Table 2.

Current methods in off-target analyses of CRISPR edited immune cells.

Off target analysis method	Definition	Pros	Cons
Cas-OFFinder (161)	It is an algorithm that searches for possible off-target sites that can be found in an already sequenced genome.	- It is not limited by the number of mismatches and the PAM sequence. - It allows alterations in PAM sequences which are differentiable with Cas9. - a rapid and highly assorted off-target searching tool available at http://www.rgenome.net/cas-offinder	- it relies on a computational method, which may result in ignoring some potential off-targets sites. - it is biased due to the assumption that off-target sequences are affiliated with the on-target site which may cause missing off-target sites in any loci throughout the genome.
SITE-Seq (selective enrichment and identification of tagged genomic DNA ends by sequencing) (159)	It is a biochemical method, using Cas9 and single-guide RNAs (sgRNAs), to recognize all the Cas9-mediated cut site sequences inside the genomic DNA.	- It allows retrieval of off-target sites with different cleavage sensitivity by utilizing a vast range of sgRNP concentrations from very low to high. - Provides guidance for precise and plenary inspection of possible off-target sites in cells by gaging the incidence of mutations and their functional cellular effects. - Production of sequencing libraries which are highly enriched for sgRNP cut sites, providing unique profiling with minimal read depth.	- DNA-repair machinery does not have a role in the process as it is performed on high molecular weight DNA.
GUIDE-seq (genome-wide, unbiased identification of DSBs enabled by sequencing) (158)	It is a PCR-based method that relies on the enteral of double-stranded oligodeoxynucleotides into the DSB caused by RNA-guided nucleases (RGN) without contributing to off-target site.	- Enables to turn out universal specificity perspective for different RGNs - Identifies the hotspots in DNA breakpoints that can take part together with RGN-induced DSBs in higher-level genomic alterations such as translocations. - Its performance on living cells enables capturing of DSBs that occur over a more extended period, thereby making it a more delicate and plenary assay.	-Relies on an integration of donor sequences, which usually happens in a low frequency. - mispriming may occur due to the annealing of PCR primers to DNA sequences apart from the ODN, resulting in PCR products that are not differentiable from products formed by primers binding to the ODN.
iGUIDE (improvement of the GUIDE-seq method) (164)	GUIDE-seq method allows mis priming artifacts to be recognizable from credible ODN integration sites by using a larger ODN (46 nt versus 34 nt).	- by using larger ODN, PCR primer binding sites can be back off from the junction of the ODN in the final PCR product and can cause mis priming events.	-It is tough to scale due to individual transfections for each target or cell source.
ChIP-seq (chromatin immunoprecipitation sequencing) (158)	It identifies the off-target binding sites by using catalytically dead Cas9 (dCas9)-gRNAs complex.	- Important for the identification of the genome-wide binding sites with dCas9 fusion proteins.	-It rarely indicates the off-target sites of cleavage caused by active Cas9 nuclease. -not effective for recognition of genome-wide, off-target cleavage sites for catalytically active RGNs. -cost and availability
CHANGE-seq (circularization for high-throughput analysis of nuclease genome-wide effects by sequencing) (160)	It is a high-throughput procedure for determining the genome-wide operations of CRISPR-Cas9 nucleases based on Tn5 mediated gDNA tagmentation in vitro.	- A simplified, susceptible, and scalable approach. - It can elucidate the genome-wide perspective of genome editing activity exquisitely sensitive. - elaborated to efficiently procreate circularized genomic DNA libraries for elucidating the genome-wide activity of genome editors by leveraging a new Tn5 tag mentation-based workflow.	-it relies on the Tn5 tagmentation of donor sequences. - Similar to SITE-Seq, the DNA repair machinery is ignored.
Churchill (162)	In clinical and population-scale genomics provides fast, decisive, scalable, and balanced parallelization tactic for the detection of human genetic mutation.	- It uses a robust comparison based on whole genome sequencing data comparing wildtype and CRISPR edited cells. - The procedure is highly scalable, authorizing full resolution of the 1000 Genomes raw sequence dataset utilizing cloud resources in a week. - It eliminates the bottlenecks of the computational sequence analysis impasse via the avail of cloud computing resources. - It matches with the amplitude of genomic data.	- Limited access to the platform and the algorithm is not publicly available yet.