The_20Scientist_20March_202019 (1)

56 THE SCIENTIST | the-scientist.com

LAB TOOLS

ADAPTED FROM AN ILLUSTRATION BY

GREG FINDLAY

Researchers were successfully inserting

short, single-stranded DNA, so why not try

making a knock-in by inserting long, sin-

gle-stranded DNA? Indeed, the approach,

which Gurumurthy calls Easi-CRISPR

(efficient additions with ssDNA inserts

-CRISPR), boosts efficiency by 2.5

times, and using single-stranded DNA

slashes the rate of off-target insertions

100-fold in cell culture (Nat Protoc

13:195–215, 2018; Nature 559:405–09,

2018). “It is quite huge,” he says. In Guru-

murthy’s lab, Easi-CRISPR has generated

a knock-in mouse line for 9 out of every

10 genes they have tried. A collaborator

has also used it in human T cells to create

CAR-T cells, patient-specific immune cells

for fighting cancer.

TRY IT:Easi-CRISPR is far from foolproof,

Gurumurthy cautions. Sometimes the tech-

nique inserts only part of the gene. Also, he

adds, it can scramble the homology arms—

the short sequences on either side of the

gene that home it to its correct target in

the genome. And some loci are inexplica-

bly more difficult to insert than others.

Few commercial vendors design and

synthesize custom long, single-stranded

DNA. Yo u can make your own, but the sta-

bility of single-stranded DNA varies; less-

stable sequences will have lower yields, so

you may need to synthesize more of them,

says Gurumurthy.

Researchers unable to insert CRISPR

into single-cell mouse embryos can pay a

core facility to make the mice with their

DNA sequence, says Gurumurthy. Core

facilities such as his charge from $5,000–

$15,000 to generate one or two breed-

ing pairs; commercial facilities charge

$20,000–$50,000, he says.

KNOCK-IN BY NUMBERS

RESEARCHER:Greg Findlay, MD/PhD

candidate in the lab of Jay Shendure, Uni-

versity of Washington

PROJECT: Findlay and his colleagues

were aiming to improve how clinicians

interpret mutations in the breast and

ovarian cancer gene BRCA1. That gene

has thousands of variants, but research-

ers don’t know how most of them affect

its function. To study the impact of

these variants, they used a knock-in

technique they developed called satu-

ration genome editing (Nature, 562,

217–22, 2018).

In an immortalized haploid human

cell line, they used C RISPR-Cas9 to

knock in 4,000 tiny variants in millions

of cells at once in vitro. The genome is

cut at the same spot in each cell, but each

cell’s genome receives a different variant.

To promote HDR, they also knocked out

the ligase4 gene, disabling the NHEJ

repair pathway—a step that yielded a

threefold gain in efficiency, Findlay says.

Finally, since all the cells’ knock-ins are

different, they sequenced the cells deeply,

covering the same genomic region mil-

lions of times, to make sure they actu-

ally knocked in the 4,000 variants they

wanted to study. They sequenced at two

time points, and deduced that the knock-

ins that didn’t come up in the sequenc-

ing at the second time point were ones

that interfered with the gene’s function,

because the cells carrying them must

have died.

TRY IT: Findlay’s team had the DNA

oligos for the 4,000 variants manufac-

tured for them on a microarray. Yo u can

buy arrays of 6,000 to 250,000 oligos,

so consider getting more bang for your

buck by combining multiple experi-

ments on the same array, says Find-

l ay. Their lab pays about $5,000 for

100,000 oligos.

This strategy comes with limita-

tions: it has so far only been used to

knock in single-nucleotide variants,

and all the edits need to be in the same

gene. The method works best when

editing a fairly narrow region of DNA,

about 110–120 base pairs, because lon-

ger DNA oligos would have too many

errors, Findlay says. It’s also important

to sequence very deeply to make sure

that you account for the full number of

variants you intended to knock in. g

BRCA1

Haploid

human cells

Genome

editing

Cell

survival

Sequencing

of variants

SNV library +

Cas9/gRNA

Cas9, multiplex repair

Library of all possible SNVs

Day 5 Day 11

SEARCH FOR SURVIVORS: Researchers

knocked in all 4,000 known single-nucleotide vari-

ants (SNVs) of the BRCA1 gene in millions of cul-

tured cells. Then, they sequenced all the variants --

once fi ve days after knocking them in, and again six

days after that. They deduced that variants that dis-

appeared on the second round of sequencing had

interfered with the gene’s function, causing the cells

that carried them to die.