A hypomorphic mutation in the mouse Csn1s1 gene generated by CRISPR/Cas9 pronuclear microinjection

A. V. Smirnov; T. Shnaider; A. N. Korablev; A. M. Yunusova; I. A. Serova; N. R. Battulin

doi:10.18699/VJ21.036

A hypomorphic mutation in the mouse Csn1s1 gene generated by CRISPR/Cas9 pronuclear microinjection

Переведенное название: Гипоморфная мутация гена Csn1s1 мыши, полученная пронуклеарной микроинъекцией CRISPR/Сas9

A. V. Smirnov, T. Shnaider, A. N. Korablev, A. M. Yunusova, I. A. Serova, N. R. Battulin

Результат исследования: Научные публикации в периодических изданиях › статья › рецензирование

Аннотация

Казеины - это основные молочные белки, которые играют важную эволюционно консервативную роль в питании. Вариации последовательности казеиновых генов влияют на состав молока у животных. Регуляторные элементы казеиновых генов можно использовать для управления экспрессией желаемых трансгенов в молоке трансгенных животных. Десятки аллелей казеина идентифицированы у коз, коров, овец, верблюдов и лошадей, и эти варианты связаны с измененной экспрессией генов и содержанием молочного белка. Большинство известных мутаций, влияющих на экспрессию генов казеина, находится в промоторных и 3’-нетранслируемых областях. Мы выполнили пронуклеарные микроинъекции с мРНК Cas9 и sgRNA против первого кодирующего экзона гена Csn1s1 мыши, чтобы ввести случайные мутации в последовательность сигнального пептида α-казеина ( Csn1s1 ) в начале гена мыши. Секвенирование мышей-основателей по Сэнгеру выявило 40 мутаций. Как и ожидалось, мутации группировались вокруг сайта разреза sgRNA (3 п. н. от PAM). Большинство мутаций представляют собой небольшие делеции (1-10 п. н.), но мы также обнаружили несколько более крупных делеций (100-300 п. н.). Функционально большинство мутаций приводило к нокаутам генов из-за сдвига рамки считывания или потери стартовых кодонов. Некоторые из мутаций представлены инделами в рамке считывания в первом кодирующем экзоне. Из них мы описываем новый гипоморфный аллель Csn1s1 ( Csn1s1 c.4-5insTCC). Мы измерили уровни белка Csn1s1 и подтвердили, что мутация отрицательно влияет на состав молока, который показывает снижение экспрессии гена на 50 % и уменьшение количества белка Csn1s1 на 40-80 % по сравнению с аллелем дикого типа. Мы предположили, что мутация влияет на стабильность транскрипта или сплайсинг по неизвестному механизму. Эта мутация потенциально может служить генетическим маркером низкой экспрессии Csn1s1.

Переведенное название	Гипоморфная мутация гена Csn1s1 мыши, полученная пронуклеарной микроинъекцией CRISPR/Сas9
Язык оригинала	английский
Номер статьи	10
Страницы (с-по)	331-336
Число страниц	6
Журнал	Vavilovskii Zhurnal Genetiki i Selektsii
Том	25
Номер выпуска	3
DOI	https://doi.org/10.18699/VJ21.036
Состояние	Опубликовано - мар 2021

Ключевые слова

Casein
CRISPR
Hypomorphic mutation
Pronuclear microinjection

Предметные области OECD FOS+WOS

4 СЕЛЬСКОХОЗЯЙСТВЕННЫЕ НАУКИ
1.06 БИОЛОГИЧЕСКИЕ НАУКИ
4.01 СЕЛЬСКОЕ ХОЗЯЙСТВО, ЛЕСОНОЕ ХОЗЯЙСТВО, РЫБНОЕ ХОЗЯЙСТВО
1.06.KM ГЕНЕТИКА И НАСЛЕДСТВЕННОСТЬ

ГРНТИ

34 БИОЛОГИЯ

Доступ к документу

10.18699/VJ21.036

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title = "A hypomorphic mutation in the mouse Csn1s1 gene generated by CRISPR/Cas9 pronuclear microinjection",

abstract = "Caseins are major milk proteins that have an evolutionarily conserved role in nutrition. Sequence variations in the casein genes affect milk composition in livestock species. Regulatory elements of the casein genes could be used to direct the expression of desired transgenes into the milk of transgenic animals. Dozens of casein alleles have been identified for goats, cows, sheep, camels and horses, and these sequence variants are associated with altered gene expression and milk protein content. Most of the known mutations affecting casein genes' expression are located in the promoter and 3'-un-translated regions. We performed pronuclear microinjections with Cas9 mRNA and sgRNA against the first coding exon of the mouse Csn1s1 gene to introduce random mutations in the α-casein (Csn1s1) signal peptide sequence at the beginning of the mouse gene. Sanger sequencing of the founder mice identified 40 mutations. As expected, mutations clustered around the sgRNA cut site (3 bp from PAM). Most of the mutations represented small deletions (1-10 bp), but we detected several larger deletions as well (100-300 bp). Functionally most mutations led to gene knockout due to a frameshift or a start codon loss. Some of the mutations represented in-frame indels in the first coding exon. Of these, we describe a novel hypomorphic Csn1s1 (Csn1s1c.4-5insTCC) allele. We measured Csn1s1 protein levels and confirmed that the mutation has a negative effect on milk composition, which shows a 50 % reduction in gene expression and a 40-80 % decrease in Csn1s1 protein amount, compared to the wild-type allele. We assumed that mutation affected transcript stability or splicing by an unknown mechanism. This mutation can potentially serve as a genetic marker for low Csn1s1 expression.",

keywords = "Casein, CRISPR, Hypomorphic mutation, Pronuclear microinjection",

author = "Smirnov, {A. V.} and T. Shnaider and Korablev, {A. N.} and Yunusova, {A. M.} and Serova, {I. A.} and Battulin, {N. R.}",

note = "Funding Information: The authors are grateful to the Centre for Genetic Resources of Laboratory Animals at the Institute of Cytology and Genetics, SB RAS (RFMEFI61914X0005 and RFMEFI61914X0010) for help with some animal procedures. The authors were supported by Russian Science Foundation (16-14-00095). Data analysis was performed on computational nodes of the Institute of Cytology and Genetics (budget project No. 0259-2021-0016). Publisher Copyright: {\textcopyright} Smirnov A.V., Shnaider T.А., Korablev A.N., Yunusova A.M., Serova I.A., Battulin N.R., 2021 This work is licensed under a Creative Commons Attribution 4.0 License",

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volume = "25",

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A hypomorphic mutation in the mouse Csn1s1 gene generated by CRISPR/Cas9 pronuclear microinjection. / Smirnov, A. V.; Shnaider, T.; Korablev, A. N.; Yunusova, A. M.; Serova, I. A.; Battulin, N. R.

В: Vavilovskii Zhurnal Genetiki i Selektsii, Том 25, № 3, 10, 03.2021, стр. 331-336.

Результат исследования: Научные публикации в периодических изданиях › статья › рецензирование

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T1 - A hypomorphic mutation in the mouse Csn1s1 gene generated by CRISPR/Cas9 pronuclear microinjection

AU - Smirnov, A. V.

AU - Shnaider, T.

AU - Korablev, A. N.

AU - Yunusova, A. M.

AU - Serova, I. A.

AU - Battulin, N. R.

N1 - Funding Information: The authors are grateful to the Centre for Genetic Resources of Laboratory Animals at the Institute of Cytology and Genetics, SB RAS (RFMEFI61914X0005 and RFMEFI61914X0010) for help with some animal procedures. The authors were supported by Russian Science Foundation (16-14-00095). Data analysis was performed on computational nodes of the Institute of Cytology and Genetics (budget project No. 0259-2021-0016). Publisher Copyright: © Smirnov A.V., Shnaider T.А., Korablev A.N., Yunusova A.M., Serova I.A., Battulin N.R., 2021 This work is licensed under a Creative Commons Attribution 4.0 License

PY - 2021/3

Y1 - 2021/3

N2 - Caseins are major milk proteins that have an evolutionarily conserved role in nutrition. Sequence variations in the casein genes affect milk composition in livestock species. Regulatory elements of the casein genes could be used to direct the expression of desired transgenes into the milk of transgenic animals. Dozens of casein alleles have been identified for goats, cows, sheep, camels and horses, and these sequence variants are associated with altered gene expression and milk protein content. Most of the known mutations affecting casein genes' expression are located in the promoter and 3'-un-translated regions. We performed pronuclear microinjections with Cas9 mRNA and sgRNA against the first coding exon of the mouse Csn1s1 gene to introduce random mutations in the α-casein (Csn1s1) signal peptide sequence at the beginning of the mouse gene. Sanger sequencing of the founder mice identified 40 mutations. As expected, mutations clustered around the sgRNA cut site (3 bp from PAM). Most of the mutations represented small deletions (1-10 bp), but we detected several larger deletions as well (100-300 bp). Functionally most mutations led to gene knockout due to a frameshift or a start codon loss. Some of the mutations represented in-frame indels in the first coding exon. Of these, we describe a novel hypomorphic Csn1s1 (Csn1s1c.4-5insTCC) allele. We measured Csn1s1 protein levels and confirmed that the mutation has a negative effect on milk composition, which shows a 50 % reduction in gene expression and a 40-80 % decrease in Csn1s1 protein amount, compared to the wild-type allele. We assumed that mutation affected transcript stability or splicing by an unknown mechanism. This mutation can potentially serve as a genetic marker for low Csn1s1 expression.

AB - Caseins are major milk proteins that have an evolutionarily conserved role in nutrition. Sequence variations in the casein genes affect milk composition in livestock species. Regulatory elements of the casein genes could be used to direct the expression of desired transgenes into the milk of transgenic animals. Dozens of casein alleles have been identified for goats, cows, sheep, camels and horses, and these sequence variants are associated with altered gene expression and milk protein content. Most of the known mutations affecting casein genes' expression are located in the promoter and 3'-un-translated regions. We performed pronuclear microinjections with Cas9 mRNA and sgRNA against the first coding exon of the mouse Csn1s1 gene to introduce random mutations in the α-casein (Csn1s1) signal peptide sequence at the beginning of the mouse gene. Sanger sequencing of the founder mice identified 40 mutations. As expected, mutations clustered around the sgRNA cut site (3 bp from PAM). Most of the mutations represented small deletions (1-10 bp), but we detected several larger deletions as well (100-300 bp). Functionally most mutations led to gene knockout due to a frameshift or a start codon loss. Some of the mutations represented in-frame indels in the first coding exon. Of these, we describe a novel hypomorphic Csn1s1 (Csn1s1c.4-5insTCC) allele. We measured Csn1s1 protein levels and confirmed that the mutation has a negative effect on milk composition, which shows a 50 % reduction in gene expression and a 40-80 % decrease in Csn1s1 protein amount, compared to the wild-type allele. We assumed that mutation affected transcript stability or splicing by an unknown mechanism. This mutation can potentially serve as a genetic marker for low Csn1s1 expression.

KW - Casein

KW - CRISPR

KW - Hypomorphic mutation

KW - Pronuclear microinjection

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