Analysis of the Role of Homology Arms in Gene-Targeting Vectors in Human Cells

Abstract

Random integration of targeting vectors into the genome is the primary obstacle in human somatic cell gene targeting. Non-homologous end-joining (NHEJ), a major pathway for repairing DNA double-strand breaks, is thought to be responsible for most random integration events; however, absence of DNA ligase IV (LIG4), the critical NHEJ ligase, does not significantly reduce random integration frequency of targeting vector in human cells, indicating robust integration events occurring via a LIG4-independent mechanism. To gain insights into the mechanism and robustness of LIG4-independent random integration, we employed various types of targeting vectors to examine their integration frequencies in LIG4-proficient and deficient human cell lines. We find that the integration frequency of targeting vector correlates well with the length of homology arms and with the amount of repetitive DNA sequences, especially SINEs, present in the arms. This correlation was prominent in LIG4-deficient cells, but was also seen in LIG4-proficient cells, thus providing evidence that LIG4-independent random integration occurs frequently even when NHEJ is functionally normal. Our results collectively suggest that random integration frequency of conventional targeting vectors is substantially influenced by homology arms, which typically harbor repetitive DNA sequences that serve to facilitate LIG4-independent random integration in human cells, regardless of the presence or absence of functional NHEJ.

Advances in the Development of Gene-Targeting Vectors to Increase the Efficiency of Genetic Modification

“…Recently, we examined the relationship between the length of homology arms and targeting efficiency using a human pre-B lymphoma cell line, Nalm-6. 20) Our data showed that the frequency of targeted integration increased with increase in arm length, but so did the frequency of random integration. 20) Therefore, shorter arms may be preferable for suppressing random integration; however, in some cases, there is no other option but to design a vector with long arms.…”

“…20) Our data showed that the frequency of targeted integration increased with increase in arm length, but so did the frequency of random integration. 20) Therefore, shorter arms may be preferable for suppressing random integration; however, in some cases, there is no other option but to design a vector with long arms. The vector design strategy is discussed in detail in Section 4.…”

“…The data showed that random integration occurs more frequently when vectors with longer arms are used, particularly when the vectors contain large amounts of repetitive DNA sequences (which include, but are not limited to, short interspersed nucleotide elements (SINEs) and long interspersed nucleotide elements (LINEs)). 20) This finding suggests that the presence of repetitive DNA sequences may promote random integration, presumably via alternative NHEJ (alt-NHEJ), which is an alternative DSB repair pathway. As the alt-NHEJ pathway typically involves 5′-end resection of a DSB (see Fig.…”

“…20) We therefore suggest that homology arms should ideally contain as few repetitive sequences as possible. In Nalm-6 cells, gene targeting is feasible even with the use of vectors with 4-5-kb homology arms.…”

Dual loss of human POLQ and LIG4 abolishes random integration

“…In several fungi, inactivation of the non-homologous end joining (NHEJ) pathway results in ∼100% gene targeting by virtue of undetectable levels of RI4, but unfortunately this is not the case in mammalian cells; for example, loss of DNA ligase IV (Lig4), which is indispensable for NHEJ5, can reduce RI of foreign DNA, but does not reduce RI of targeting vectors harbouring long homology arms678. Thus, the exact contribution of NHEJ to RI is still uncertain and undoubtedly an NHEJ-independent route for RI does exist.…”