Systematic optimization and prediction of cre recombinase for precise genome editing in mice DOI Creative Commons

Valerie Erhardt,

Elli Hartig,

Kristian Lorenzo

и другие.

Genome biology, Год журнала: 2025, Номер 26(1)

Опубликована: Апрель 4, 2025

The Cre-Lox system is a powerful tool in mouse genetics, enabling precise spatiotemporal control of gene expression and conditional knockout models. Since its development, it has transformed genome editing by facilitating targeted deletions, translocations, inversions, complex modifications-double-floxed inverse orientation. Its utility extends beyond mice to rats, pigs, zebrafish. However, challenges such as high costs, lengthy timelines, unpredictable recombination remain, highlighting the need for ongoing improvements enhance efficiency, reliability, applicability across genetic In this study, we perform systematic analysis Cre-mediated mice, creating 11 new strains with alleles at Rosa26 locus, using C57BL/6J background. Factors influencing efficiency include inter-loxP distance, mutant loxP sites, zygosity, chromosomal location, breeder age. Our results demonstrate that choice Cre-driver strain plays significant role efficiency. Optimal achieved when sites are spaced less than 4 kb 3 kb. Complete fails wildtype ≥ 15 or lox71/66 7 best observed breeders aged 8-20 weeks heterozygous floxed alleles. remains indispensable engineering, offering flexibility standalone applications integrating CRISPR-based methods expand utility. Despite challenges, our findings provide framework optimizing recombination. By refining strategies, knowledge enhances experimental precision, improves reproducibility, ultimately reduces time cost modification.

Язык: Английский

A Myosin Nanomotor Essential for Stereocilia Maintenance Expands the Etiology of Hereditary Hearing Loss DFNB3 DOI Creative Commons

Ghazaleh Behnammanesh,

Abigail K. Dragich,

Xiayi Liao

и другие.

bioRxiv (Cold Spring Harbor Laboratory), Год журнала: 2025, Номер unknown

Опубликована: Фев. 21, 2025

ABSTRACT Cochlear hair cells transduce sound using stereocilia, and disruption to these delicate mechanosensors is a significant cause of hearing loss. Stereocilia architecture dependent upon the nanomotor myosin 15. A short isoform (MYO15A-2) drives stereocilia development by delivering an elongation-promoting complex (EC) tips, alternatively spliced long (MYO15A-1) tunes postnatal size in shorter which possess mechanosensitive ion channels. Disruption functions causes two distinct pathologies, underly human autosomal recessive non-syndromic loss DFNB3. Here, we characterize new isoform, MYO15A-3, that increases expression as developmental MYO15A-2 wanes reciprocally. We show critical EC initially delivered MYO15A-2, followed handover continues deliver EC. In Myo15a-3 mutant mouse, develop normally with correct targeting, but lack postnatally do not maintain their adult architecture, leading progressive conclude MYO15A-3 delivers are both required integrity. Our results add spectrum pathology underlying DFNB3 reveal molecular mechanisms necessary for resilient during life.

Язык: Английский

Процитировано

1
Systematic optimization and prediction of cre recombinase for precise genome editing in mice DOI Creative Commons

Valerie Erhardt,

Elli Hartig,

Kristian Lorenzo

и другие.

Genome biology, Год журнала: 2025, Номер 26(1)

Опубликована: Апрель 4, 2025

The Cre-Lox system is a powerful tool in mouse genetics, enabling precise spatiotemporal control of gene expression and conditional knockout models. Since its development, it has transformed genome editing by facilitating targeted deletions, translocations, inversions, complex modifications-double-floxed inverse orientation. Its utility extends beyond mice to rats, pigs, zebrafish. However, challenges such as high costs, lengthy timelines, unpredictable recombination remain, highlighting the need for ongoing improvements enhance efficiency, reliability, applicability across genetic In this study, we perform systematic analysis Cre-mediated mice, creating 11 new strains with alleles at Rosa26 locus, using C57BL/6J background. Factors influencing efficiency include inter-loxP distance, mutant loxP sites, zygosity, chromosomal location, breeder age. Our results demonstrate that choice Cre-driver strain plays significant role efficiency. Optimal achieved when sites are spaced less than 4 kb 3 kb. Complete fails wildtype ≥ 15 or lox71/66 7 best observed breeders aged 8-20 weeks heterozygous floxed alleles. remains indispensable engineering, offering flexibility standalone applications integrating CRISPR-based methods expand utility. Despite challenges, our findings provide framework optimizing recombination. By refining strategies, knowledge enhances experimental precision, improves reproducibility, ultimately reduces time cost modification.

Язык: Английский

Процитировано

0