 돕기 위하여 바람직한 실시예를 제시한다. 그러나 하기의 실시예는 본 발명을 보다 쉽게 이해하기 위하여 제공되는 것일 뿐, 하기 실시예에 의해 본 발명의 내용이 한정되는 것은 아니다.Below, preferred embodiments are presented to aid understanding of the present invention. However, the following examples are provided only to make the present invention easier to understand, and the content of the present invention is not limited by the following examples.
[실시예][Example]
실시예 1. 실험준비 및 방법Example 1. Experimental preparation and method
1-1. 동물모델 및 시약의 준비1-1. Preparation of animal models and reagents
본 발명에 사용된 모든 마우스들은 표준 마우스 케이지(16 x 36 x 12.5 cm)에서 태어났으며, 음식과 물에 자유롭게 접근할 수 있는 환경에서 사육하였다. 마우스들은 3-4 주에 젖을 뗀 다음, 22 ± 1℃의 온도에서 12시간의 암/명 주기로 케이지 당 최대 4마리의 동성 마우스와 함께 사육하였고, 모든 절차는 대구경북과학기술원(Daegu Gyeongbuk Institute of Science and Technology, DGIST)의 동물 관리 및 이용위원회의 승인을 받아 수행되었다. 상기 마우스 중, PER2::LUC knock-in 마우스는 Joseph Takahashi(Proc. Natl. Acad.Sci. USA 101, 5339-5346 (2004))에게 제공받았으며, Cas9-expressing 마우스는 Feng Zhang(Cell 159, 440-455 (2014))에게 제공받아 사용하였다.All mice used in the present invention were born in standard mouse cages (16 x 36 x 12.5 cm) and raised in an environment with free access to food and water. Mice were weaned at 3-4 weeks and then housed with up to four same-sex mice per cage under a 12-h dark/light cycle at a temperature of 22 ± 1°C. All procedures were performed at the Daegu Gyeongbuk Institute of Science and Technology. The study was conducted with the approval of the Animal Care and Use Committee of the National Institute of Science and Technology (DGIST). Among the above mice, the PER2::LUC knock-in mouse was provided by Joseph Takahashi (Proc. Natl. Acad.Sci. USA 101, 5339-5346 (2004)), and the Cas9-expressing mouse was provided by Feng Zhang (Cell 159, 440). -455 (2014)) was provided and used.
1-2. 세포의 준비1-2. Preparation of cells
본 발명에 사용된 세포들은 기존에 알려진 방법에서 약간의 변형(Mol. Cells 42, 418-425 (2019))된 방식으로 배양하였다. Neuro2a 및 Neuro2a Cas9-GFP-Hygorres (CSC-RO0033, Genecopeoia, Rockville, MD, USA) 세포는 1Х 페니실린(penicillin)/스트렙토마이신(streptomycin)(Capricorn Scientific, Ebsdorfergrund, Germany), 10mg/mL의 하이그로마이신(hygromycin)(H-34274, Merck Millipore, Burlington, MA, USA) 및 10 %의 소태아혈청(fetal bovine serum, FBS) (Hyclone)을 포함하고 있는 DMEM(Dulbecco's modified Eagle medium (Hyclone, Chicago, IL, USA)) 배지에서 37℃의 온도와 5%의 CO2 조건에서 배양하였다.The cells used in the present invention were cultured using a method slightly modified from a previously known method (Mol. Cells 42, 418-425 (2019)). Neuro2a and Neuro2a Cas9-GFP-Hygor res (CSC-RO0033, Genecopeoia, Rockville, MD, USA) cells were treated with 1Х penicillin/streptomycin (Capricorn Scientific, Ebsdorfergrund, Germany), 10 mg/mL Hygor. DMEM (Dulbecco's modified Eagle medium (Hyclone, Chicago, USA) containing hygromycin (H-34274, Merck Millipore, Burlington, MA, USA) and 10% fetal bovine serum (FBS) (Hyclone). IL, USA)) cultured in medium at a temperature of 37°C and 5% CO 2 .
Cas9:EGFP를 안정적으로 발현하는 Neuro2a 세포(이하, Neuro2a-Cas9)는 형질주입(transfection) 시약인 리포펙타민 2000(Lipofectamine 2000) (#11668019, Thermo Scientific, Waltham, MA, USA)을 제조사의 지침(Cosmo Genetech, Seoul, South Korea)에 맞게 활용하여 pAAV-U6-sgCRY-hSyn-mCherry, 또는 pAAV-U6-sgLacZ-hSyn-mCherry로 형질주입 하였고, 돌연변이 여부는 제조사(Integrated DNA Technologies, Coralville, IA, USA)의 지침에 따른 Surveyor 돌연변이 분석을 수행하여 확인하였다.Neuro2a cells stably expressing Cas9:EGFP (hereinafter referred to as Neuro2a-Cas9) were transfected with the transfection reagent Lipofectamine 2000 (#11668019, Thermo Scientific, Waltham, MA, USA) according to the manufacturer's instructions. (Cosmo Genetech, Seoul, South Korea) was transfected with pAAV-U6-sgCRY-hSyn-mCherry, or pAAV-U6-sgLacZ-hSyn-mCherry, and mutations were checked by the manufacturer (Integrated DNA Technologies, Coralville, IA). , USA) was confirmed by performing Surveyor mutation analysis according to the guidelines.
1-3. 플라스미드 및 AAV 바이러스의 준비1-3. Preparation of plasmids and AAV viruses
sgRNA의 DNA 올리고머는 CRISPR-Cas9의 위치 선택 알고리즘인 CHOPCHOP(Nucleic Acids Res. 42,W401-407; 10.1093/nar/gku410 (2014))과 클로닝용 어댑터를 함께 사용하여 Bionics(Seoul, South Korea)에서 상업적으로 제작한 것을 사용하였고, 어닐링을 수행한 다음, sgRNA를 포함하는 DNA 단편을 sgRNA 발현 벡터인 pAAV-hU6-gRNA-hSyn-mCherry-KASH에 클로닝 하였다. 플라스미드의 서열은 생어 시퀀싱(Sanger sequencing)(Macrogen, Seoul, South Korea)을 수행하였다.The DNA oligomer of sgRNA was produced at Bionics (Seoul, South Korea) using CHOPCHOP (Nucleic Acids Res. 42,W401-407; 10.1093/nar/gku410 (2014)), a site selection algorithm for CRISPR-Cas9, and an adapter for cloning. A commercially produced one was used, annealing was performed, and the DNA fragment containing the sgRNA was cloned into the sgRNA expression vector, pAAV-hU6-gRNA-hSyn-mCherry-KASH. The sequence of the plasmid was subjected to Sanger sequencing (Macrogen, Seoul, South Korea).
본 발명에서 사용된 AAV는 삼중 형질감염(triple transfection) 방법을 사용하여 제작하였고, 이렇게 제작된 AAV를 요오딕사놀(iodixanol) 구배 방법(Nat. Protoc. 1, 1412-1428 (2006))을 활용하여 수득하였다. 모든 AAV 벡터들을 종이에 처리한 다음, qPCR로 정량화한 결과, 1012 내지 1013 viral genome copies/milliliter(GC/mL) 수준(AAV-DJ-hU6-sgLacZ-hSyn-mCherry-KASH 1.8 Х 1013 GC/mL; AAV-DJ-CSAC-Crys-hSyn-mCherry-KASH 1.1 Х 1013 GC/mL)으로 정제된 것을 확인할 수 있었다.The AAV used in the present invention was produced using a triple transfection method, and the AAV produced in this way was produced using the iodixanol gradient method (Nat. Protoc. 1, 1412-1428 (2006)). It was obtained. All AAV vectors were processed on paper and quantified by qPCR, resulting in a level of 10 12 to 10 13 viral genome copies/milliliter (GC/mL) (AAV-DJ-hU6-sgLacZ-hSyn-mCherry-KASH 1.8 Х 10 13 It was confirmed that it was purified (GC/mL; AAV-DJ-CSAC-Crys-hSyn-mCherry-KASH 1.1 Х 10 13 GC/mL).
1-4. 통계 분석1-4. statistical analysis
카이-제곱 주기도(Chi-square periodogram) 분석은 기존에 알려진 R(R Core Team. R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria.)의 xsp 패키지를 활용하여 수행하였고, Real time bioluminescence는 cosinor 절차(Chronobiologia 6, 305-323 (1979) 및 Biol. Rhythm Res. 38, 275-325; 10.1080/09291010600903692 (2007))로 수행하였다.Chi-square periodogram analysis was performed using the previously known xsp package of R (R Core Team. R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria.) , Real time bioluminescence was performed by the cosinor procedure (Chronobiologia 6, 305-323 (1979) and Biol. Rhythm Res. 38, 275-325; 10.1080/09291010600903692 (2007)).
실시예 2. 생체시계(circadian clock)의 조절을 위한 조성물의 제작Example 2. Preparation of a composition for regulating the circadian clock
2-1. 생체시계 관련 유전자 조절을 위한 sgRNA의 설계2-1. Design of sgRNA for regulation of circadian clock-related genes
생체시계의 필수적인 분자 구성을 knock-out할 수 있는 sgRNA를 설계하기 위해 CHOPCHOP을 활용하여 생체시계관련 핵심인자로 알려진 유전자(Cry1, Cry2)를 타겟팅할 수 있는 sgRNA를 예측해본 다음, 격자이동 넌센스(Frame shift-mediated nonsense) 돌연변이를 극대화 할 수 있도록 도 1a에 나타낸 바와 같이, 초기 엑손을 표적으로 하는 3가지의 sgRNA를 선택하여 설계하였다.In order to design sgRNAs that can knock-out the essential molecular components of the biological clock, CHOPCHOP was used to predict sgRNAs that could target genes (Cry1, Cry2) known to be key factors related to the biological clock, and then grid shift nonsense ( To maximize frame shift-mediated nonsense) mutations, three sgRNAs targeting early exons were selected and designed, as shown in Figure 1a.
상기 3가지 sgRNA를 U6 프로모터 구동 sgRNA 발현 벡터에 클로닝 한 다음(도 1b), 돌연변이 유도 효율을 평가하였으며 각각의 sgRNA를 SpCas9 발현 플라스미드를 활용하여 Neuro2a 신경모세포종 세포주를 형질감염 시켰다. 세포주에서 DNA를 추출해낸 다음, Surveyor nuclease mismatch cleavage assay를 활용하여 DNA의 돌연변이율을 확인하였다. 그 결과, 도 1c에 화살표로 표시한 바와 같이, Cry1(sgCry1)을 표적으로 하는 sgRNA를 발현하는 플라스미드로 형질감염된 Neuro2a 세포의 게놈 DNA는 상기 분석에서 모두 절단된 DNA 밴드를 생성하는 것을 확인할 수 있었다. Cry2(sgCry2)를 표적으로 하는 그룹에서도 절단이 일어나는 것을 확인할 수 있었으나, Cry1을 표적으로 하는 sgRNA를 사용한 그룹만큼 많은 절단은 관찰할 수 없었다.The above three sgRNAs were cloned into the U6 promoter-driven sgRNA expression vector (Figure 1b), then the mutation induction efficiency was evaluated, and each sgRNA was transfected into the Neuro2a neuroblastoma cell line using the SpCas9 expression plasmid. After extracting DNA from the cell line, the mutation rate of the DNA was confirmed using the Surveyor nuclease mismatch cleavage assay. As a result, as indicated by the arrow in Figure 1c, the genomic DNA of Neuro2a cells transfected with a plasmid expressing sgRNA targeting Cry1 (sgCry1) was confirmed to generate cleaved DNA bands in the above analysis. . It was confirmed that cleavage occurred in the group targeting Cry2 (sgCry2), but as much cleavage could not be observed as in the group using sgRNA targeting Cry1.
단일 AAV는 최대 3개의 sgRNA 발현 카세트를 수용할 수 있기에 Cry1과 Cry2를 동시에 녹아웃 할 수 있도록 강한 돌연변이 유도 효율을 가지는 Cry1 타겟팅 sgRNA(sgCry1-2)와 중간 수준의 돌연변이 유도 효율을 가지는 Cry2(sgCry2-1 및 sgCry2-2)를 타겟으로 하는 sgRNA를 활용하여 AAV를 제작하였다. 본 발명에서 활용된 sgRNA의 구조는 하기 표 1에 나타내었다.A single AAV can accommodate up to three sgRNA expression cassettes, allowing for simultaneous knockout of Cry1 and Cry2, including a Cry1-targeting sgRNA (sgCry1-2) with strong mutagenesis efficiency and Cry2 (sgCry2-) with a moderate mutagenesis efficiency. AAV was produced using sgRNA targeting 1 and sgCry2-2). The structure of sgRNA used in the present invention is shown in Table 1 below.
2-2. 제작한 sgRNA의 단백질 발현 조절 효과 확인2-2. Confirmation of protein expression regulation effect of produced sgRNA
상기 실시예 2-1에서 제작한 sgRNA 및 이를 탑재한 AAV의 단백질 발현 조절 효과를 확인하기 위해 Neuro2a-Cas9에 도 2a에 표시된 카세트를 포함하는 플라스미드로 형질감염 시킨 다음, 세포에서 단백질 추출물을 분리하였고, 이렇게 분리해낸 단백질 수준을 측정하기 위해, 세포들을 인산염 완충 식염수(phosphate-buffered saline)로 2회 세척한 다음, 방사선 면역침전 분석(radioimmunoprecipitation assay(RIPA) 버퍼(#9806, Cell Signaling, Danvers, MA, USA))에 초음파 처리(sonication)를 수행하여 용해시키고, 얼음에서 30분동안 배양하였다. 이렇게 수득한 용해물은 Laemmli 샘플 버퍼(#NP0007, Invitrogen, Waltham, MA, USA)에서 5분 끓인 다음 원심분리하여 상청액을 수득하였다.To confirm the effect of sgRNA produced in Example 2-1 and AAV carrying it on protein expression regulation, Neuro2a-Cas9 was transfected with a plasmid containing the cassette shown in Figure 2a, and then protein extract was separated from the cells. To measure the level of the separated proteins, the cells were washed twice with phosphate-buffered saline and then subjected to radioimmunoprecipitation assay (RIPA) buffer (#9806, Cell Signaling, Danvers, MA. , USA), were dissolved by sonication, and incubated on ice for 30 minutes. The lysate thus obtained was boiled in Laemmli sample buffer (#NP0007, Invitrogen, Waltham, MA, USA) for 5 minutes and then centrifuged to obtain a supernatant.
단백질은 4-15%의 트리스-글라이신(tris-glycine, TG) 젤(#45101080003-2, SMOBIO, Hsinchu, Taiwan)에서 분리한 다음, 트리스-글라이신 버퍼에서 폴리비닐리덴플루오라이드(polyvinylidene fluoride, PVDF)막으로 전기전달(electro-transferred) 하였다. 막은 트윈-20(Tween-20, TTBS) 및 1%의 소 혈청 알부민(bovine serum albumin, BSA) 처리된 상기 버퍼에서 차단하였고, 밤새 트윈-20(Tween-20, TTBS) 및 1%의 소 혈청 알부민(bovine serum albumin, BSA)에서 4℃의 온도로 1차 항체인 Cry1(#AF3764-SP, R&D Systems, Minneapolis, MN, USA), Cry2(#HPA037577, Atlas Antibodies, Bromma, Sweden) 및 β-actin-HRP(#SC-47778, Santa Cruz, Dallas, TX, USA)와 함께 배양하였다. 2차 항체로는 Anti-Rabbit(SKU # 31460, Thermo Scientific) 또는 antigoat(Jackson Laboratory, Bar Harbor, ME, USA)를 사용하였고, 강화된 화학발광(Enhanced Chemiluminescence, ECL) 선택 용액(GE Healthcare)과 ECL Pico System(ECL-PS100, Dongin, Seoul, Korea)을 활용하여 단백질을 검출하였다.Proteins were separated on a 4-15% tris-glycine (TG) gel (#45101080003-2, SMOBIO, Hsinchu, Taiwan) and then incubated with polyvinylidene fluoride (PVDF) in tris-glycine buffer. ) Electricity was transferred to the membrane. The membrane was blocked in the buffer treated with Tween-20 (TTBS) and 1% bovine serum albumin (BSA), and incubated with Tween-20 (TTBS) and 1% bovine serum overnight. Primary antibodies Cry1 (#AF3764-SP, R&D Systems, Minneapolis, MN, USA), Cry2 (#HPA037577, Atlas Antibodies, Bromma, Sweden) and β- were incubated in bovine serum albumin (BSA) at a temperature of 4°C. Cultured with actin-HRP (#SC-47778, Santa Cruz, Dallas, TX, USA). Anti-Rabbit (SKU # 31460, Thermo Scientific) or antigoat (Jackson Laboratory, Bar Harbor, ME, USA) was used as the secondary antibody, and enhanced chemiluminescence (ECL) selection solution (GE Healthcare) was used. Proteins were detected using the ECL Pico System (ECL-PS100, Dongin, Seoul, Korea).
그 결과, 도 2b에 나타낸 바와 같이, sgCrys를 발현하는 플라스미드로 형질감염된 Neuro2a-Cas9 세포의 Cry1 및 Cry2 단백질 수준은 대조군에 비해 현저하게 낮게 나타남을 확인할 수 있었다. As a result, as shown in Figure 2b, it was confirmed that the Cry1 and Cry2 protein levels of Neuro2a-Cas9 cells transfected with a plasmid expressing sgCrys were significantly lower than those of the control group.
실시예 3. 본 발명 AAV 벡터 시스템의 생체시계 관련 인자 조절 효과 확인Example 3. Confirmation of the effect of the AAV vector system of the present invention on regulating biological clock-related factors
상기 실시예 2에서 제작한 다중 sgRNA를 탑재한 AAV 벡터 시스템(이하, CSAC)의 효과를 확인하기 위해, 상기 실시예 1-2에서 준비한 PER2 및 LUCIFERASE 융합 단백질 및 Cas9(PER2::LUC;Cas9)을 지속적으로 발현하는 시교차상핵 절편의 바이오형광 진동(oscillation of bioluminescence)을 확인하기 위해 도 3a에 나타낸 대로 실험을 설계하였다. 바이오형광 진동은 시교차상핵(Suprachiasmatic nucleus, SCN) 절편을 활용하여 수행하였고, 이때 시교차상핵 절편의 배양은 기존에 알려진 방식에서 약간 수정(Exp. Mol. Med. 52, 473-484 (2020))된 방법을 활용하여 준비하였다. 시교차상핵의 준비를 위해 1 주령된 mPer2::LUC; Cas9 마우스를 희생시킨 다음, 뇌를 신속하게 제거하여 얼음 위에서 식혔다. 0.01M HEPES와 36mM D-glucose가 보충된 Gey 's Balanced Salt Solution(GBSS)에 뇌를 적신 다음, 5 % CO2 및 95 % O2로 구성된 공기를 처리하였다. 뇌는 Leica VT1000 S vibratome(Leica, Wetzlar, Germany)을 사용하여 400μm 두께의 조각으로 관상 절단하였으며, 절단된 슬라이스를 배양 삽입막(Millicell-CM; Millipore, Bedford, MA, USA)에 유지하였다가, 37℃의 배양 배지(50 % minimum essential medium, 25 % GBSS, 25 % horse serum, 36 mM glucose 및 1Х antibiotic-antimycotic)에 살짝 잠기게 하였다. In order to confirm the effect of the AAV vector system (hereinafter CSAC) carrying the multiple sgRNA produced in Example 2, the PER2 and LUCIFERASE fusion protein and Cas9 (PER2::LUC;Cas9) prepared in Example 1-2 were used. An experiment was designed as shown in Figure 3a to confirm the oscillation of bioluminescence in the suprachiasmatic nucleus section that continuously expresses. Biofluorescence oscillation was performed using suprachiasmatic nucleus (SCN) sections, and the culture of the suprachiasmatic nucleus sections was slightly modified from the previously known method (Exp. Mol. Med. 52, 473-484 (2020) ) was prepared using the method described above. For preparation of the suprachiasmatic nucleus, 1-week-old mPer2::LUC; Cas9 mice were sacrificed, and then brains were quickly removed and cooled on ice. The brain was soaked in Gey's Balanced Salt Solution (GBSS) supplemented with 0.01M HEPES and 36mM D-glucose, and then treated with air consisting of 5% CO 2 and 95% O 2 . The brain was cut coronally into 400-μm-thick slices using a Leica VT1000 S vibratome (Leica, Wetzlar, Germany), and the cut slices were maintained on a culture insert membrane (Millicell-CM; Millipore, Bedford, MA, USA). The cells were gently submerged in culture medium (50% minimum essential medium, 25% GBSS, 25% horse serum, 36mM glucose, and 1Х antibiotic-antimycotic) at 37°C.
상기와 같은 방식으로 준비된 시교차상핵 절편은 실험에 사용되기 전에 2주 이상 배양하였고, 생물발광을 정략적으로 측정하기 위해, 시교차상핵 절편을 36℃의 온도조건에서 0.3mM D-luciferin(Promega, Madison, WI, USA)을 포함하는 1mL 배양 배지를 넣은 35mm 페트리 접시에서 보관하였다. 빛 방출(Light emission)은 접시-타입 바퀴달린 발광계(luminometer)(AB-2550 Kronos-Dio; ATTO, Tokyo, Japan)를 사용하여 10분 간격으로 1분 동안 측정하였다. The suprachiasmatic nucleus sections prepared in the same manner as above were cultured for more than 2 weeks before being used in the experiment, and in order to quantitatively measure bioluminescence, the suprachiasmatic nucleus sections were incubated with 0.3mM D-luciferin (Promega, Madison, WI, USA) were stored in a 35 mm Petri dish containing 1 mL of culture medium. Light emission was measured for 1 minute at 10-minute intervals using a dish-type wheeled luminometer (AB-2550 Kronos-Dio; ATTO, Tokyo, Japan).
달리 언급이 없는 한, 절편 배양을 위해 사용된 모든 배양 배지 및 보충제는 Thermo-Fisher Scientific에서 구입한 것을 사용하였고, 시교차상핵 절편은 기존에 알려진 바(Neuroendocrinology, 10.1159/000505922 (2020))와 같이, 안정화한지 7일 후, 바이러스를 활용하여 형질 도입하였다. AAV 바이러스들을 상기 35mm 페트리접시에 있는 시교차상핵 절편의 표면에 직접 떨어뜨린 다음, 15일 배양하고, 세포의 생물 발광을 확인하였다.Unless otherwise stated, all culture media and supplements used for section culture were purchased from Thermo-Fisher Scientific, and suprachiasmatic nucleus sections were cultured as previously known (Neuroendocrinology, 10.1159/000505922 (2020)). , 7 days after stabilization, transduction was performed using the virus. AAV viruses were dropped directly onto the surface of the suprachiasmatic nucleus section in the 35 mm Petri dish, cultured for 15 days, and bioluminescence of cells was confirmed.
그 결과, 도 3b에 나타낸 바와 같이, 시교차상핵 절편에서 sgRNA의 발현이 잘되고 있음을 알려주는 광범위한 형광을 확인할 수 있었다. 대조군인 sgLacZ를 발현하는 시교차상핵 외식편(explants)은 도 3c에 나타낸 바와 같이, PER2에 대해서 강력한 일주기 진동을 보여줌을 확인할 수 있었으나, 본 발명의 CSAC-Crys를 형질감염 시킨 그룹에서는 도 3d에 나타낸 바와 같이, PER2의 바이오형광 진동이 심하게 저해된 것을 확인할 수 있었다. As a result, as shown in Figure 3b, a wide range of fluorescence was confirmed in the suprachiasmatic nucleus section, indicating that sgRNA was well expressed. It was confirmed that control suprachiasmatic nucleus explants expressing sgLacZ showed strong circadian oscillations for PER2, as shown in Figure 3c, but in the group transfected with CSAC-Crys of the present invention, Figure 3d As shown, it was confirmed that the biofluorescence oscillation of PER2 was severely inhibited.
상기와 같은 결과가 본 발명의 CSAC의 시교차상핵 세포의 손상 또는 PER2 발현 조절 유전자의 억제에 기인한 것이 아닌, 생체시계를 조절한다는 사실을 명확히 하기 위해서 아데닐일 사이클레이스 활성화제(adenylyl cyclase activator)인 포스콜린(forskolin)을 처리하였다. 그 결과, 모든 그룹에서 포스콜린의 처리는 CSAC와 관계없이 PER2::LUC 발현을 강력하게 유도하는 것을 확인할 수 있었으며, PER2 :: LUC 진동을 통계적으로 분석해 보았을 때, 도 3e에 나타낸 바와 같이, CSAC-Crys가 PER2 :: LUC 진동의 진폭을 현저하게 억제하고, 도 3f에 나타낸 바와 같이, 강인성(robustness)도 억제하는 것을 확인할 수 있었다. 다만, 진동 기간의 경우 도 3g에 나타낸 바와 같이, 유의한 변화가 발생하지 않음을 확인할 수 있었다.In order to clarify that the above results are not due to damage to the suprachiasmatic nucleus cells of the CSAC of the present invention or to inhibition of the PER2 expression regulatory gene, but to the fact that it regulates the biological clock, adenylyl cyclase activator Phosphorus was treated with forskolin. As a result, it was confirmed that forskolin treatment strongly induced PER2::LUC expression in all groups regardless of CSAC, and when PER2::LUC oscillation was statistically analyzed, as shown in Figure 3e, CSAC It was confirmed that -Crys significantly suppressed the amplitude of PER2::LUC oscillation and, as shown in Figure 3f, also suppressed robustness. However, in the case of the vibration period, it was confirmed that no significant change occurred, as shown in Figure 3g.
본 발명자들은 상기와 같은 발견을 통해 본 발명의 CSAC가 생체 외(ex vivo) 분자 시계를 효과적으로 감쇠시킨다는 사실을 확인할 수 있었다.Through the above findings, the present inventors were able to confirm that CSAC of the present invention effectively attenuates the molecular clock ex vivo.
실시예 4. 본 발명 AAV 벡터 시스템의 생체내 생체시계 조절 효과Example 4. In vivo biological clock regulation effect of the AAV vector system of the present invention
4-1. 본 발명 AAV 벡터 시스템의 생체 내 주입4-1. In vivo injection of the AAV vector system of the invention
생체내의 CSAC의 효과를 확인하기 위해 마우스에 정위주입법(Stereotaxic injection)으로 CSAC를 주입하는 실험을 진행하였다. 수술은 무균기술을 활용하여 수행되었고, 구체적으로 케타민(ketamine, 100mg/kg)과 자일라이진(xylazine, 10mg/kg)을 사용하여 마취시킨 마우스를 정위 장치(stereotaxic apparatus, (RWD Life Science, Shenzhen, China))에 넣은 다음, Nanoliter 주입기 시스템(WPI, Sarasota, FL, USA)을 사용하여 0.1 μL/min의 속도로 AAV 시교차상핵 절편(AP(Anteroposterior) = -0.8 mm, ML(Mediolateral) = ±0.22 mm(bregma 기준), DV(Dorsoventral) = -5.85 mm(brain surface 기준))에 주입하였다(도 4a). To confirm the effect of CSAC in vivo, an experiment was conducted in which CSAC was injected into mice using stereotaxic injection. The surgery was performed using aseptic technique, and specifically, mice anesthetized using ketamine (100 mg/kg) and xylazine (10 mg/kg) were used in a stereotaxic apparatus (RWD Life Science, Shenzhen). , China) and then injected into AAV suprachiasmatic nucleus sections (Anteroposterior (AP) = -0.8 mm, Mediolateral (ML) = 0.1 μL/min using a Nanoliter injector system (WPI, Sarasota, FL, USA). It was injected at ±0.22 mm (based on bregma), DV (dorsoventral) = -5.85 mm (based on brain surface) (Figure 4a).
4-2. 시교차상핵 부분이 감염된 마우스의 선별4-2. Selection of mice infected with suprachiasmatic nucleus region
상기 실시예 4-1에서 주입한 본 발명 CSAC의 주입여부 확인을 위해, 바이러스의 충분한 발현을 위해 3주 이상 사육한 마우스를 대상으로 하여 4%의 파라 포름알데히드(paraformaldehyde)를 심장내 관류한 다음, 4℃의 온도에서 밤새 고정시켰다. Leica VT1000 S vibratome(Leica, Wetzlar, Germany)를 활용하여 조직을 관상(coronal)의 50-μm 두께 절편으로 잘라내었다. 핵은 4'6'-diamidino-2-phnylindole(DAPI)를 활용하여 염색하였고, 이미지는 Nikon C2+ 공초점 현미경 시스템을 활용하여 촬영한 다음, Fiji(Nat.Methods 9, 676-682 (2012))를 활용하여 선형조정하였다. 그 결과, 도 4b에 나타낸 바와 같이, 성공적으로 마우스 내에 주입된 사실을 확인할 수 있었다. 마우스의 운동 활성 및 온도 확인을 위해 도 4c(대조군) 및 4d(Crys)에 나타낸 바와 같이, CSAC에 의해 시교차상핵 부분이 형질감염된 마우스만 선별하여 다음 실험에 사용하였다.To confirm whether the CSAC of the present invention injected in Example 4-1 was injected, mice raised for more than 3 weeks to ensure sufficient expression of the virus were intracardially perfused with 4% paraformaldehyde. , and fixed overnight at a temperature of 4°C. The tissue was cut into coronal 50-μm thick sections using a Leica VT1000 S vibratome (Leica, Wetzlar, Germany). Nuclei were stained using 4'6'-diamidino-2-phnylindole (DAPI), and images were taken using a Nikon C2+ confocal microscope system and then analyzed in Fiji (Nat.Methods 9, 676-682 (2012)). Linear adjustment was performed using . As a result, as shown in Figure 4b, it was confirmed that the injection was successfully administered into the mouse. To check the locomotor activity and temperature of the mice, as shown in Figures 4c (Control) and 4d (Crys), only mice in which the suprachiasmatic nucleus was transfected by CSAC were selected and used in the next experiment.
4-3. 마우스의 일반운동 활성(locomotor activity) 및 온도 확인4-3. Check the mouse's locomotor activity and temperature
상기 4-2에서 선별해낸 마우스를 대상으로 하여 운동 활성 및 체온을 측정하기 위해, 무선송신기 기반 원격 측정 시스템인 E-mitter(Starr Life Science, Oakmont, PA, USA)를 활용하였으며, E-mitter는 복강내 케타민(ketamine, 100mg/kg)과 자일라이진(xylazine, 10mg/kg)의 투여로 인해 전신 마취된 마우스의 피부 아래에 무균 기술을 활용하여 이식하였다. E-mitter가 이식된 마우스는 12시간의 규칙적인 명암주기 조건으로 1주일의 회복기간을 주었으며, E-mitter가 감지한 마우스의 활동 및 온도 데이터는 수신기(ER-4000 engergizer receiver, Starr Life Science)로 송신되었고, 데이터 수집 및 디지털 변환은 VitalView 소프트웨어(Starr Life Science)를 사용하여 6분 마다 수행하였다.To measure motor activity and body temperature of the mice selected in 4-2 above, E-mitter (Starr Life Science, Oakmont, PA, USA), a wireless transmitter-based telemetry system, was used. It was transplanted using aseptic technique under the skin of mice under general anesthesia due to intraperitoneal administration of ketamine (100 mg/kg) and xylazine (10 mg/kg). Mice implanted with an E-mitter were given a recovery period of one week under regular 12-hour light/dark cycle conditions, and the activity and temperature data of the mouse detected by the E-mitter were recorded using a receiver (ER-4000 engergizer receiver, Starr Life Science). Data collection and digital conversion were performed every 6 minutes using VitalView software (Starr Life Science).
① 상기와 같은 방법을 통해 운동 활동을 측정한 결과, 도 5a에 나타낸 바와 같이, 일주기 운동 활동 형광 단백질(mCherry)을 발현하는 AAV가 주입된 대조군 동물에서는 명 조건 및 암 조건에서 모두 잘 정렬된 운동활동이 관찰되었으며, 일정한 암 조건(constant darkness, DD)에서 대조군 동물은 24시간 보다 약간 짧은 생체시계에 따른 각성(wake) 및 휴식(resting) 주기를 보여주었다. 그러나, 도 5b에 나타낸 바와 같이, CSAC-Crys를 주사한 마우스는 일정한 암 조건에서 일주기 리듬 이상(arrhythmicity)을 보여주는 것을 확인할 수 있었다.① As a result of measuring locomotor activity through the above method, as shown in Figure 5a, in control animals injected with AAV expressing circadian locomotor activity fluorescent protein (mCherry), well-aligned cells were observed in both light and dark conditions. Motor activity was observed, and control animals under constant darkness (DD) showed wake and rest cycles according to the biological clock slightly shorter than 24 hours. However, as shown in Figure 5b, it was confirmed that mice injected with CSAC-Crys showed arrhythmicity under certain dark conditions.
본 발명자들은 상기 데이터를 토대로 하여 상기 실시예 1-4의 카이-제곱 주기도 분석을 수행해 보았을 때, 본 발명의 CSAC가 일주기 운동활동을 방해한다는 사실을 명확히 할 수 있었는데, 구체적으로 대조군 동물에서 평균 Qp 값은 도 5c에 나타낸 바와 같이 정상적인 일주기 운동활동의 전형적인 형태인 24시간 즈음에 피크를 보여줌을 확인할 수 있었으나, 도 5d(CSAC-Crys 투여 그룹)은 어느 시점에서도 유의한 수치를 보여주지 않았다.When the present inventors performed the chi-square periodogram analysis of Examples 1-4 based on the above data, they were able to clarify that the CSAC of the present invention interferes with circadian motor activity, specifically, the average As shown in Figure 5c, the Qp value was confirmed to peak around 24 hours, a typical form of normal circadian motor activity, but Figure 5d (CSAC-Crys administration group) did not show significant values at any time point. .
② 상기와 같은 방법으로 온도를 측정하였을 때에도, 상기 운동활동과 거의 유사한 결과를 관찰할 수 있었는데, 구체적으로 도 6a에 나타낸 바와 같이, 대조군 동물은 일정한 암 조건 및 명/암 조건에서 모두 전형적인 온도 변화를 보여줬으나, CSAC-Crys를 투여한 그룹에서는 도 6b에 나타낸 바와 같이, 체온의 일정한 일주기 변화를 보여주지 않았다.② Even when temperature was measured in the same manner as above, almost similar results to the above-mentioned motor activity could be observed. Specifically, as shown in Figure 6a, control animals showed typical temperature changes in both constant dark conditions and light/dark conditions. However, the group administered CSAC-Crys did not show consistent circadian changes in body temperature, as shown in Figure 6b.
상기와 같은 온도 데이터는 카이-제곱 주기도 분석을 수행하였을 때, 운동활동 데이터와 유사하게 대조군의 경우 평균 Qp 값이 24시간 즈음에 피크를 보여줌으로써 일주기의 전형적인 형태를 가지고 있음을 확인할 수 있었으나, 도 6c(CSAC-Crys 투여 그룹)는 어느 시점에서도 유의한 수치를 보여주지 않았기에, 본 발명의 CSAC를 처리한 그룹은 일정한 일주기 변화를 보여주지 않음을 확인할 수 있었다.When chi-square periodogram analysis was performed on the above temperature data, it was confirmed that, similar to the exercise activity data, the average Qp value in the control group peaked around 24 hours, showing a typical circadian pattern. Since Figure 6c (CSAC-Crys administration group) did not show significant values at any time, it was confirmed that the group treated with CSAC of the present invention did not show constant circadian changes.
4-4. 마우스의 일반운동 활성(locomotor activity) 및 온도의 평균 확인4-4. Check the average locomotor activity and temperature of the mouse
상기 4-3의 실험예에서 얻어진 데이터를 토대로 하여, 명/암 조건(LD)에서 마우스의 평균 활동(도 7a) 및 평균 온도(도 7b)를 측정하고, 일정한 암 조건(DD)에서 마우스의 평균 활동(도 7c) 및 평균 온도(도 7d)를 측정하였다. 그 결과, 대조군에 비해서, 본 발명의 CSAC를 투여한 그룹에서도 평균 활동 및 평균 온도의 유의한 차이가 나지 않음을 확인할 수 있었다.Based on the data obtained in the experimental example of 4-3 above, the average activity (FIG. 7a) and average temperature (FIG. 7b) of mice were measured under light/dark conditions (LD), and the average activity of mice was measured under constant dark conditions (DD). Average activity (Figure 7C) and average temperature (Figure 7D) were measured. As a result, it was confirmed that compared to the control group, there was no significant difference in average activity and average temperature in the group administered CSAC of the present invention.
본 발명자들은 상기와 같은 결과를 통해 본 발명의 CSAC는 평균적인 운동활동이나 온도에 영향을 주지 않으면서도 효과적으로 생체시계를 조절하여 생체 주기적인 특징(활동 및 온도의 변화)을 저해한다는 사실을 확인하였다.Through the above results, the present inventors confirmed that the CSAC of the present invention effectively regulates the biological clock and inhibits circadian characteristics (changes in activity and temperature) without affecting average motor activity or temperature. .
상기 진술한 본 발명의 설명은 예시를 위한 것이며, 본 발명이 속하는 기술분야의 통상의 지식을 가진 자는 본 발명의 기술적 사상이나 필수적인 특징을 변경하지 않고서 다른 구체적인 형태로 쉽게 변형이 가능하다는 것을 이해할 수 있을 것이다. 그러므로 이상에서 기술한 실시예들은 모든 면에서 예시적인 것이며 한정적이 아닌 것으로 이해해야만 한다.The description of the present invention stated above is for illustrative purposes, and a person skilled in the art to which the present invention pertains can understand that it can be easily modified into other specific forms without changing the technical idea or essential features of the present invention. There will be. Therefore, the embodiments described above should be understood in all respects as illustrative and not restrictive.
<110> Institute for Basic Science Daegu Gyeongbuk Institute of Science and Technology <120> Composition for inducing disturbance of circadian clock and model for disturbing circadian clock made using same <130> PD21-017 <160> 3 <170> KoPatentIn 3.0 <210> 1 <211> 23 <212> RNA <213> sgCry 1-2 <400> 1 ggtcgaggat atagacgcag cgg 23 <210> 2 <211> 23 <212> RNA <213> sgCry 2-1 <400> 2 ctgtgggcat caaccgatgg agg 23 <210> 3 <211> 23 <212> RNA <213> sgCry 2-2 <400> 3 tcggttgatg cccacagacg agg 23 <110> Institute for Basic Science Daegu Gyeongbuk Institute of Science and Technology <120> Composition for inducing disturbance of circadian clock and model for disturbing circadian clock made using same <130>PD21-017 <160> 3 <170> KoPatentIn 3.0 <210> 1 <211> 23 <212> RNA <213> sgCry 1-2 <400> 1 ggtcgaggat atagacgcag cgg 23 <210> 2 <211> 23 <212> RNA <213> sgCry 2-1 <400> 2 ctgtgggcat caaccgatgg agg 23 <210> 3 <211> 23 <212> RNA <213> sgCry 2-2 <400> 3 tcggttgatg cccacagacg agg 23
Claims (14)
상기 생체시계 관련 유전자는 Cry1(Cryptochrome Circadian Regulator 1) 또는 Cry2(Cryptochrome Circadian Regulator 2)인 것을 특징으로 하며,
상기 가이드 RNA는 상기 생체시계 관련 유전자의 엑손 1을 표적하는, 가이드 RNA.A guide RNA targeting a biological clock-related gene, consisting of a base sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3,
The biological clock-related gene is characterized as Cry1 (Cryptochrome Circadian Regulator 1) or Cry2 (Cryptochrome Circadian Regulator 2),
The guide RNA targets exon 1 of the circadian clock-related gene.
상기 서열번호 1로 이루어진 가이드 RNA는 Cry1를 표적하는 것을 특징으로 하는, 가이드 RNAAccording to paragraph 1,
The guide RNA consisting of SEQ ID NO: 1 is a guide RNA characterized in that it targets Cry1.
상기 서열번호 2 및 서열번호 3으로 이루어진 가이드 RNA는 Cry2를 표적하는 것을 특징으로 하는, 가이드 RNA.According to paragraph 1,
Guide RNA consisting of SEQ ID NO: 2 and SEQ ID NO: 3 is characterized in that it targets Cry2.
상기 벡터는 아데노연관바이러스(Adeno-associated virus; AAV) 벡터인 것을 특징으로 하는, 가이드 RNA 벡터.According to clause 5,
A guide RNA vector, characterized in that the vector is an Adeno-associated virus (AAV) vector.
상기 가이드 RNA 벡터는 Cry1 및 Cry2를 동시에 표적하는 것을 특징으로 하는, 가이드 RNA 벡터.According to clause 5,
The guide RNA vector is characterized in that it simultaneously targets Cry1 and Cry2.
Cas9(CRISPR associated protein 9) 핵산분해효소;를 포함하는, 생체시계 교란 유도용 CRISPR/Cas9 복합체.The guide RNA vector of claim 5; and
A CRISPR/Cas9 complex for inducing circadian clock disruption, including Cas9 (CRISPR associated protein 9) nuclease.
상기 생체시계 관련 유전자는 Cry1(Cryptochrome Circadian Regulator 1) 또는 Cry2(Cryptochrome Circadian Regulator 2)인 것을 특징으로 하는, 생체시계 교란 유도된 동물 모델.An animal model induced by circadian clock disruption in which circadian clock-related genes are knocked out in the suprachiasmatic nucleus of wild type,
An animal model induced by circadian clock disruption, wherein the circadian clock-related gene is Cry1 (Cryptochrome Circadian Regulator 1) or Cry2 (Cryptochrome Circadian Regulator 2).
상기 생체시계 교란 유도된 동물 모델은 생체시계 관련 유전자가 발현하는 단백질의 수준이 감소하는 것을 특징으로 하는, 생체시계 교란 유도된 동물 모델. According to clause 11,
The biological clock disruption-induced animal model is characterized by a decrease in the level of proteins expressed by biological clock-related genes.
(a) 제 11항의 생체시계 교란 유도된 동물모델에 후보물질을 처리하는 단계; 및
(b) 후보물질이 처리된 생체시계 교란 유도된 동물모델을 무처리 대조군과 비교하여 주기적 특성의 회복 여부를 확인하는 단계.A method for screening circadian clock correction agents, comprising the following steps:
(a) treating the candidate material in the biological clock disruption-induced animal model of Article 11; and
(b) Comparing the biological clock disruption-induced animal model treated with the candidate substance with the untreated control group to determine whether cyclical characteristics are recovered.
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| JP2011036143A (en) | 2009-08-06 | 2011-02-24 | Hitoshi Okamura | Medicine for body dysrhythmia and method for screening the same |
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