Science – RNA-Guided Human Genome Engineering via Cas9
Bacteria and archaea have evolved adaptive immune defenses termed clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems that use short RNA to direct degradation of foreign nucleic acids. Here, we engineer the type II bacterial CRISPR system to function with custom guide RNA (gRNA) in human cells. For the endogenous AAVS1 locus, we obtained targeting rates of 10 to 25% in 293T cells, 13 to 8% in K562 cells, and 2 to 4% in induced pluripotent stem cells. We show that this process relies on CRISPR components, is sequence-specific, and upon simultaneous introduction of multiple gRNAs, can effect multiplex editing of target loci. We also compute a genome-wide resource of ~190k unique gRNAs targeting ~40.5% of human exons. Our results establish an RNA-guided editing tool for facile, robust, and multiplexable human genome engineering.
CRISPR needs 20 base pairs of RNA for targeting. The previous best genetic engineering method TALE Nuclease used 2000 base pairs for targeting and was about 0.37% accurate for targeting.
CRISPR is 100 times easier to create the targeting and 10 to 20 times more effective at targeting.
Beneficial gene therapy for HIV cure in phase 2 human trials
There is a phase 2 clinical trial to genetically modify t-cells to create immunity to HIV. Tim Brown, the famous “Berlin patient” and first person cured of HIV. Brown’s visit and Sangamo’s clinical trial results draw attention to human gene therapy with beneficial mutations. In Sangamo’s case, its scientists generate mutations in the CCR5 gene in human CD4 T cells that conferred resistance to HIV-1, the most common strain of the virus. Brown was cured when he received donated CD4 T cells with a naturally occurring CCR5 mutation. The Richmond, Calif.-based Sangamo has touted results at the one-year clinical trial endpoint: in five of nine subjects, CD4 T cell counts persisted a year after infusion at greater than 500 cells/mm3, the accepted threshold for initiation of HAART therapy.
CRISPR will allow large scale genetic editing for a lot of beneficial mutations.
Mutations for viral immunity, longevity and other mass changes to stem cells extracted and modified and then reintroduced to the body.
Thousands of changes could be made and then copied for millions of stem cells.
This method could be used for a transhuman future with radical life extension and other genetic and epigenetic changes.
George Church described these goals in his latest book Regenesis and a Discovery Magazine article.
34 pages of supplemental information.
We sought to generate a set of gRNA gene sequences that maximally target specific
locations in human exons but minimally target other locations in the genome. Maximally efficient targeting by a gRNA is achieved by 23nt sequences, the 5’‐most 20nt of which exactly complement a desired location, while the three 3’‐most bases must be of the form NGG. Additionally, the 5’‐most nt must be a G to establish a pol‐III transcription start site. However, according to (4), mispairing of the six 5’‐most nt of a 20bp gRNA against its genomic target does not abrogate Cas9‐mediated cleavage so long as the last 14nt pairs properly, but mispairing of the eight 5’‐most nt along with pairing of the last 12 nt does, while the case of the seven 5‐most nt mispairs and 13 3’ pairs was not tested. To be conservative regarding off‐target effects, we therefore assumed that the case of the seven 5’‐most mispairs is, like the case of six, permissive of cleavage, so that pairing of the 3’‐most 13nt is sufficient for cleavage. To identify CRISPR target sites within human exons that should be cleavable without off‐target cuts.
To assess targeting at a gene level, we clustered RefSeq mRNA mappings so that any two RefSeq accessions (including the gene duplicates we distinguished in (ii)) that overlap a merged exon region are counted as a single gene cluster, the 189864 exonic specific CRISPR sites target 17104 out of 18872 gene clusters (~90.6% of all gene clusters) at a multiplicity of ~11.1 per targeted gene cluster.
As we gather information on CRISPR performance at our computationally predicted
human exon CRISPR target sites, we plan to refine our database by correlating performance with factors we expect to be important, such as base composition and secondary structure of both gRNAs and genomic targets and the epigenetic state of these targets in human cell lines for which this information is available.
Finally, we also incorporated these target sequences into a 200bp format that is
compatible for multiplex synthesis on DNA arrays (14, 46). Our design allows for targeted retrieval of a specific or pools of gRNA sequences from the DNA array based oligonucleotide pool and its rapid cloning into a common expression vector (fig. S11A, Table S2). Specifically we tested this approach by synthesizing a 12k oligonucleotide pool from CustomArray Inc. (Table S3). Furthermore, as per our approach we were able to successfully retrieve gRNAs of choice from this library. We observed an error rate of ~4 mutations per 1000bp of synthesized DNA
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