CRISPR-Cas-mediated targeted genome editing in human cells

Area of Science:

• Molecular Biology
• Genetics
• Biotechnology

Background:

• The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated (Cas) systems are bacterial defense mechanisms.
• These systems utilize RNA-guided nucleases to target and degrade foreign genetic material.

Purpose of the Study:

• To present a protocol for using the Streptococcus pyogenes type II CRISPR-Cas9 system for genome editing in human cells.
• To enable sequence-specific DNA cleavage and subsequent genomic alterations.

Main Methods:

• Designing and generating custom guide RNA (gRNA) expression constructs.
• Delivering Cas9 protein and gRNA components into human cells.
• Inducing genomic alterations through nonhomologous end joining (NHEJ) or homologous recombination (HR) with a donor sequence.

Main Results:

• Demonstration of sequence-specific targeting of any genomic sequence with the N₁₉NGG motif.
• Successful implementation of multiplexed gRNA expression for targeting multiple sites.
• Efficient delivery of Cas9-gRNA into human cells for genome engineering.

Conclusions:

• The CRISPR-Cas9 system provides a highly customizable and synthesizable RNA-guided genome engineering platform.
• This technology offers a versatile tool for multiplexed human genome modification.
• The protocol facilitates precise sequence-specific alterations in the human genome.