Biology subjects

Gurumurthy, C.

Publications and source records attributed to Gurumurthy, C..

3 recordsLinked to original sources

bioRxiv2018

Re-Evaluating One-step Generation of Mice Carrying Conditional Alleles by CRISPR-Cas9-Mediated Genome Editing Technology

CRISPR-Cas9 gene editing technology has considerably facilitated the generation of mouse knockout alleles, relieving many of the cumbersome and time-consuming steps of traditional mouse embryonic stem cell technology. However, the generation of conditional knockout alleles remains an important challenge. An earlier study reported up to 16% efficiency in generating conditional knockout alleles in mice using 2 single guide RNAs (sgRNA) and 2 single-stranded oligonucleotides (ssODN) (2sgRNA-2ssODN). We re-evaluated this method from a large data set generated from a consortium consisting of 17 transgenic core facilities or laboratories or programs across the world. The dataset constituted 17,887 microinjected or electroporated zygotes and 1,718 live born mice, of which only 15 (0.87%) mice harbored 2 correct LoxP insertions in cis configuration indicating a very low efficiency of the method. To determine the factors required to successfully generate conditional alleles using the 2sgRNA-2ssODN approach, we performed a generalized linear regression model. We show that factors such as the concentration of the sgRNA, Cas9 protein or the distance between the placement of LoxP insertions were not predictive for the success of this technique. The major predictor affecting the methods success was the probability of simultaneously inserting intact proximal and distal LoxP sequences, without the loss of the DNA segment between the two sgRNA cleavage sites. Our analysis of a large data set indicates that the 2sgRNA-2ssODN method generates a large number of undesired alleles (>99%), and a very small number of desired alleles (<1%) requiring, on average 1,192 zygotes.

genetics↗

bioRxiv2017

Correction of autoimmune IL2RA mutations in primary human T cells using non-viral genome targeting

Human T cells are central to physiological immune homeostasis, which protects us from pathogens without collateral autoimmune inflammation. They are also the main effectors in most current cancer immunotherapy strategies1. Several decades of work have aimed to genetically reprogram T cells for therapeutic purposes2-5, but as human T cells are resistant to most standard methods of large DNA insertion these approaches have relied on recombinant viral vectors, which do not target transgenes to specific genomic sites6, 7. In addition, the need for viral vectors has slowed down research and clinical use as their manufacturing and testing is lengthy and expensive. Genome editing brought the promise of specific and efficient insertion of large transgenes into target cells through homology-directed repair (HDR), but to date in human T cells this still requires viral transduction8, 9. Here, we developed a non-viral, CRISPR-Cas9 genome targeting system that permits the rapid and efficient insertion of individual or multiplexed large (>1 kilobase) DNA sequences at specific sites in the genomes of primary human T cells while preserving cell viability and function. We successfully tested the potential therapeutic use of this approach in two settings. First, we corrected a pathogenic IL2RA mutation in primary T cells from multiple family members with monogenic autoimmune disease and demonstrated enhanced signalling function. Second, we replaced the endogenous T cell receptor (TCR) locus with a new TCR redirecting T cells to a cancer antigen. The resulting TCR-engineered T cells specifically recognized the tumour antigen, with concomitant cytokine release and tumour cell killing. Taken together, these studies provide preclinical evidence that non-viral genome targeting will enable rapid and flexible experimental manipulation and therapeutic engineering of primary human immune cells.

genetics↗

bioRxiv2017

In situ genome editing method suitable for routine generation of germline modified animal models

Animal genome engineering experimental procedures involve three major steps: isolation of zygotes from pregnant females; microinjection of zygotes, and; transfer of injected zygotes into recipient females, that have been practiced for over three decades. The laboratory set ups intending to performing these procedures require to have sophisticated equipment as well as highly skilled technical personnel. Because of these reasons, animal transgenesis experiments are typically performed at centralized core facilities in most research organizations. We recently showed that all three steps, of animal transgensis, can be bypassed using a method termed GONAD (Genome-editing via Oviductal Nucleic Acids Delivery), by directly electroporating genome editing components into zygotes in situ. Although our first report demonstrated the genome-editing capability, its efficiency was lower than the standard methods using microinjection. Here we investigated critical parameters of GONAD to make it suitable for creating animal models of large genomic deletions, single nucleotide corrections and long sequence insertions. The efficiency of genome editing in the improved GONAD (i-GONAD) method reached to the levels comparable to traditional microinjection methods. The streamlined parameters, and the simplified experimental steps, in the i-GONAD method makes it suitable for routine genome editing applications performed both at centralized facilities as well as at the laboratories that lack highly skilled personnel and the sophisticated equipment.

genomics↗