A few key players in the biotechnology space with a high number of patents related to CRISPR are: CRISPR Therapeutics, Caribou Biosciences and Intellia Therapeutics, while those in pharmaceuticals and drug discovery space are Regeneron Pharmaceuticals, Editas Medicine, Beam Therapeutics and Metagenomi, among many others. Regeneron Pharmaceuticals, most well-known for its AAV vectors for gene delivery, in collaboration with Intellia Therapeutics is actively pursuing development of CRISPR/Cas9-based therapeutics for hemophilia A and B, 402 a type of rare bleeding disorder. Regeneron Pharmaceuticals appears to also be engaged in collaboration with Mammoth Biosciences 404 co-founded by the Nobel Laureate and CRISPR pioneer Jennifer Doudna. genome engineering applications, as it may result in the targeting and editing of off-target sites. Numerous studies have reported off-target activity at sites ranging from a single base mismatch to sites containing multiple consecutive mismatches, or even nucleotide insertions or deletions. Regardless of the mismatch tolerance of CRISPR/Cas9, most potential off-target sites do not result in dsDNA cleavage and gene editing. This might be due to existing intrinsic checkpoints in the DNA binding and cleavage mechanisms of Cas9. Notably, high-throughput profiling studies exploring off-target effects have shown that their frequency is consistently lower in vivo as compared with isolated genomic DNA. • PAM requirement: Another limitation of the technology is the requirement for a PAM near the target site, which restricts its targeting scope. SpCas9 is one of the most extensively used Cas9s with a relatively short PAM recognition site -5′NGG3′ (N is any nucleotide).