Tutorial: design and execution of CRISPR in vivo screens

Area of Science:

• * Molecular Biology
• * Genomics
• * Mammalian Genetics

Background:

• * CRISPR screening has revolutionized genetic research, enabling large-scale functional genomics.
• * In vivo applications are crucial for understanding gene function within a whole organismal context.
• * Challenges in in vivo screening require careful methodological considerations.

Purpose of the Study:

• * To provide a comprehensive tutorial on CRISPR in vivo screening methodologies.
• * To guide researchers in designing and executing effective in vivo screens in mice.
• * To highlight potential pitfalls and solutions for successful CRISPR in vivo screening.

Main Methods:

• * Detailed explanation of CRISPR tools and their applications in vivo.
• * Guidance on designing transplantation-based and direct in vivo screens.
• * Instruction on technical execution, sequencing, computational analysis, and data interpretation.

Main Results:

• * Demonstration of interrogating diverse in vivo phenotypes, including organ development and oncogenesis.
• * Strategies for overcoming common in vivo screening limitations like delivery and library bottlenecking.
• * Successful application of CRISPR screening to address complex biological questions.

Conclusions:

• * CRISPR in vivo screening offers powerful new avenues for dissecting complex organismal phenotypes.
• * Methodological rigor is essential to avoid screening failures and ensure valid conclusions.
• * Emerging applications, such as genetic interaction screening, promise further advancements.