High-fidelity CRISPRCas9 nucleases with no detectable genome-wide off-target effects

By: Benjamin P. Kleinstiver, Vikram Pattanayak, Michelle S. Prew1, Shengdar Q. Tsai, Nhu T.

Nguyen, Zongli Zheng & J. Keith Joung

CRISPR-Cas9s tendency to not target the right gene can severely hinder research. This

report focuses on the effects of Cas9 mutants on the editing process, specifically SpCas9. The

authors hypothesize that the SpCas9-sgRNA complex contains more energy that it needs, which

causes it to use that energy on off-target sites, and by disrupting the DNA (mutation). By doing

this the SpCas9 will be altered in terms of energy, but it will still contain enough to carry out on-

target activity but will not have enough for off-target activity. The authors first tested their

hypothesis by creating 15 SpCas9 variants with single, double, triple, and quadruple alanine

substitutions and assessed it with the wild-type SpCas9 using a gene for a fluorescent protein

(EGFP) to mark any changes. These variants had the same amount of activity as the wild-type.

The authors substituted alanine in the residues of these variants. To assess the amount of activity

at off-target sites, the authors used EGFP again and used all substitutes. They found that the

quadruple substitute (SpCas9-HF1) had the best results, so they tested it further. This variant

demonstrated over 70% of the on-target activity of the wild type. The authors then tested this

variant on human genes (EMX1, FANCF, RUNX1, and ZSCAN2). Including the wild-type,

seven out of the eight variants tested had variable off-target sites on each gene. The only variant

that did not have any off-target sites was the one used for FANCF site 4. No new off-target sites

were created with any of these variants. In order to make sure this was valid, the authors

extended the experiment to measure the frequency of off-target activity. These experiments

concluded that both the wild type and the variation were able to function as replicates, the wild
type has high off-target activity, and it is difficult to distinguish the off-target indels for the

variations at the off-target sites. There were two sites that were off-target by only a few, but it

was hard to tell if it was a sequencing error or it was actually reduced by the complex. Next, this

variation was tested in a genome-wide experiment that involved a human gene called VEGFA.

SpCas9 was able to reduce off-target sites and on-target activity was not affected by the sgRNAs

being used to target the different VEGFA sites. The authors then tested another hypothesis that

involved combining SpCas9-HF1 with another SpCas9 variant (D1135E) and combining another

with a truncated (shortened) sgRNA sequence, respectively. The first experiment showed that

combining these two variants caused SpCas9-HF1 to retain most of its on-target activity while

truncating the sgRNA in the second experiment decreased SpCas9s ability to retain on-target

activity. Three other variants were created (SpCas9-HF2-HF3-HF4) and tested to see if they

could reduce off-target activity at the same sites that HF1 was tested on. On the FANCF site,

HF4 was able to reduce off-target activity and increase specificity, and on the VEGFA site, HF2

increased specificity. More research will be needed in order to create a strategy for making high-

fidelity Cas9 variants.

Creating and evaluating accurate CRISPR-Cas9 scalpels for genomic surgery

By: Mehmet Fatih Bolukbasi, Ankit Gupta & Scot A Wolfe

The CRISPR-Cas9 system is only found in a small number of bacteria species and is an

amazing adaptation for these species, as they can effectively fend off viruses. This system has

also been adapted for gene editing in other species, mainly mammalian species. This can be done

by coding an sgRNA sequence that will guide the Cas9 to its target where it deletes the gene by

cutting it via DSBs. Cas9 has also been effective in inserting a new gene using a template that

has been provided. This process is called a homology-directed-repair (HDR). When using Cas9,

it is important to make it as precise as possible in order to prevent any unwanted mutations that

can potentially alter the function of the gene. Predicting off-target frequency and sites are

possible with licensing. The information provided for this process are from cell cultures and

previous experiments. In order for a DNA sequenced to be licensed, there has to be a favorable

PAM sequence and a successful separation of the DNA strands based on Watson-Crick base

pairing. The PAM sequence is essential in the structure of the complex because it prevents it

from forming a heteroduplex structure with the DNA. Profiles have shown that the activity level

of Cas9 is affected by the number and position of the mismatch. There are some specific sites

that seem to have higher off-target activity than others.

There are several ways to calculate the number of off-target sites. Before sequencing was

developed, computational algorithms were used to detect any off-target sites. The algorithms

used today are practical for finding sequences that can reduce off-target activity. However, these

algorithms lead to a decrease in the density of available target sequences, and more future work
would be needed to find more sequences. In order to make accurate programs, computational

algorithms will need to be combined with GUIDE-seq. Another method used for determining off-

target sites involves using viruses and high-throughput, genome-wide translocation sequencing to

find breaks that were made by Cas9. Researchers can also manipulate the specificity of Cas9 by

using the PAM-interacting domain. When used with SpCas9, it also increases the genome-wide

precision of this variant, making it ideal for all kinds of treatments.