Efficient Generation of Single- and Multigene CRISPR-Cas9 Knockout in Human Cells

The CRISPR-Cas9 system has revolutionized genome editing, but generating multiple gene knockouts (multi-KOs) remains challenging. This study presents an improved method for efficient generation of multi-KOs using sgRNA DNA strands and the CRISPR/Cas9 system. The method was tested on genes involved in the apoptosis pathway of HEK293T cells. The crRNA for each target gene was designed from Exon 1, near the starting codon ATG, such that a small deletion or insertion in this region would lead to a premature STOP codon and functional knockout. The T7 endonuclease I assay confirmed the high editing efficiency of sgRNAs in both single- and multiple-gene knockouts. Deep sequencing analysis revealed frameshift mutations and premature stop codons, indicating successful loss-of-function mutations. Using the sgRNA DNA strands provided a more uniform and efficient editing profile compared to other methods. This improved protocol offers a simpler, faster, and cost-effective approach for generating multigene knockout clones, with implications for functional genomics, therapeutic development, and synthetic biology.