CRISPR Landing Pads for Large DNA Payload Insertion

The method divides the genome editing process into two distinct stages: first, CRISPR nucleases insert small landing pad sequences (approximately 100 bp or less) at target genomic locations; second, separate recombinase or integrase enzymes insert large DNA payloads (greater than 100 bp) into these pre-established landing pads. This segmentation allows each enzymatic system to operate within its optimal size range while achieving the combined capability of high-precision targeting and large payload insertion.

The CRISPR-mediated insertion of landing pads serves as a preliminary action that prepares the genomic target sites before the actual large payload insertion. By first establishing recognition sequences (landing pads) at the desired genomic locations, the system creates ready-made docking sites that subsequent recombinase or integrase enzymes can recognize and utilize for inserting large DNA sequences, thereby enabling payloads much larger than what CRISPR alone could insert.

The complex genome editing process is segmented into two independent, sequential steps performed with different enzymatic systems. The first step uses high-throughput CRISPR-Cas nucleases to insert landing pads, while the second step uses lower-throughput recombinases or integrases to insert large payloads. This segmentation allows each step to be optimized independently and simplifies the overall process design compared to attempting to use a single system for both tasks.

The landing pad sequence serves as an intermediary element that bridges the two different enzymatic systems. It is first inserted by CRISPR nucleases and then serves as the recognition target for subsequent recombinase or integrase-mediated payload insertion. This intermediary structure enables compatibility between the high-throughput CRISPR system and the large-payload-capable recombinase/integrase systems, thereby managing process complexity while achieving large payload insertion.

The method segments the editing process into two independent phases that can be performed in parallel across multiple target loci. In the first phase, multiple CRISPR guide RNAs direct simultaneous insertion of landing pads at numerous genomic locations across the cell population. In the second phase, recombinase or integrase systems simultaneously insert large payloads into these distributed landing pads. This segmented approach maintains precision at each target site while enabling high-throughput multiplex editing across the genome.

The landing pad sequence design provides universality across multiple target loci. Each landing pad contains standardized recognition sequences for recombinases or integrases, allowing the same payload insertion machinery to operate at numerous different genomic locations. This universal design enables precise, identical editing operations to be performed simultaneously at multiple targets throughout the genome, maintaining edit precision while achieving high productivity through multiplexing.