Lentiviruses have been widely used as a means of transferring exogenous DNAs into human cells to treat various genetic diseases. Lentiviral vectors are fundamentally integrated into the host genome, but their integration sites are generally unpredictable, which may increase the uncertainty for their use in therapeutics. To determine the viral integration sites in the host genome, several PCR-based methods have been developed. However, the sensitivities of the PCR-based methods are highly dependent on the primer sequences, and optimized primer design is required for individual target sites. In order to address this issue, we developed an alternative method for genomewide mapping of viral insertion sites, named CReVIS-seq (CRISPR-enhanced Viral Integration Site Sequencing). The method is based on the sequential steps: fragmentation of genomic DNAs, in vitro circularization, cleavage of target sequence in a CRISPR guide RNA-specific manner, highthroughput sequencing of the linearized DNA fragments in an unbiased manner, and identification of viral insertion sites via sequence analysis. By design, CReVIS-seq is not affected by biases that could be introduced during the target enrichment step via PCR amplification using site specific primers. Furthermore, we found that multiplexed CReVIS-seq, using collections of different single-guide RNAs (sgRNAs), enables simultaneous identification of multiple target sites and structural variations (i.e., circularized viral genome), in both single cell clones and heterogeneous cell populations.
The clustered regularly interspaced short palindromic repeats (CRISPR) system is a family of DNA sequences originally discovered as a type of acquired immunity in prokaryotes such as bacteria and archaea. In many CRISPR systems, the functional ribonucleoproteins (RNPs) are composed of CRISPR protein and guide RNAs. They selectively bind and cleave specific target DNAs or RNAs, based on sequences complementary to the guide RNA. The specific targeted cleavage of the nucleic acids by CRISPR has been broadly utilized in genome editing methods. In the process of genome editing of eukaryotic cells, CRISPR-mediated DNA double-strand breaks (DSB) at specific genomic loci activate the endogenous DNA repair systems and induce mutations at the target sites with high efficiencies. Two of the major endogenous DNA repair machineries are non-homologous end joining (NHEJ) and homology-directed repair (HDR). In case of DSB, the two repair pathways operate in competition, resulting in several possible outcomes including deletions, insertions, and substitutions. Due to the inherent stochasticity of DSB-based genome editing methods, it was difficult to achieve defined single-base changes without unanticipated random mutation patterns. In order to overcome the heterogeneity in DSB-mediated genome editing, novel methods have been developed to incorporate precise single-base level changes without inducing DSB. The approaches utilized catalytically compromised CRISPR in conjunction with base-modifying enzymes and DNA polymerases, to accomplish highly efficient and precise genome editing of single and multiple bases. In this review, we introduce some of the advances in single-base level CRISPR genome editing methods and their applications.
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